MICROSCOPY RESEARCH AND TECHNIQUE 32~459-497 (1995)

Spermatogenesis in Nonmammalian Vertebrates
JEFFREY PUDNEY
Fearing Re...
460

J. PUDNEY

cold-blooded (poikilotherm) or warm-blooded (homeotherm).
Since the structural association between germ ce...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

B

$.

dG

+@

sG--@

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Fig. 3. Schematic diagram showing general feature...
462

J. PUDNEY

when these cysts rupture and the released spermatozoa
must then pass down the lobule, through the immature...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

463

Fig. 4. Amphioxus testis. A Germinal epithelium with a layer of
immature...
464

J. PUDNEY

Fig. 5. Polyspermatocystic testis of Myxine sp.

goes early stages of oogenesis. The posterior region of
t...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

465

Fig. 6. Germinal zone of S . acunthias testis showing cysts a t various ...
466

J. PUDNEY

Fig. 7.
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

containing a specific number of germ cells and Sertoli
cells. Initially, mito...
468

J. PUDNEY

Fig. 8. Secondary spermatogonia possess irregularly shaped nuclei with clumps of chromatin and
contain num...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

469

Fig. 9. Primary spermatocytes have round nuclei with evenly dispersed ch...
470

J. PUDNEY

Fig. 10. Spermiogenesis in testis of S . acanthias. A Round spermatids with spherical nuclei and abundant ...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

471

Fig. 11. Spermiogenesis in S. acanthias. A: Acrosome initially develops ...
472

J. PUDNEY

Fig. 12. Development of filamentous bundles in spermatids of S.
acanthias. A Fibrous bundles associated wi...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

473

Fig. 13. A characteristic feature of spermatids is the formation of a lo...
474

J. PUDNEY

Fig. 14. Spermiogenesis in S. acanthias. A Acrosome forms a loose cap over the elongated tip of the
nucleu...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

475

B

Fig. 15. Cross-section through a bundle of mature spermatids located ...
476

J. PUDNEY

with transfer of spermatophores to the efferent ducts.
The remaining Sertoli cells either become incorpora...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

cies a t the electron microscope level, e.g., Lebistes reticulatus (Asai, 197...
478

J. PUDNEY

Fig. 18. Various stages of spermatogenesis in L.macrochrius. A
Cyst containing primary spermatocytes. B: R...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

and in some cases to surround the periphery of the
nucleus; (2) an intercentr...
480

J. PUDNEY

tei, 1984) and I . punctatus (Poirier and Nicholson,
1982). The centrioles lie parallel to one another and...
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES

481

Fig. 19. Organization of N . maculosus testis. A Seminiferous lobules co...
482

J. PUDNEY

spermatozoa embedded in their apical cytoplasm (Fig.
19E). Sertoli cells are either sloughed with the sper...
Fig. 20. Low power electron micrographs of N. maculosus testis
showing (A) Primary spermatogonia (SP)with irregularly shap...
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
Spermatogenesis in Nonmammalian Vertebrates
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Spermatogenesis in Nonmammalian Vertebrates

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Spermatogenesis in Nonmammalian Vertebrates

  1. 1. MICROSCOPY RESEARCH AND TECHNIQUE 32~459-497 (1995) Spermatogenesis in Nonmammalian Vertebrates JEFFREY PUDNEY Fearing Research Laboratory, Brigham and Women's Hospital, Haruard Medical School, Boston, Massachusetts 02115 KEY WORDS Anamniotes, Spermatocysts, Amniotes ABSTRACT Spermatogenesis appears to be a fairly conserved process throughout the vertebrate series. Thus, spermatogonia develop into spermatocytes that undergo meiosis to produce spermatids which enter spermiogenesis where they undergo a morphological transformation into spermatozoa. There is, however, variation amongst the vertebrates in how germ cell development and maturation is accomplished. This difference can be broadly divided into two distinct patterns, one present in anamniotes (fish, amphibia) and the other in amniotes (reptiles, birds, mammals). For anamniotes, spermatogenesis occurs in spermatocysts (cysts) which for most species develop within seminiferous lobules. Cysts are produced when a Sertoli cell becomes associated with a primary spermatogonium. Mitotic divisions of the primary spermatogonium produce a cohort of secondary spermatogonia that are enclosed by the Sertoli cell which forms the wall of the cyst. With spermatogenic progression a clone of isogeneic spermatozoa is produced which are released, by rupture of the cyst, into the lumen of the seminiferous lobule. Following spermiation, the Sertoli cell degenerates. For anamniotes, therefore, there is no permanent germinal epithelium since spermatocysts have to be replaced during successive breeding seasons. By contrast, spermatogenesis in amniotes does not occur in cysts but in seminiferous tubules that possess a permanent population of Sertoli cells and spermatogonia which act as a germ cell reservoir for succeeding bouts of spermatogenic activity. There is, in general, a greater variation in the organization of the testis and pattern of spermatogenesis in the anamniotes compared to amniotes. This is primarily due to the fact there is more reproductive diversity in anamniotes ranging from a relatively unspecialized condition where gametes are simply released into the aqueous environment to highly specialized strategies involving internal fertilization. These differences are obviously reflected in the mode of spermatogenesis and this is particularly true of the stage of spermiogenesis where the morphology of the species-specific spermatozoon is determined. Moreover, unlike amniotes, many anamniotes display a spermatogenic wave manifest, depending upon the species, either at the level of the cyst or seminiferous lobule. This variation in the organization of the testis makes certain anamniotes perfect models for investigating germ cell development and maturation. For instance, the presence of a spermatogenic wave provides an opportunity to manually isolate discrete germ cell stages for analysis of specific Sertoli/germ cell interactions. Furthermore, for many anamniotes, germ cells mature in association with a morphologically poorly developed Sertoli cell. This seeming independence of Sertoli cell regulation allows the in vitro culture of isolated germ cells of some species of anamniotes through several developmental stages. Thus, due either to the anatomical organization of the testis, or structural simplicity of the germinal units, nonmammalian vertebrates can provide excellent experimental animal models for investigating many basic problems of male reproduction. 0 1995 Wiley-Liss, Inc. INTRODUCTION Throughout the vertebrate series it would appear that germ cell development and maturation proceeds in a similar fashion. Thus, spermatogonial stem cells divide to produce generations of spermatogonia which enter the spermatogenic cycle. Spermatogonia develop into spermatocytes which undergo meiosis to produce spermatids. These spermatids then proceed through a morphological metamorphosis during spermiogenesis to form spermatozoa. When, however, spermatogenesis is compared between the different classes of vertebrates it is obvious this process can be accomplished in a variety of ways. These differences in the pattern of 0 1995 WILEY-LISS, INC. spermatogenesis depend primarily on several parameters which are not necessarily mutually exclusive; (1) the organization of the testis, i.e., the anatomical disposition of the primary germinal compartment; (2) the relationship between germ cells and Sertoli cells during the spermatogenic cycle; (3) whether fertilization is external or internal; and (4) whether the species is Received May 31, 1994; accepted in revised form August 12, 1994. Address reprint requests to Dr. Jeffrey Pudney, Fearing Research Laboratory, Brigham and Women's Hospital, 250 Longwood Avenue, Boston, MA 02115.
  2. 2. 460 J. PUDNEY cold-blooded (poikilotherm) or warm-blooded (homeotherm). Since the structural association between germ cells and Sertoli cells during spermatogenesis largely dictates the morphological appearance of the germinal compartment, these two factors will be discussed together. THE ORGANIZATION OF THE TESTIS AND SERTOLUGERM CELL RELATIONSHIPS It is now well established that Sertoli cells play a pivotal role during spermatogenesis. A recent review is available dedicated to the Sertoli cell in nonmammalian vertebrates (Pudney, 1993). For this present report, therefore, information on the Sertoli cell will be restricted to aspects of this cell which affect or impinge on the pattern of spermatogenesis in different classes of nonmammalian vertebrates. THE VERTEBRATE TESTIS In the phylum, chordata, nonmammalian vertebrates include the jawless fishes, lampreys and hagfish (Agnatha); cartilaginous fishes, sharks, rays, and chimeras (Chondrichthys); the bony fishes (Osteichthys); newts, salamanders, frogs, toads, and caecilians (Amphibia); turtles, snakes, lizards, crocodiles, alligators (Reptilia), and the birds (Aves). In the protochordate Amphioxus the testes are formed from metamerically arranged coelomic pouches (Fig. 1).As vertebrates evolved, however, these rows of testes tended to form localized compact organs. In most vertebrates the testes are paired except for the Agnatha in which fusion of the testes has occurred and there are several species of bony fishes which possess a single testis. The shape of the vertebrate testis varies from extremely elongated, as in most cartilaginous and bony fishes, to round or oval as in many amphibia, reptiles, and birds. For some urodelean amphibia and bony fishes, however, the testes are composed of distinct lobes. In nonmammalian vertebrates the testes lie within the body attached by a mesorchium to the body wall. Usually the testes are isolated structures but can be associated with other tissue elements such as hemopoietic tissue, the epigonal organ which invests the testes in selachians or the fat body attached to the testes of anuran amphibia. An interesting feature in bony fishes is the occurrence of ambisexual species which possess both an ovary and a testis (Fig. 2). These gonads may be present simultaneously or develop sequentially. Those species in which the testis develops first are termed protandrous, while those where the ovary develops first are protogynous. The vertebrate testis is composed of two distinct compartments. One consists of the interstitial tissue, containing blood vessels, lymphatics and Leydig cells which secrete the male hormones or androgens. The second is the seminiferous compartment which contains the germinal epithelium defined as a tripartite structure consisting of an acellular basement membrane, germ cells and Sertoli cells (Grier, 1993). The association of germ cells with a somatic element, i.e., Sertoli cell of the germinal epithelium, is an evolution- Fig. 1. Amphiorus testes develop from coelomic pouches. This testis is filled with mature spermatozoa. Fig. 2. Ambisexual teleost containing an ovary (0)and testis (T). ary conserved feature of the vertebrate testis and even occurs in the testis of Amphioxus (Pudney, 1993). ORGANIZATION OF THE TESTIS Within the vertebrate series the general structural appearance of the testis can be divided into two distinct patterns, one present in the anamniotes (fish, amphibia) the other in amniotes (reptiles, birds, mammals). Anamniotes In these nonmammalian vertebrates, the unit of spermatogenesis is a spermatocyst, usually referred to as a cyst (von La Valette, St. George, 1876). Incipient
  3. 3. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES B $. dG +@ sG--@ @ + Fig. 3. Schematic diagram showing general features of cyst formation in anamniotes. A: Initially a Sertoli cell (SC) becomes associated with a primary spermatogonium (PG). B: The Sertoli cell completely surrounds the germ cell to produce a nascent cyst. C: The primary spermatogonium undergoes repeated mitotic divisions to produce secondary spermatogonia (SG) enclosed by Sertoli cytoplasm which forms the wall of the cyst. D Germ cell maturation then proceeds with the eventual production of a n isogeneic clone of spermatozoa which are often orientated with their heads towards the Sertoli cell nucleus. E: Spermiation is accomplished by rupture of the cyst. F: The Sertoli cell then undergoes degeneration. (Reproduced from Pudney, 1993, with permission of Cache River Press.) cysts are formed when a primary spermatogonium becomes engulfed by a Sertoli cell (Fig. 3). The primary spermatogonium then undergoes numerous mitotic divisions to produce secondary spermatogonia. The cyst is now composed of a mass of germ cells surrounded by Sertoli cytoplasm which forms the wall of the cyst. Germ cell maturation then occurs with formation of an isogeneic clone of spermatozoa which are released by rupture of the cyst wall. Following spermiation, in most anamniotes, Sertoli cells usually degenerate. In bony fishes and amphibia, cysts develop within larger structures that develop from sperm collecting ducts. In the literature, these blind-ending pouches have been referred to as either lobules, tubules, ampullae, or follicles. Recently, a consistent and unifying terminology has been proposed for describing the anamniote testis (Grier, 1993). In this study, the lobule was defined as the anatomically correct term for applying to the germinal compartment of the testis in most bony fishes and all amphibia. 46 1 Studies on the morphology of the testis of chondrichthyan fishes have also described the germinal compartment as consisting of cysts contained within lobules (or follicles or ampullae). This organization of the testes in these species, however, has been recently shown to be anatomically incorrect (Grier, 1992). This study was based on the observation that the germinal comDartment in the vertebrate testis is composed of a i e p i thelium (germinal) which by definition rests on a basement membrane that sequesters it from the surrounding interstitial tissue. Called the extracellular matrix hypothesis, it was shown that, chronologically, the production of a basement membrane by the germinal compartment differed in the testes of vertebrate groups. Utilizing this hypothesis, it was reported that a major factor in how the anamniote testis is organized was when the germinal elements became isolated from the interstitial tissue, by the secretion of a basement membrane, to form a separate germinal compartment. Thus, in the cartilaginous fishes, when germ cells (primary spermatogonia) and precursor somatic (Sertoli) cells initially come together they lie free within the interstitial tissue. These cells are not enclosed by a basement membrane and so do not represent a germinal compartment. Then the basement membrane is secreted t o surround and isolate the Sertoli cell and germ cells (now secondary spermatogonia) from the interstitial tissue to produce a distinct germinal compartment, the spermatocyst. In the chondrichthyan testis, therefore. cvsts are not Dresent within lobules. but occur as isolatid structure; Since the chondrichthyan testis is composed of a mass of cysts, it is called polyspermatocystic (Grier, 1992). Similarly, the agnathan testis is also made up of numerous individual cysts, i.e., polyspermatocystic. The composition of cysts, however, differs between the two vertebrate classes. Cysts in the agnathan testis all contain a single generation of isogeneic germ cells. By contrast, the chondrichthyan cysts contain many (although fixed in number) Sertoli cells, each of which is associated with a clone of germ cells. These individual Sertoli cells plus their cohort of germ cells present within a cyst have been called spermatoblasts (Callard, 1991). The term spermatoblast was initially used to define a functional unit of spermatogenesis consisting of a Sertoli cell with its associated germ cells within the mammalian seminiferous epithelium (Von Ebner, 1871). It should be noted that in the chondrichthyan testis, each cyst is composed of spermatoblasts which all contain germ cells at roughly the same stage of development. For the agnatha, most species of bony fishes, and anuran amphibia, the cysts are stationary within the testis. By contrast, in the chondrichthyes, several species of bony fishes and urodelean amphibia, cysts move through the testis as they mature during spermatogenesis. The direction of this cystic migration, however, differs amongst these nonmammalian vertebrates. In urodeles, cysts form in the region of the lobule proximal to the central sperm collecting duct. During spermatogenesis, as cysts mature they move distally towards the blind end of the lobule. Spermiation occurs
  4. 4. 462 J. PUDNEY when these cysts rupture and the released spermatozoa must then pass down the lobule, through the immature region, to enter the sperm duct. For certain species of bony fishes, however, the reverse occurs with nascent cysts located at the blind end of the lobule which then migrate, as they mature, down towards the sperm duct. On reaching the sperm duct these cysts break down to release spermatozoa either as free entities or as naked (spermatozeugmata) or encapsulated (spermatophores) bundles. In the chondrichthyes it is the cyst which migrates through the testis. Developing cysts originate in a germinal zone, the location of which varies in the testes of different chondrichthyan species (Pratt, 1988). For example, in the spiny dogfish Squalus acanthias, the germinal zone is found on the dorsolateral surface of the gonad. During spermatogenesis, as cysts mature, they migrate radially through the testis t o the opposite side by which time they are filled with spermatozoa. Spermiation then occurs and the cysts degenerate. This pattern of cyst development has been called diametrical since it occurs across the diameter of the testis (Pratt, 1988). It should be mentioned that the migration of cysts in lobules of bony fishes and urodeles or testes of chondrichthyans does not occur by movement of the cysts themselves. Cysts are passive structures and it is presumably the continual formation of cysts and/or growth and elongation of sperm ducts which cause their migration either along the lobule or through the testis. Also, during spermatogenesis, in some anamniotes, e.g., urodeles, although all cysts contain synchronously developing germ cells, the lobule itself could consist of different regions composed of cysts at different stages of maturation, i.e., an immature zone with spermatogonial cysts and a mature zone with spermatogenically active cysts. Furthermore, the stage of maturation reached by cysts in the mature zone may vary from lobule to lobule within the same testis, resulting in production of a spermatogenic wave. A spermatogenic wave also occurs in the chondrichthyes that involves the progressive maturation of cysts as they migrate through the testis. This results in a distinct zonation of spermatogenesis in the testis of these species. Amniotes In amniotes there is no cystic form of spermatogenesis and therefore the testis has been defined as postspermatocystic (Grier, 1993). Instead, the seminiferous epithelium is composed of a stable population of Sertoli cells associated with successive stages of germ cell maturation. For all amniotes the germinal compartment is composed of seminiferous tubules which are single structures except for birds in which extensive branching may occur. The number and length of these tubules vary greatly among anamniotes. The immature germ cells, the spermatogonia, are located a t the base of the seminiferous epithelium while spermatocytes and spermatids occur at successively higher levels. Mature, elongate spermatids border the lumen of the tubules into which they are released during spermiation. Therefore, unlike the anamniote testis where cysts contain germ cells a t the same stage of develop- ment, in the amniote testis there is a stratification of germ cells at different phases of maturation within the seminiferous epithelium. Furthermore, in anamniotes, following spermiation, Sertoli cells undergo degeneration, whereas in amniotes, Sertoli cells represent a permanent feature of the seminiferous epithelium. There is, in general, less variation in the organization of the testis and pattern of spermatogenesis in the amniotes compared to the anamniotes. SEASONAL CYCLES In vertebrates the pattern of spermatogenesis varies according to whether: (1) species are cold-blooded (poikilotherm), e.g., fish, amphibia, reptiles, or warmblooded (homeotherm), e.g., birds, mammals; and (2) reproduction is seasonal. Poikilotherm vertebrates undergoing reproductive seasonal cycles exhibit postnuptial spermatogenesis. This is because spermatogenesis begins soon after the conclusion of the breeding season. In contrast, spermatogenesis essentially stops in homeothermic vertebrates following the breeding season. For these species, therefore, spermatogenesis is prenuptial because recrudescence does not occur until the start of the next breeding season. In general, all feral vertebrates exhibit a seasonal cycle of reproductive activity. This is more pronounced for species that inhabit temperate zones than those living in more stable habitats, e.g., tropical, or benthic environments. For temperate poikilotherms which practice postnuptial spermatogenesis, germ cell development begins in the summer and then proceeds either slowly throughout winter, or ceases completely during winter to be reinitiated in the spring. In homeotherms, following the breeding season, there is complete testicular involution and regression of the germinal epithelium with spermatogonia (usually) as the remaining germ cells. It should be pointed out that few descriptive studies have been carried out on the changes the testes of nonmammalian vertebrates undergo throughout their annual reproductive cycles. All amniotes possess a permanent seminiferous epithelium which contains a resident population of Sertoli cells and a constant population of germ cells, i.e., primary spermatogonia which act as stem cells and are responsible for annual reinitiation of spermatogenesis. By contrast, the only anamniotes reported to possess a permanent germinal epithelium are anurans (Burgos and Vitale-Calpe, 196713; Lofts, 1984). This is because, although the apical Sertoli cytoplasm is shed at spermiation, the basal portion remains intact and regenerates from one breeding season to the next. For other anamniotes there is degeneration of cysts following spermiation and so these germinal units have to be constantly replenished. Recent observations, however, suggest that a permanent germinal epithelium occurs in some teleosts with persistence of a few Sertoli cells and germ cells (spermatogonia) from one breeding season to the next (Grier, 1993). Several reviews are available on the organization of the testis in nonmammalian vertebrates (Callard, 1991; Grier, 1993; Pilsworth and Setchell, 1981; Pudney, 1987; Roosen-Runge, 1977).
  5. 5. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 463 Fig. 4. Amphioxus testis. A Germinal epithelium with a layer of immature germ cells at the periphery (arrowheads) and mature spermatozoa in the lumen. B: Spermatogonia are poorly differentiated with few organelles except for several large mitochondria. C: Sper- matozoa appear primitive and possess a small acrosome (arrowhead), and a single large mitochondrion (MI at the base of the nucleus and surrounding the flagellum. EXTERNAL VERSUS INTERNAL FERTILIZATION In general, nonmammalian vertebrates that practice external fertilization produce a larger number of poorly differentiated spermatozoa compared t o those which engage in internal fertilization. The spermatozoa of external fertilizing species may be considered primitive, but a number also produce gametes with highly specialized features such as the acrosomal filament of petromyzonids (Nicander and Sjoden, 1971). Also, spermatozoa of teleosts could be considered primitive since they lack an acrosome. The absence of this structure, however, is due to the method of fertilization in which spermatozoa gain access to the ovum via a micropyle, thus making the presence of an acrosome obsolete. trioles orientated roughly a t 90" to each other, one of which (presumably the distal centriole) gives rise to the long flagellum. The flagellum contains a typical axoneme consisting of a 9 + 2 arrangement of doublets. A single large mitochondrion is located a t the base of the nucleus and appears to partially surround the centriole region (Fig. 4). It would appear that spermatogenesis proceeds until the testis is filled with mature spermatozoa (see Fig. 1)at which time the gonad (presumably) ruptures to release the gametes to the external environment. PROTOCHORDATES In Amphioxus, the testes occur as a metamerically arranged series along the length of the body (Fig. 1). Each testis contains a typical germinal epithelium with layers of developing germ cells present a t the periphery and mature spermatozoa occupying the central cavity of the testis (Fig. 4A). Germ cells develop in association with Sertoli cells (see Pudney, 1993). Spermatogonia appear poorly differentiated with nuclei containing finely dispersed chromatin, apparently lacking a distinct nucleolus and few cytoplasmic organelles except for several large mitochondria with lamelliform cristae (Fig. 4B). The spermatozoa posses a rounded nucleus with clumps of condensed chromatin and a small, cap-shaped acrosome located on the anterior pole (Fig. 4C). This acrosome appears to be separated from the nucleus by a clear zone containing a flocculent, homogenous substance. The posterior pole of the nucleus contains a fossa which houses two cen- AGNATHA The agnatha consist of the lampreys (Petromyonidae) and hagfish (Myxinoidae). Presumably due to the difficulty in obtaining specimens and lack of commercial interest in these fish, few studies are available on spermatogenesis in this group of vertebrates. Lampreys spend a number of years as larvae, called ammocetes (which is a nonparasitic feeding stage) before they metamorphose into mature animals. Although gender is established in the larval stage, gonadal growth is postmetamorphosis and in some species is accomplished in a short period of time, i.e., 6-9 months. Mitotic activity of germ cells begins following metamorphosis, and cysts develop (Larsen, 1965; Weisbart et al., 1978). As spermatogenesis proceeds, the testis becomes a mass of cysts containing spermatozoa. The mature testis eventually fills the body cavity since, as lampreys spawn once and then die, other body organs atrophy. Most myxinoid species do not possess a discrete breeding season since they live in a constant environment in the sea at 50-100 meters or more. Hagfish juvenile males are progynous because during sexual differentiation the anterior portion of the gonad under-
  6. 6. 464 J. PUDNEY Fig. 5. Polyspermatocystic testis of Myxine sp. goes early stages of oogenesis. The posterior region of the gonad remains sexually indifferent until, a t a late stage in growth, cysts develop containing spermatogonia. At this phase the anterior ovarian structures generally undergo degeneration. In both myxinoids and petromyzonids the testis is composed of a mass of cysts, i.e., polyspermatocystic (Grier, 1992; Fig. 5). Sperm ducts are lacking in agnathans and spermatozoa are shed into the body cavity where they exit to the outside via the abdominal pore. In myxinoids, germ cell development is synchronous within individual cysts although different stages of spermatogenesis can be found between cysts (Dodd and Sumpter, 1984). An early light microscope study on spermatogenesis in Myxine glutinosa is available (Cunningham, 1891). It has been reported that the most primitive of polyspermatocystic testis occurs in the hagfish Eptatretus stouti (Grier, 1993). This is a mesenteric type of testis with regions containing spermatocysts which are interconnected by bridges of tissue identical to the mesorchium. Nascent cysts are derived from isolated spermatogonia and Sertoli cells lying free within the interstitial tissue (Tsuneki and Gorbman, 1977). Prior to meiosis a basement membrane is produced to isolate Sertoli cells and spermatogonia from the interstitial tissue, resulting in the formation of cysts. Few electron microscopic studies have been carried out on the agnathan testis and so information on the ultrastructure of spermatogenesis is lacking. Several reports, however, are available on the fine structure of spermatozoa in a few lamprey species, Lampetra fluuiatilis (Nicander and Sjoden, 1971) and Lampetra planeri (Follenius, 1965; Stanley, 1967). These descriptions are extremely similar for both species. The long nucleus is rod-shaped and possesses an acrosomal complex. This complex consists of a simple acrosomal vesicle positioned on the tip of the nucleus, a dense subacrosomal ring and an axial nuclear canal lined by invaginated nuclear membrane that runs the length of the nucleus and into the flagellum. This endonuclear canal contains an undulating central fiber that extends into the flagellum where it is bound by apposing inner and outer nuclear membranes. Apparently, during fertilization, an acrosome reaction occurs, resulting in the extrusion of the long, preformed central fiber. This fiber, enclosed by the plasma membrane of the spermatozoon, has been referred to as the acrosomal tubule or filament (Nicander and Sjoden, 1971).The extension of the central fiber is thought to be involved in spermatozoal penetration of the egg. Lamprey spermatozoa do not possess a discernible midpiece. The nuclear implantation fossa contains 2 centrioles both aligned nearly parallel with the longitudinal axis of the spermatozoon. One centriole forms the basal body, from which the axoneme of the flagellum develops, while the other remains as an accessory centriole. The flagellum contains a typical axoneme consisting of a 9 -t2 arrangement of doublets. The doublets possess coarse fibers apposed to their outer surfaces. A number of elongate mitochondria are located along the proximal region of the flagellum. CHONDRICHTHYES The cartilaginous fishes are composed of two unequal subclasses, the Elasmobranchii (sharks, dogfish, skates, rays) with approximately 600 species and the Holocephali (ratfish) with only 28 known species. As for agnathans this group of vertebrates has received little attention from reproductive biologists. The detailed process of spermatogenesis is, therefore, only known for a few species of elasmobranchs and even less for holocephalans. It is apparent from the literature that cyst development and spermatogenesis, at least as reported at the light microscope level, is fairly similar for several different species of chondrichthyans: Cetorhinus maximus (Matthews, 1950); it should be mentioned that in this account an error in observation was made by Matthews in that cells described as “spermatogonia” represent Sertoli cells while the “primary spermatocytes” correspond to spermatogonia: Hydrolagus colliei (Stanley, 1963);Scyliorhinus caniculus (Dobson and Dodd, 1977; Mellinger, 1965; Stanley, 1966); Torpedo marmorata (Stanley, 1966); Squalus acanthias (Callard et al., 1985; Holstein, 1969) and Heterodontus portusjacksoni (Jones and Jones, 1982). The nuclear changes associated with germ cell development and maturation have been described for several species of elasmobranchs (Moore, 1895) In S. acanthias, gonocytes and undifferentiated Sertoli cell precursors are located in the germinal zone on the rostra1 aspect of the testis. Nascent cysts form when these Sertoli cells engulf primary spermatogonia within cytoplasmic processes (Fig. 6A). According to the extracellular matrix hypothesis (Grier, 19921,spermatocysts are not produced until the basement membrane is formed which isolates the Sertoli/germ cell unit from the interstitial tissue. By the time this happens both cell types have undergone several mitotic divisions resulting in the formation of a spermatocyst consisting of a single layer of Sertoli cells and secondary spermatogonia (Fig. 6B). Cysts then be-
  7. 7. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 465 Fig. 6. Germinal zone of S . acunthias testis showing cysts a t various stages of development. A: Cysts form when a Sertoli cell (arrow) engulfs a gonocyte (arrowhead). Nascent cysts are composed of primary spermatogonia (P) surrounded by Sertoli cells ( ) B: Fully S. formed cyst with a lumen and basal layer of secondary spermatogonia and Sertoli cells. One cyst (C) has become attached to the system of sperm excurrent ducts (arrowhead). come attached to the system of sperm ducts in the testis (Fig. 6B). Sertoli and germ cells undergo repeated mitotic divisions to form a mass of cells within the cyst, which now contains a lumen. These cells eventually become organized into a peripheral layer of secondary spermatogonia surrounded by Sertoli cytoplasm with Sertoli nuclei bordering the lumen of the cyst (Fig. 7A). For T. marmorata, however, Sertoli and germ cells form less conspicuous concentric layers (Stanley, 1966). At this stage the number of secondary spermatogonia approximates the number of Sertoli cells in the cyst. Sertoli cells now cease dividing, which heralds the appearance of spermatoblasts within the cyst. This occurs when each Sertoli cell encompasses a single spermatogonium with cytoplasmic processes (or possibly a clone of spermatogonia connected by intercellular bridges) which effectively isolates germ cells from each other. The spermatogonia continue to divide, causing the cyst to increase in size. Identifiable spermatoblasts, consisting of a Sertoli cell plus its cohort of isogeneic germ cells, can now be identified in cysts (Fig. 7B). During this period, Sertoli nuclei enlarge and move from their adlumenal position towards the periphery of the cyst wall. By the time this migration of Sertoli nuclei has been accomplished meiosis has occurred and spermiogenesis proceeds. It is interesting to note that during the initial development of cysts, mitotic divisions appear to occur within groups of spermatogonia (Fig. 7A). These groups could possibly represent clones of germ cells connected by cytoplasmic bridges. During meiosis, however, division appears to be initiated by a group of germ cells and then proceeds as a wave to involve other germ cells present in the cyst (Fig. 7C). During spermiogenesis there is a great deal of translocation of germ cells within the spermatoblast. This eventually results in a single layer of spermatids lining the inner face of the spermatoblast with their tails projecting into the spermatoblast cavity (Fig. 7D,E). How this movement of germ cells within the spermatoblast is accomplished is unknown. The cyst still possesses a patent lumen which is bound by the apical margins of the Sertoli cells. During spermiogenesis, spermatids elongate and begin to form loose bundles within the spermatoblast with their tails now projecting into the lumen of the cyst (Fig. 7F). Spermatids progressively form tighter bundles as they mature, until a t the end of spermiogenesis they have aggregated into a closely packed mass located in a pocket of apical Sertoli cytoplasm (Fig. 7G-I). Spermiation involves release of the mature gametes along with the apical portion of Sertoli cytoplasm. Spermatozoa exit the cyst via the sperm duct which has now acquired a patent opening. Following spermiation the Sertoli cells forming the cyst undergo fatty degeneration. There are species-specific variations in chondrichthyan testicular organization (Hoar, 1969). This includes major differences in the location of the germinal zone and consequently migration of spermatocysts through the testis for different species (Pratt, 1988). Quantitative estimates of spermatogenesis are facilitated in chondrichthyans since germ cells can be easily identified and hence counted in cysts as they mature. The number of divisions spermatogonia undergo are species-specific. Thus, S. acanthias has 13 spermatogonial divisions, two of them type A and eleven involving type B spermatogonia (Holstein, 1969). The number of mitotic divisions carried out by both Sertoli cells and spermatogonia are fixed, resulting in cysts
  8. 8. 466 J. PUDNEY Fig. 7.
  9. 9. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES containing a specific number of germ cells and Sertoli cells. Initially, mitotic divisions are synchronized between spermatogonia and Sertoli cells through the first 9 divisions (Stanley, 1966). Sertoli cells then cease dividing, at which time the cyst contains approximately 500 of these cells. Spermatogonia continue to undergo four more cycles of mitoses before they enter meiosis. The end product of all these divisions is 64 spermatids associated with each Sertoli cell (Stanley, 1966). This results roughly in 32,000 spermatozoa per mature cyst. For Sphyrna tiburo, it has been estimated that a mature spermatocyst contains approximately 460 spermatoblasts, each possessing 64 spermatozoa resulting in 29,000 spermatozoa per spermatocyst (Parsons and Grier, 1992). A rare case of germ line chromatin diminution has been reported to occur during spermatogenesis in H . colliei (Stanley et al., 1984). At metaphase I in meiosis of primary spermatocytes, a mass of heterochromatin accumulates at the side of the metaphase plate. This clump of heterochromatin is eventually passed into one of the secondary spermatocytes during cytokinesis at anaphase I. As the nuclear membrane is restored, a double membrane (possibly of nuclear origin) forms around the mass of heterochromatin which is then termed the chromatin diminution body. By the end of the second meiotic division this body ends up in the cytoplasm of one of the four spermatids where it remains unchanged until midspermiogenesis. At this time the chromatin body is discarded by means of apocrine exocytosis and is engulfed by the Sertoli cell. Seasonal changes in spermatogenesis in elasmobranchs have been reviewed (Dodd and Sumpter, 1984; Parsons and Grier, 1992; Wourms, 1977). Due to the difficulty in obtaining sufficient numbers of specimens throughout the reproductive cycle, few species, mainly restricted to sharks, e.g., S. acanthias (Simpson and Wardle, 1967)S. tiburo (Parsons and Grier, 1992),have been completely and adequately studied. For many species a seasonal cycle of spermatogenesis manifests itself by the development of a zone of breakdown. This zone, consisting of degenerating cysts, was first described in S. acanthias (Simpson and Wardle, 1967). This zone develops annually, in the spring, due to spermatogonial degeneration and proceeds to migrate 467 through the testis. Since the zone represents an absence of germ cells, when the next breeding season occurs there is a short arrest in spermatogenic activity. In contrast, a zone of degeneration is absent from S. tiburo (Parson and Grier, 1992). For this shark there is widespread complete regression of the testis which only contains spermatogonial cysts during the period of testicular involution. Few electron microscopic studies have been carried out on the chondrichthyan testis. Ultrastructurally, the primary spermatogonia of S. acanthias are characterized by large, spherical, pale-staining nuclei with few chromatin clumps and a paucity of organelles in the cytoplasm. Secondary spermatogonia appear smaller in size with irregularly shaped, dark staining nuclei containing many clumps of chromatin and possess many mitochondria, plus profiles of both smooth (SER) and rough (RER) endoplasmic reticulum (Fig. 8). Cytoplasmic bridges were observed connecting groups of secondary spermatogonia (Fig. 8). These connections are maintained by clones of germ cells throughout spermatogenesis (Fig. 9). Small desmosome-like junctions were often detected between spermatogonia and Sertoli cells. At later stages of spermatogenesis, however, no junctional specializations were observed between Sertoli cells and germ cells. Primary spermatocytes appear cytologically more differentiated than spermatogonia. They possess large spherical nuclei with finely dispersed chromatin and the cytoplasm contains numerous mitochondria, cisterns of SER, a well-developed Golgi apparatus as well as an assortment of vacuoles and dense bodies (Fig. 9). An occasional lipid droplet and centriole were observed in some spermatocytes. An excellent detailed study of the fine structure of spermiogenesis has been carried out for S. suckleyi (Stanley, 1971a,b). Round spermatids contain spherical nuclei with fine granular chromatin. A number of mitochondria and a well-developedGolgi apparatus as well as an extensive system of SER is present in the cytoplasm (Fig. 10A). There appears to be a distinct polarization of some organelles such as the Golgi apparatus which appear to be located adjacent to the developing flagellum (Fig. 10B). A bundle of juxtanuclear filaments is commonly detected in round spermatids (Fig. lOC). Interestingly, the nuclear envelope next to the Golgi apparatus exhibits structural modifications, related to adhesion of the acrosome to the nucleus, before the appearance of the acrosomal vesicle (Stanley, 1971a). In this region Fig. 7. Cyst development in the testis of S. acanthias. A: Mitotic the nuclear membranes become closely apposed and activity of both germ cells and Sertoli cells results in growth of the cyst which is now composed of several basal layers of secondary sper- hence appear dense in appearance under the electron matogonia surrounded by Sertoli cytoplasm. Nuclei of the Sertoli cells microscope (Fig. 10D). This membrane modification, (arrowheads) form a distinct layer bordering the lumen. Note: mitotic therefore, is the initial event that gives nuclear polardivisions appear t o occur amongst distinct groups of germ cells. B: Spermatoblast formation occurs when the cytoplasm of a single Ser- ity and is unusual since normally this results from the toli cell (arrowheads) surrounds and isolates a cohort of secondary formation of the acrosome. The acrosomal vesicle despermatogonia. C Meiosis occurs as a wave that progresses around velops from the Golgi apparatus located close to the the cyst. D: and E: Spermatoblasts sectioned longitudinally (D) and developing flagellum and becomes attached to the transversely (E) containing a cohort of isogeneic spermatids in the dense nuclear membrane (Fig. 11A). As the acrosome lumen. Arrowheads indicate the Sertoli cytoplasm forming the walls of the spermatoblast. F-I: These micrographs illustrate the progres- vesicle enlarges to form the acrosome it actually insive condensation of elongating spermatids (F)into loosely organized dents upon the nucleus which accommodates by formbundles with their heads orientated toward the basement membrane ing a shallow depression (Fig. 11B). An acrosomal of the cyst (G) that coalesce into closer packed bundles (HI to evengranule does not appear to develop (Fig. 11B). The nutually form a tight mass of mature spermatids embedded in Sertoli clear envelope continues to condense over the surface of cytoplasm (I).
  10. 10. 468 J. PUDNEY Fig. 8. Secondary spermatogonia possess irregularly shaped nuclei with clumps of chromatin and contain numerous organelles and are connected by cytoplasmic bridges (arrowhead) S . acunthias. the nucleus while at the same time fibrous material becomes deposited along the outer nuclear membrane to form the fibrous nuclear sheath (Fig. 1lC). Eventually this sheath will cover most of the nucleus except for the posterior pole. There also appears to be a distinctive pattern of chromatin condensation as spermatids mature. This begins with aggregation of the chromatin into coarse granules (see Fig. 10A) which possibly is initiated in the anterior pole of the nucleus beneath the acrosome. This process continues but seems to be limited to the central region of the nucleus, resulting in a peripheral zone of nucleoplasm virtually devoid of chromatin granules (see Figs. 10A and 1 1 0 . At a later stage of nuclear condensation, the chromatin granules become transformed into a mass of randomly orientated short chromatin fibers (see Fig. 14A). Initially the centrioles lie close to the Golgi apparatus and near the periphery of the cell (Fig. 11A). For S. suckleyi it was reported that as the acrosome develops, the nucleus undergoes a rotation of approximately 180" that results in the acrosome becoming located at roughly the opposite pole within the cell in respect to the developing flagellum (Stanley, 1971a). This now defines the anterior pole of the cell, containing the acrosome, and posterior pole containing the flagellum. At this stage two filamentous bundles, one striated and one nonstriated, become associated with both distal and proximal centrioles (Fig. 12A). These fibrous bundles are precursors of the axial midpiece rod (a characteristic feature of chondrichthyan spermatozoa) that will eventually connect the spermatid nucleus to the developing flagellum. A dense material is also deposited around the flagellum where it projects from the spermatid that presumably represents the ring centriole or annulus (Fig. 12A). For S. acanthias, however, it appears that nuclear rotation occurs at a later stage of spermatid differentiation since the fibrous bundles, plus a developing flagellum, could still be detected in a lateral position (with respect to the nucleus) within the cell (Fig. 12A). Following nuclear rotation, these fibrous bundles increase in length to eventually insert in a deep fossa located at the posterior pole of the nucleus (Fig. 12B). With further elongation of the spermatid the bundles of filaments forming the axial component of the midpiece become twisted upon each other. This spiraling, however, appears to only affect the nonstriated fibrous bundle which wraps itself around the striated bundle of filaments (Fig. 12C). A characteristic feature of round spermatids is the formation of a long cytoplasmic projection (Fig. 13A).This process contains
  11. 11. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 469 Fig. 9. Primary spermatocytes have round nuclei with evenly dispersed chromatin, abundant organelles and are connected by cytoplasmic bridges (arrow). Also present in this micrograph is a portion of the Sertoli cytoplasm (arrowheads) forming the walls of the spermatoblast S. acunthius. a system of fine filaments with an accumulation of dense material a t the tip. Often these projections can be seen to indent the Sertoli cytoplasm of the spermatoblast (Fig. 13B). These structures, therefore, would seem to act as some kind of anchoring device. As the spermatid develops the nucleus becomes elongated. The acrosome now forms an elongated cap over the pointed tip of the nucleus (Fig. 14A). Interestingly, the outer acrosomal membrane is closely associated with what appears to be a number of electron-dense bodies that are evenly spaced along this membrane. A short, subacrosomal rod (perforatorium) develops to occupy the space between the nucleus and acrosome (Fig. 14A). The subacrosomal rod is attached at the base of the acrosome and extend towards, and slightly beyond the tip of the nucleus. The nuclear chromatin has now condensed to form a mass of coarse strands orientated along the longitudinal axis of the nucleus (Fig. 14A). During nuclear condensation the nuclear volume decreases from about 180 p3 to about 10 p3 and the sur. face area from around 154 p' to 58 p2 (Stanley, 1971b). It was proposed that the excess nuclear envelope was discarded by the release of vesicles from the posterior margin of the nuclear membrane. For S. acanthias the posterior region of the nucleus is dilated and devoid of chromatin and encloses the proximal portion of the fibrous bundles (see Fig. 12B). Moreover, the inner nuclear membrane facing the fibrous bundle is rich in nuclear pores (see Fig. 12B). This development is reminiscent of the redundant nuclear envelope present in mammalian spermatozoa. With further maturation of the spermatid, the elongate nucleus assumes a helical shape and a t this time the fibrous nuclear sheath disappears. This spiralling of the nucleus also includes the chromatin which becomes orientated into a helical configuration (Fig. 14B). This helical pattern begins posteriorly to advance anterially and is accompanied by a transient progressive loosening of the nuclear membrane adjacent to the chromatin just acquiring a spiral pattern (Fig. 14B; Stanely, 1971b). As nuclear elongation and chromatin condensation is occurring, the two fibrous elements of the axial midpiece rod fuse. At the same time elongate mitochondria aggregate around the axial midpiece rod to form a mitochondrial sheath that now delineates the midpiece. These mitochondria are associated with glycogen granules and are enclosed by a fibrous midpiece sheath. The posterior end of the midpiece rod inserts on the basal body (distal centriole) of the flagellum. At doublets 3 and 8 the fibrous material surrounding the centrioles extends down as two longitudinal columns and accompanies the axoneme the length of the flagellum (Fig. 15). As spermatids elongate, the cytoplasm is displaced posteriorly and is reflected to form a sleeve of cytoplasm surrounding the proximal portion of the tail. For
  12. 12. 470 J. PUDNEY Fig. 10. Spermiogenesis in testis of S . acanthias. A Round spermatids with spherical nuclei and abundant organelles present in lumen of the spermatoblast. Note the pattern of chromatin condensation which occurs centrally within the nucleus leaving a peripheral ring of nucleoplasm virtually devoid of chromatin granules. B: Golgi apparatus associated with the developing flagellum. C : A long bundle of filaments present in the cytoplasm of round spermatids. D Nuclear membrane adjacent the Golgi apparatus condenses (arrowheads) and becomes modified for attachment of the acrosome. Arrows point to what appear to be acrosomal vesicles budding from the Golgi apparatus S. acanthias.
  13. 13. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 471 Fig. 11. Spermiogenesis in S. acanthias. A: Acrosome initially develops in the vicinity of the developing flagellum. B Acrosomal vesicle attached to nucleus lacks an acrosomal granule. C:Fibrous sheath (arrowheads) deposited along outer surface of nucleus. Note the pattern of chromatin condensation into a mass of coarse granules that occurs centrally producing a clear peripheral zone of nucleoplasm. some species this remnant cytoplasm is discarded during spermiation, e.g., S. suckleyi (Stanley, 1971b) whereas for others it is retained, e.g., H . portusjacksoni (Jones et al., 1984). During the process of spermiogenesis the early round spermatids are located in the lumen of the spermatoblast. As spermatids undergo elongation, however, they become embedded within individual recesses formed by Sertoli cytoplasm. At this stage the 64 spermatids are loosely arranged but as the germ cells mature they move closer and closer together to eventually form a compact bundle that apparently resides in a single pocket formed by Sertoli cytoplasm (Fig. 15). The mechanisms involved in bundle formation of chondrichthyan spermatozoa has been investigated in the ratfish H . colliei (Stanley and Lambert, 1985). It was shown that when elongate spermatids indent the Sertoli cell to occupy a cytoplasmic recess, filaments present in Sertoli cytoplasm become concentrated around the developing germ cell. As spermatids elongate further, these filaments condense into bundles which become aligned parallel to the long axis of each spermatid and extend down to the base of the Sertoli cell. Eventually a filamentous sheath is formed immediately surrounding the acrosome and anterior nuclear region of the spermatid. It was suggested that a close transmembrane adherence existed between the Sertoli cell filaments and tip of the spermatid acrosome. Through the use of NBD-Phallacidin and specific antibodies to actin and myosin it was shown the filamen- tous sheath contained both contractile proteins. This suggested that during the formation of spermatid bundles the system of acto-myosin represented a contractile structure involved in the orientation of spermatid nuclei towards the periphery of the spermatoblast. It was postulated these filaments were analogous to those developed between Sertoli cells and spermatids in the mammalian testis. Initially described by Brokelmann (1961) and called junctional specializations (Flickinger and Fawcett, 19671,they are now commonly referred to as ectoplasmic specializations (Russell, 1977). Compaction of the spermatids into tight bundles was thought to occur by a mechanism of endocytosis of Sertoli plasma membrane located between the germ cells. This uptake of Sertoli plasma membrane would cause the spermatids to move closer together, resulting in their coalescence. Stability of the individual spermatids, as this process occurred, was maintained by an extracellular dense material present between the tip of the acrosome and Sertoli plasma membrane which was thought to act as an adhesive substance. A recent study has reported on the fate of mitochondria during spermiogenesis in H . colliei (Stanley and Lambert, 1990). Apparently there is fragmentation of elongate mitochondria into smaller mitochondria. These mitochondria then either migrate to surround the midpiece of the developing spermatid or become enclosed in large vacuoles. These vacuoles are limited by a double membrane (with ribosomes along the inner membrane). The vacuoles containing excess mitochon-
  14. 14. 472 J. PUDNEY Fig. 12. Development of filamentous bundles in spermatids of S. acanthias. A Fibrous bundles associated with developing flagellum. The dense material (arrowheads) deposited at the cell membrane in the vicinity of the flagellum represents the ring centriole or annulus. Note that in this spermatid, nuclear rotation has not yet occurred since although the acrosome is well-developed the flagellum projects laterally from the cell and has not yet moved to the posterior pole of the nucleus. B The striated (arrowheads) and nonstriated bundles of filaments insert in a deep fossa at the posterior pole of the nucleus. Note that the posterior region of the nucleus that surrounds the filamentous bundles is dilated, devoid of chromatin with numerous nuclear pores in the inner membrane that faces the bundles of filaments. C: This micrograph shows the nonstriated bundle of filaments (arrowheads) is twisted around the striated bundle of filaments.
  15. 15. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 473 Fig. 13. A characteristic feature of spermatids is the formation of a long cytoplasmic process. A: This process contains an array of fine filaments. B: Often these processes indented the Sertoli cytoplasm forming the wall of the spermatoblast and may represent an anchoring device S. ucanthius. dria are eliminated and phagocytosed by the Sertoli cell. The morphology of chondrichthyan spermatozoa is conservative and appears similar for those species which have been studied, e.g., the sharks S. suckleyi (Stanley, 1965a, 1964,1971b);H . portusjacksoni (Jones et al., 1984); Prionace glauca, Carchurhinus falciformis, Centroscymnus owstoni and Chlamydoselachus anguineus (Hara and Tanaka, 1990); rays Dasyatis kuhlii and Dasyatis garouaensis (Hara and Tanaka, 1990) and the chimaera H . colliei (Stanley, 1965a, 1983) and Chimaera phantasma (Hara and Tanaka, 1990). The acrosome tends to be small and elongated with a deep posterior indentation that fits loosely over the pointed tip of the nucleus. The long nucleus is attached to the flagellum via the midpiece which contains the midpiece rod surrounded by a small number of mitochondria. The anterior of the midpiece rod inserts into the nuclear implantation fossa while the centriole complex is positioned at the posterior end of the midpiece rod. Unlike many other vertebrates, therefore, flagellar elements are not present in the midpiece of chondrichthyan spermatozoa. The flagellum is composed of a typical axoneme consisting of a 9 + 2 array of doublets plus a characteristic feature of chondrichthyan spermatozoa, the longitudinal columns (Fig. 15). Species differences in spermatozoon morphology primarily involve flagellar structures. Thus, in H . colliei it was reported that the proximal centriole is absent from the basal body (Stanley, 1965a, 1983).Major variations, however, involve the number and appearance of the longitudinal columns (Hara and Tanaka, 1990). In sharks the flagellum is symmetrical with a central ax- oneme and two equal-sized, oval longitudinal columns a t doublets 3 and 8. Spermatozoa of rays also have two longitudinal columns but these are round in cross-section. For chimeric species, however, the development of the longitudinal column adjacent doublet 8 is suppressed, appearing only as a short rod, whereas the one associated with doublet 3 extends the length of the flagellum (Stanley, 1983; Hara and Tanaka, 1990). This arrangement results in an asymmetric axoneme in spermatozoa of the chimera. Glycogen granules apparently are commonly present in chondrichthyan spermatozoa. For elasmobranchs this occurs as a small number of granules dispersed between the mitochondria (Stanley, 1971b). In H. colliei, however, abundant glycogen granules were detected along the length of the flagellum (Stanley, 1983). A characteristic feature of chondrichthyan spermatozoa is their helical shape. The long, pointed head appears spirally twisted giving it a corkscrew appearance. This helical twisting also involves the acrosome, and elements of both the midpiece, and flagellum. In S. acanthias the axoneme is straight but the microtubules of the doublets follow a helical course (Stanley, 1971b). By contrast, in H . colliei both axoneme and microtubules exhibit a spiral pattern along the length of the flagellum (Stanley, 1983). How spiralization of these components of the spermatozoa is accomplished is unknown. The helical configuration of the head and flagellar elements in chondrichthyan spermatozoa are related to motility. Forward progression in these spermatozoa is not achieved by lateral bending of the flagellum but by rotating around their long axis. The function of the longitudinal columns in chondrichthyan
  16. 16. 474 J. PUDNEY Fig. 14. Spermiogenesis in S. acanthias. A Acrosome forms a loose cap over the elongated tip of the nucleus and a perfortorium (arrowhead) develops in the subacrosomal space. Note chromatin is organized as a mass of fibers orientated parallel to the long axis of the nucleus. B Elongate spermatid showing helical pattern of chromatin fibers and loosening of the nuclear membrane adjacent regions of chromatin acquiring a spiral configuration. spermatozoa is unknown although it has been suggested they may restrict lateral movement of the flagellum allowing the spermatozoa to progress by revolving about its long axis (Stanley, 1971b). OSTEICHTHYES The osteichthyes are composed of two classes of bony fishes, the Crossopterygii and Actinopterygii. The Crossopterygii include the Dipnoi (lung fishes) and Coelacanthini, represented by a living fossil, the coelacanth (Latimeria). The Actinopterygii consist of the Chondrostei (sturgeons), Holostei (bowfin, garpike) and the Teleostei. Of all these groups, the Teleostei are by far the most important and extensive with more than 20,000 extant species. Despite the economic importance of teleosts and their ready availability as research subjects, the reproductive biology of relatively few species has been adequately studied. The bony fishes are a very diverse group of vertebrates with extremes of reproductive strategies ranging from external to internal fertilization. This results in great variation in the organization of the testis. Due to this, the nomenclature describing the different structural arrangements of the teleost testis has been somewhat confusing and inconsistent (Grier, 1981). To rectify this condition a more uniform nomenclature has been proposed, based on the distribution of spermatogonia within the seminiferous compartment (Grier et al., 1980; Grier, 1981, 1993). The teleost testis is organized as a system of either anastomosing tubules, possibly restricted to lower fish or lobules present in other species (Grier, 1993). Furthermore, both types of testes can be further defined by the distribution of spermatogonia within the tubules or lobules (Grier et al., 1980; Grier, 1981). First, there is the unrestricted spermatogonial testis type characterized by the presence of spermatogonia along the entire length of the tubule or lobule (Fig. 16A). During the nonbreeding season, primary spermatogonia and Sertoli cells occur as solid cords within the tubule or lobule which contains a vestigial lumen. With the onset of spermatogenesis, cysts develop and line the tubules or lobules which now have a patent lumen. Spermiation results in the breakdown of the Sertoli cells which form the walls of the cyst to release spermatozoa into the lumen. This unrestricted spermatogonial testis type is found in most species of teleost. The second pattern of organization found in teleosts is the restricted spermatogonial testis type. This describes the situation where primary spermatogonia are confined to the distal region of the lobule only (Fig. 16B). During spermatogenesis the primary spermatogonia associate with Sertoli cells to form cysts. As germ cells mature, cysts migrate down the lobule toward the efferent sperm ducts. By the time spermatozoa have formed, the cyst is located near the sperm duct. Sper-
  17. 17. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 475 B Fig. 15. Cross-section through a bundle of mature spermatids located in a pocket formed by the Sertoli cytoplasm. This section passes through the axoneme illustrating the presence of the longitudinal columns (arrowheads) located at doublet 3 and 8. S. acunthias. miation ensues with release of spermatozoa into the sperm duct. Sertoli cells, depending on the species, either degenerate or become incorporated into the epithelium of the efferent ducts. The restricted spermatogonial testis type is only found in one group of teleosts, the Atheriniformes. Although Atheriniforme species all possess a common testicular structure, there is great diversity in their reproductive biology depending upon whether fertilization is external or internal. Atherinimorph teleosts practicing external fertilization include Fundulus heteroclitus (Selman and Wallace, 1986) and Oryzias latipes (Gresik et al., 1973). For these species, spermatogenesis is a fairly simple process. Primary spermatogonia occur at the blind end of the lobule and undergo several mitotic divisions before becoming associated with Sertoli cells to form cysts. As cysts mature they move down the lobule towards the efferent sperm ducts. Unlike other teleost species, during maturation of these cysts, there is little differentiation of Sertoli cells which remain remarkably undeveloped through the process of spermatogenesis. At the completion of spermatogenesis, cysts are located in the lobular region proximal to the efferent ducts. The Sertoli cells, forming the cyst, then fuse with epithelial cells lining the efferent ducts which results in release of the contained spermatozoa. Those atherinimorph species which engage in internal fertilization demonstrate a more complex process of spermatogenesis. An adaptation for this reproductive Fig. 16. Schematic diagrams illustrating spermatogenesis in the Osteichthyes. A: Unrestricted spermatogonial testis type characterized by the presence of spermatogonia (PG) along the length of the lobule. Cysts form when a Sertoli cell (SC) surrounds a primary spermatogonium. With the onset of spermatogenesis, cysts (C) mature and line the lobule. Spermiation occurs with rupture of the cyst and liberation of spermatozoa (SP)into the lobule lumen. B: Restricted spermatogonial testis type where spermatogonia (PG) are confined to the distal end of the lobule. Cysts form when a Sertoli cell (SC) surrounds a primary spermatogonium. As cysts (C) mature they migrate down the lobule towards the efferent ducts (ED). By the time cysts reach the efferent duct they contain spermatozoa. Spermiation results when the cyst fuses with the efferent duct. (Adapted from Grier, 1993.) strategy is the production of spermatozoa packaged either in naked (spermatozeugmata) or encapsulated (spermatophores) bundles which are introduced into the female at insemination. This results in complex morphological relationships developing between spermatids and Sertoli cells not seen in atherinimorph species with external fertilization, e.g., Lebistes reticulatus (Vaupel, 1929) Poecilia reticulata (Billard, 1970a) Poecilia latipinna (Grier, 1975a) Mollienisia latipinna (Van den Hurk et al., 1975). Only one atherinimorph species, Horaichthys setnai, produces true spermatophores, whereas all others develop spermatozeugmata (Grier et al., 1981). Spermatophores develop within cysts and the walls of these structures, which encapsulate the spermatozoa, are formed by a Sertoli cell secretion. The process of spermatophore formation has been described for H . setnai (Grier, 1984).Briefly, spermatogenesis is initiated and proceeds in a similar fashion to that occurring in species with external fertilization until the stage of spermiogenesis is reached. At this time the Sertoli cell hypertrophies, progressively acquiring a columnar shape, and becomes highly secretory. The released secretion then self-assemblesto form the capsule of the spermatophore which surrounds the spermatozoa within the cyst. Rupture of the cyst occurs
  18. 18. 476 J. PUDNEY with transfer of spermatophores to the efferent ducts. The remaining Sertoli cells either become incorporated into the efferent ducts or are sloughed and degenerate. Spermatozeugonata formation has been reported for Lebistes reticulatus (Winge, 1922; Vaupel, 1929) Poecilia latipinna (Grier, 1975a) and Mollienisia latipinna (Van den Hurk et al., 1975). As for other atherinimorphs, spermatogenesis proceeds normally up to spermiogenesis. During this phase the Sertoli cells hypertrophy, acquire a columnar shape and in these poeciliid species spermatids align themselves with their heads abutting the apical Sertoli cytoplasm to form the spermatozeugmata. For goodeide, however, i t is the developing flagellum that becomes associated with Sertoli cells, e.g., Ameca splendens, Ataenobius toweri, Characodon lateralis, Xenotoca eiseni (Grier et al., 1978). Spermatozoa are not held together by junctional specializations with Sertoli cells. Instead, Sertoli cell projections anchor spermatozoa at the lumenal margin of the cyst. When the mature cyst contacts the efferent duct, spermiation occurs and the contained spermatozeugmata are released. Seasonal reproduction is common for teleosts and the testis may exhibit large variations in size and histological appearance. The length of time following completion of spermatogenesis and spawning may vary from species to species (Borg, 1982; Ruby and McMillan 1970) or may occur directly following production of spermatozoa (Henderson, 1962). For some tropical species of teleosts there is no apparent seasonal fluctuation in spermatogenesis. There is diversity in the germ cells composing the regressed teleost testis. For many species the quiescent testis contains mainly spermatogonia (Shrestha and Khanna, 1978); for others either primary spermatocytes (Ahsan, 19661, spermatids (Rosenblum et al., 1987) or spermatozoa (Borg 1982; Ruby and McMillan, 1970). At the light microscope level the process of spermatogenesis has been described for a number of teleost species, e.g., Ictalurus nebulosus (Rosenblum et al., 1987); Couesius plumbeus (Ahsan, 1966); Gasterosteus aculeatus (Borg, 1982); Eucalia inconstans (Ruby and McMillan, 1970); Tandanus tandanus (Davis, 1977); Sciaenops ocellatus (Grier et al., 1987). In general, spermatogenesis is very similar for all these species of teleosts. For most teleosts spermatogonia are the prevalent germ cell present in the regressed testis. As recrudescence ensues, spermatogonia undergo repeated mitotic divisions and eventually become associated with Sertoli cells to form cysts. Spermatogenesis then proceeds and cysts containing maturing germ cells are distributed along the length of the lobule (Fig. 17). Eventually the cysts become filled with spermatozoa which are released into the lumen at spermiation resulting from rupture of the cyst. Few cysts can now be detected and the lobules contain scattered spermatogonia which will serve as stem cells for the next cycle of spermatogenesis. To reflect these changes in spermatogenesis, the following terms have been proposed to describe the lobules: production lobules to describe the lobule or part that contains maturing cysts, with or without mature spermatozoa in the lumen; transitional Fig. 17. Unrestricted spermatogonial testis type seminiferous lobule during the breeding season with a patent lumen and lined by cysts containing maturing germ cells in the testis of the teleost Pugellus sp. lobule to describe lobules containing spermatozoa with few scattered cysts; and storage lobule to describe lobules packed with spermatozoa and containing no cysts (Grier et al., 1987). Germ cell renewal in the lobules has been investigated in several species. It is generally accepted that a permanent population of primordial stem germ cells exist in the lobule wall. During each annual cycle of spermatogenesis these cells undergo mitosis to produce spermatogonia. The remaining stem cells provide a reservoir for future spermatogenic cycles. Other studies, however, have suggested there is a n annual migration of stem cells into the lobules from a n interstitial location. Migratory germ cells have been described in the interstitial tissues of C. plumbeus (Ahsan, 1966); E . inconstans (Ruby and McMillan, 1970); and Salmo gairdneri (Hurk et al., 1978). None of these studies were able to identify the original source of these germ cells and the morphological evidence (light microscopy), illustrating the entry of germ cells into the lobules is not convincing. A number of reports are available describing different aspects of spermatogenesis in various teleost spe-
  19. 19. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES cies a t the electron microscope level, e.g., Lebistes reticulatus (Asai, 1971; Gronberg and Telkka, 1968; Gronberg and Wartiovaara, 1972; Mattei and Boisson, 1966; Mattei et al., 1967);Oligocottus maculosus (Stanley, 1969); Upeneus prayensis (Boisson et al., 1969); Pimephales notatus (Schjeide et al., 1972); Albula vulpes (Mattei and Mattei, 1973); Gambusia affinis (Grier, 1975b);Poecilia latipinna (Grier, 1973, 1975a); Trichurus lepturus (Mattei and Mattei, 1976a); Anguilla australis schmidtii and A. dieffenbachii (Todd, 1976); Oryzias latipes (Grier, 1976; Hamaguchi, 1987); Lepodogaster lepadogaster (Mattei and Mattei, 1978a); Liza aurata (Brusle, 1981); Lepomis macrochirus (Sprando et al., 1988); Salmo gairdneri (Billard, 1972, 1983);Poecilia reticulatus (Billard and Flechon, 1969); Platichthys flesus (Jones and Butler, 1988a,b); Thymallus thymallus (Lahnsteiner et al., 1991); Rhodeus sericeus sinensis (Guan and Afzelius, 1991); Blennius pholis (Silveira et al., 1990);Ophidion sp (Mattei et al., 1989);8 species of blennidae (Lahnsteiner and Patzner, 1990);Paroncheilus sp (Mattei and Mattei, 1984);Zctalurus punctatus (Poirier and Nicholson, 1982);Brachydanio rerio (Kessel et al., 1983); a large number of cyprinids (Clerot, 1976; Thiaw et al., 1986; Thiaw and Mattei, 1989); several species of gobies (Fishelson et al., 1990). In general, the fine structure of germ cell development is very similar for all species studied. The spermatogonia are characterized by large regular nuclei usually containing a distinct nucleolus. In P. latipinna and P. notatus, spermatogonia contain annulate lamellae and the mitochondria are associated with a dense material of nuclear origin (Grier, 1975a; Schjeide et al., 1972). In several species of cyprinids, groups of mitochondria were located at specific sites in the cytoplasm of spermatogonia (Clerot, 1976). These mitochondria were related with a dense material (cement). Annulate lamellae were also often detected associated with this intermitochondrial cement. Nuage has been reported to occur in spermatogonia of B . pholis (Silveira et al., 1990)and 0. latipes (Hamaguchi, 1987).Spermatocytes possess a large oval nucleus with coarse chromatin and a distinct Golgi apparatus (Fig. 18A). Abundant microtubules occur in spermatocytes of some cyprinodonts (Thiaw and Mattei, 1989) Centriolar division occurs in spermatocytes. Round spermatids contain a number of mitochondria dispersed throughout the cytoplasm with two centrioles located a t the periphery of the cell (Fig. 18B). Annulate lamellae have been described in spermatids of B. pholis (Silveira et al., 1990), and glycogen granules and mitochondria1 cement in spermatids of blennidae (Lahnsteiner and Patzner, 1990). The distal centriole lies closer to the plasma membrane than the proximal centriole which is usually aligned at roughly 90” with respect to the distal centriole. It has been reported that the distal centriole appears to be anchored to the plasma membrane by a number of fibers (Mattei and Mattei, 1976a, 1978a). Usually a well-developed Golgi apparatus is associated with the centrioles. The flagellum then begins to develop from the distal centriole. Flagella, however, have been detected in spermatogonia and spermatocytes (Billard and Flechon, 1969). In several teleost species it has been shown that 477 nuclear pores are distinct in young spermatids but disappear when the flagellum begins to develop (Grier, 1975b; Gronberg and Wartiovaara, 1972). As round spermatids mature there occurs a distinct spatial arrangement of organelles that will eventually define the structural appearance of the resultant spermatozoon. Thus, the centrally located nucleus moves to occupy a position close to the cell surface that (usually) is adjacent to the Sertoli cell cytoplasm. Then the two centrioles plus the developing flagellum migrate from their position at the periphery of the cell to become located close to the nucleus. During this transloction the distal centriole carries the flagellum through the cytoplasm, resulting in the formation of a cytoplasmic canal (Fig. 18C). The flagellum is covered by reflected plasma membrane and is therefore separated from the rest of the cell body (Fig. 18C). In some teleost species the nucleus undergoes approximately a 90” rotation and centrioles become located within a nuclear depression. For other species, this nuclear rotation does not occur and centrioles do not become associated with a nuclear depression. At this time the previously dispersed mitochondria also move to become preferentially associated with the developing flagellum. Due to the presence of the cytoplasmic canal these mitochondria do not, as in many other vertebrates, come into close physical contact with the flagellum. For some species a submitochondrial net, consisting of filaments enclosed between two membranes, develops between the mitochondria and plasma membrane of the cytoplasmic canal (Dadone and Narbaitz, 1967; Grier, 1975b). A different location of mitochondria has been described where these organelles surround the base of the nucleus in P. flesus (Jones and Butler, 1988a) and a number of species of blennidae (Lahnsteiner and Patzner, 1990). The mitochondria occupied recesses developed by the nucleus. In several species of gobies it has been reported that small mitochondria present in round spermatids disappear to be replaced by 2 large mitochondria (Fishelson et al., 1990). Also in these teleosts a peculiar system of alternating RER and SER was described that formed rings in the cytoplasm. As the spermatids matured, however, these rings of ER disappeared. A stranger location of mitochondria occurs in B . pholis with several of these organelles located at the anterior pole of the nucleus, resulting in the absence of a midpiece (Silveira et al., 1990). In T . thymallus the mitochondria of the midpiece becomes fused to form a chondriosome which is wound around the proximal region of the axoneme (Lahnsteiner et al., 1991). In L . lepadogaster it has been reported that a regional differentiation occurs at the anterior surface of spermatid nuclei (Mattei and Mattei, 1978a). In this area a number of lamellae occur dispersed between the thickened inner and outer nuclear membranes which were termed “lamelles perinucleaires intermembranaires.” It was suggested this represented an atavistic development of the acrosome which has been lost in the evolution of the teleosts. Also in S. gairdneri, an abortive acrosomal vesicle seems to develop that disappears later in spermiogenesis (Billard, 1983). The association of the developing flagellum with the
  20. 20. 478 J. PUDNEY Fig. 18. Various stages of spermatogenesis in L.macrochrius. A Cyst containing primary spermatocytes. B: Round spermatids possess bullet-shaped nuclei with a deep implantation fossa (arrowhead) at the posterior pole. A cross-sectional profile of the proximal centriole (arrow) can be seen in this spermatid. C: Flagellum located in the cytoplasmic canal. (Micrographs courtesy of R.L. Sprando and L.D. Russell.) nucleus signals chromatin condensation as well as ini- cent condensed chromatin remains intact. Eventually tiating morphological changes, e.g., elongation, that the nuclear membrane reforms to preferentially eneventually result in the species-specific shape of the close only the condensed chromatin, thus eliminating head of the mature spermatozoon. It has been shown, in excess membrane. several species, that the condensation of chromatin in Concomitant with changes in nuclear volume, as the nuclei of spermatids, as they mature, occurs in a spermatids mature, is also a reduction in cell volume. specific pattern that often begins in the region adjacent For many teleosts this excess cytoplasm is sloughed, to the developing flagellum (Boisson et al., 1968b;Rus- towards the end of spermiogenesis, as a residual body sell, 1988; Sprando et al., 1988; Stanley, 1969). A fine which is usually engulfed by the Sertoli cell. For L. structural study of nuclear differentiation during sper- macrochirus, however, the process of elimination of exmiogenesis has been undertaken for Oncorhynchus tra cytoplasm is accomplished by the formation of tshawytscha (Zirkin, 1975).The nuclei of early sperma- membrane-bound cytoplasmic packets which migrate tids contain randomly distributed chromatin fibers to the cell surface and are extruded (Sprando et al., similar to other (somatic) cells. As spermatids mature 1988). It was estimated that during spermiogenesis in the chromatin progresses to highly orientated thick fi- L. macrochirus, both nuclear and cytoplasmic volumes bers and unusual, irregular ribbon-like elements. Nu- of spermatids were reduced some 80% and 92%, respecclei of mature spermatozoa were extremely dense and tively, resulting in a total reduction of 87% in cell volshowed little substructure. It was suggested these ume. structural changes were related to the process of reFollowing division of the centriole and initiation of placement of nuclear histones by protamines. flagellum development, a transitory intercentriolor As the chromatin condenses there is obviously a lamellated body appears in many species, e.g., L. reticgreat reduction in the volume of the nucleus. This re- ulatus (Asai, 1971; Gronberg and Telkka, 1968; Mattei sults in a large area of redundant nuclear membrane. and Mattei, 1966); U. prayensis (Boisson et al., 1969); The removal of this excess membrane has been de- P. Zatipinna (Grier, 1973, 1975a); 0. latipes (Grier, scribed in several teleost species. This can involve the 1976). This structure has been suggested to be derived release of small vesicles from either the entire surface, from satellites associated with the proximal centriole e.g., 0. maculosus (Stanley, 1969)or base, e.g.,P. flesus (Grier, 1973, 1976). The intercentriolar lamellated (Jones and Butler, 1988a) of the nucleus as the chro- body accompanies the centrioles as they migrate from matin condenses. A more interesting process, however, the cell periphery to the nucleus. The implantation has been described for L. macrochirus (Sprando et al., fossa, therefore, houses a centriolar complex consisting 1988). Apparently the nuclear membrane associated of: (1)a proximal centriole from which, for various spewith clear, chromatin-free nucleoplasm undergoes in- cies, microtubules radiate towards the nucleus to attermittent breakdown. The nuclear membrane adja- tach to the nuclear membrane of the implantation fossa
  21. 21. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES and in some cases to surround the periphery of the nucleus; (2) an intercentriolar lamellated body disposed between the proximal and distal centriole; and (3) a distal centriole (basal body) which forms the axoneme of the flagellum. It has been reported that the microtubles emanating from the proximal centriole serve to stabilize the nucleus prior to chromatin condensation as well as the relationship between the centriolar complex and nucleus during nuclear morphogenesis and development of the implantation fossa (Grier, 1973).The microtubles are transient and disappear as soon as the implantation fossa has formed but before chromatin condensation has ceased. For most species the intercentriolar lamellated body appears during spermiogenesis. In a few other species, it has been reported to arise in spermatocytes (Grier, 1975b; Gronberg and Telkka, 1968). The proximal centriole and intercentriolar lamellated body are, for many species, temporary structures and degenerate a t a later stage of spermiogenesis. A few species, however, retain either the lamellated body, e.g., Pantodon buchholzi (VanDeurs and Lastein, 1973) or proximal centriole, e.g., 0. latipes (Grier, 1976). The exact function of the intercentriolar lamellated body is unknown. As yet, ectoplasmic specializations have not been observed between spermatids and Sertoli cells in the teleost testis (Sprando and Russell, 1987a). It was suggested that since spermatids did not enter into close physical contact with Sertoli cells, ectoplasmic specializations are not developed. Occasional “desmosomelike” structures have been reported between spermatocytes and Sertoli cells in L. macrochirus (Sprando and Russell, 198713). A rather remarkable observation has been reported for L . lepadogaster during spermatogenesis (Mattei and Mattei, 1978a). It was reported that mature spermatozoa were commonly seen penetrating spermatocytes and spermatids. Usually the spermatozoa penetrated the cytoplasm but occasionally also passed through the nucleus. A total of 6 spermatozoa were found penetrating a single spermatid. Apparently the embedded spermatozoa did not interfere with maturation of the germ cells and were released when the residual cytoplasm was shed. Another unusual report concerns a number of species of blennidae where immature spermatids were released from the cyst to mature in the testicular gland and/or spermatic ducts (Lahnsteiner and Patzner, 1990). The fine structure of spermatozoa of many species of Osteichthyes has been reported. For the Crossopterygii, information is available on the ultrastructure of several species of lungfish, e.g., Protopterus annectans (Boisson and Mattei, 1965a) and Neoceratodus forsteri (Jespersen, 1971). The head of the spermatozoon consists of a very elongated nucleus with a small anterior acrosome. Two rod-shaped structures extend from the tip of the acrosome, through the nucleus, to terminate in the cytoplasm. Each rod is enveloped by nuclear membrane. The midpiece contains two perpendicularly aligned centrioles. The proximal centriole is attached to the posterior margin of the nucleus, while the distal centriole gives rise to the flagellum. The axoneme is 479 composed of a typical 9 + 2 arrangement of doublets. Along the entire length of the flagellum two broad extensions or “wings” are present which contain a dense material. For Actinopterygii, the fine structure of spermatozoa has been studied for the Holostein Lepisosteus osseus (Afzelius, 1978).In this species the spermatozoa are not highly differentiated and may be considered primative, since they lack an acrosome, have a short midpiece with a single ring-shaped mitochondrion and lateral ridges or “wings” on the flagellum that are roughly in the plane of the two central doublets of the axoneme. The two centrioles are aligned perpendicular to each other and the proximal centriole is joined to a striated fibrous body which projects into the posterior fossa of the nucleus. A short cytoplasmic sleeve surrounds the intital portion of the flagellum which contains a 9 + 2 arrangement of doublets. The spermatozoa of the Teleostei, however, have been best studied at the electron microscope level, e.g., Cnesterodon decemmaculatus (Sotelo and TrujilloCenoz, 1958);L . reticulatus (Mattei and Boisson, 1966); J . lineata (Dadane and Narbaitz, 1967); Oligocottus maculosus (Stanley, 1969); Dactylopterus uolitans (Boisson et al., 1968a);P. reticulata (Billard, 1970b);F . heteroclitus, Cyprinodon variegatus (Yasuzumi, 1971); 0. latipes (Grier, 1976). Spermatozoa of species which practice external fertilization, e.g., 0. latipes are not highly developed morphologically and consist of either a round, bullet-shaped or slighly elongated head; a simple centriolar complex; few mitochondria; a poorly differentiated midpiece; and usually a lateral head to tail articulation. By contrast, species with internal fertilization produce more morphologically complex spermatozoa, e.g., P. reticulatus which have elongate heads, a highly modified centriolar complex, a well-developed midpiece associated with a large number of mitochondria, and caudal articulation of the head with the tail. The number of mitochondria associated with the midpiece of teleost spermatozoa varies from species to species, e.g., 4 in L. aurata (Brusle, 1981), and 6-10 for L . lepodogaster (Mattei and Mattei, 1978a). In some species a submitochondrial net is present, e.g., J . lineata (Dadone and Narbaitz, 1967). Species variation also exists in the morphology of the midpiece. For instance, in L. lepadogaster a portion of the midpiece is devoid of mitochondria (Mattei and Mattei, 197813). The flagellum of most species contains a typical 9 + 2 axoneme of doublets and often develops lateral ridges (“wings”) along the entire length. The axoneme of some other species, however, is composed of atypical 9 + 0 organization of doublets, e.g., Lampanyctus sp. (Mattei and Mattei, 1976b); Anguilla anguilla (Billard and Ginsburg, 1973; Ginsburg and Billard, 1972);Albula vulpes (Mattei and Mattei, 1973);A. australis schmidtii and A. dieffenbachii (Todd, 1976); Lycodontis afer (Boisson et al., 1967). Species variation also exists in the number of flagella developed by spermatozoa. Biflagellated spermatozoa are produced by Porichthys notatus (Stanley, 1965b);I . punctatus (Yasuzumi, 1971);Lampanyctus sp. (Mattei and Mattei, 1976b);L . lepadogaster (Mattei and Mattei, 1974,1978b);Paroncheilus sp. (Mattei and Mat-
  22. 22. 480 J. PUDNEY tei, 1984) and I . punctatus (Poirier and Nicholson, 1982). The centrioles lie parallel to one another and each participates in the development of an individual flagellum. Although a large number of teleosts, representing different families, produce biflagellated spermatozoa, the evolutionary significance (if any) of this feature is unknown. A more bizarre condition exists in Gymnarchus niloticus (Mattei et al., 1967b)and a number of species of mormyres (Mattei et al., 1972b) which produce aflagellated spermatozoa. Spermiogenesis appears normal but neither of the two centrioles, which lie between the nucleus and mitochondria, develops a flagellum. It was suggested these gametes carry out ameoboid movement. In a large number of different cyprinodonts it was reported there was great variation in the presence and absence of the dynein arms of axonemal doublets (Thiaw et al., 1986). Despite these differences all spermatozoa were found to be motile. A number of teleost species produces morpholgoically bizzare spermatozoa. In L. afer (Boisson et al., 1967) the proximal centriole develops to produce an intracellular flagellum. Furthermore, one of the proximal centriole microtubles becomes closely associated with the nuclear membrane to extend along the periphery of the nucleus. A similar condition occurs in A. uulpes (Mattei and Mattei, 1973) where the nuclear membrane penetrates the proximal centriole, causing disruption and dislocation of the microtubular triplets which become associated with the nuclear membrane. These then extend through the cytoplasm, associated with a projection of the nuclear membrane, to form what has been termed a pseudoflagellum. Furthermore, a large mitochondrion is not associated with the centrioles but is located at the anterior of the spermatozoon towards the lateral aspect of the nucleus. The spermatozoa of P. buchholzi are extremely aberrant (Van Deurs and Lastein, 1973). The centriolar complex consists of a single centriole and a lamellated cap-shaped body situated between the centriole and the nucleus. The midpiece is composed of 9 helical mitochondria1 threads, formed by end-to-end fusion of small, single mitochondria that alternate with 9 helical dense fibers. Posterior to the midpiece is a structure called the fenestrated sheath composed of inner and outer membranes penetrated by pores. This sheath surrounds the initial portion of the axoneme. Many species of anguillidae also produce extremely morphologically exotic spermatozoa, e.g., A. anguilla (Billard and Ginsburg, 1973; Ginsburg and Billard, 1972); A. australis schmidtii and A. diefenbachii, (Todd, 1976). The spermatozoa have a sickle- or crescent-shaped head tapering to a narrow neck region and a long flagellum. A short striated rod or appendage projects from the posterior region of the head near the origin of the flagellum. The proximal centriole develops and divides into two groups of 4 and 5 microtubular triplets which then course anteriorly along either the inner concave or outer concave aspects of the nucleus. A midpiece is not present because the single mitochondrion becomes displaced (in these species) to the anterior pole of the nucleus. The nucleus may partly surround the mitochondrion that lies in an indentation of its concave side. The presence of this mitochondrion, at the tip of the spermatozoon, gives a bulbous protuburance to the gamete. Patches of regular particles have been detected by freeze-fracture in the cell membrane covering the anterior head of spermatozoa in B . rerio (Kessel et al., 1983) and R. sericeus sinensis (Guan and Afzelius, 1991). It was suggested these regions may act as recognition andlor adhesion sites allowing spermatozoa t o fuse with the ova. AMPHIBIA The amphibia are composed of three extant orders: the Anura (frogs, toads) is the largest and more than two-thirds of amphibians belong to this group; Urodelia (newts, salamanders) and Apoda (caecilians) a small group of legless amphibians. Amphibians are the most primitive of terrestrial vertebrates and most species need to return to an aqueous environment to breed. Morphologically, the amphibian testis is similar to that of teleosts. Spermatogenesis occurs in cysts present in a seminiferous compartment called lobules. Based on spermatogonial distribution, three types of testes have been described for amphibia (Grier, 1993): one, in urodeles, in which the testis is divided into lobes and new lobules form from primary spermatogonia and their associated Sertoli cells within connections of the testicular lobes, e.g., Taricha granulosa. The other occurs in urodeles that do not have lobed testes and here primary spermatogonia and Sertoli cells are located in the lobular region proximal to the sperm duct, e.g., Necturus maculosus (Fig. 19A). The third is found in anuran species in which the primary spermatogonia are present along the length of the lobule (Fig. 24). Most amphibian species undergo a postnuptial spermatogenic cycle. Initiation of spermatogenesis occurs soon after spawning and is completed quickly, resulting in spermatozoa being retained within the testis prior to the next breeding season. Temperate amphibians undergo an annual spermatogenic cycle with maturation of spermatozoa occurring in the winter followed by spermiation in the spring breeding season. Urodeles Many urodeles exhibit a spermatogenic wave in the testis during the breeding season. This results in a spatial and temporal segregation of germ cells. Spermatogenesis begins at the caudal end of the testis and progresses towards the cephalic end. Lobules in sequential stages of maturation, therefore, can be found along the length of the testis with the least mature located in the cephalic region. Furthermore, spermatogenesis is confined to regions of the seminiferous lobule distal t o the main sperm duct. Lobular regions proximal to the sperm duct contain primary spermatogonia which act as a reservoir for future waves of spermatogenesis (Fig. 19B). In many urodeles, therefore, there is a maturational wave of spermatogenesis occurring longitudinally in a caudacephalic direction, plus a proximal-to-distal cycle of differentiating germ cells which eventually form the maturing segments of the seminiferous lobules which initiate seasonal testicular recrudescence. Spermatogenesis and cyst development has
  23. 23. SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES 481 Fig. 19. Organization of N . maculosus testis. A Seminiferous lobules contain an immature proximal segment composed of cysts filled with spermatogonia. Arrowheads point to sperm duct. B: Primary spermatogonia are large cells with irregularly shaped nuclei and undergo mitotic activity t o produce secondary spermatogonia. C: Mitotic divisions of spermatogonia result in growth of the cysts. D: Cross- section of seminiferous lobule containing cysts filled with secondary spermatogonia. Arrowheads point to Sertoli cytoplasm that forms the walls of the cysts. Note absence of a lumen in the lobule. E As sper: matogenesis proceeds, cysts migrate towards the distal end of the lobule where spermiation occurs by rupture of the cysts resulting in the lumen of the lobule containing bundles of spermatozoa. been described a t the light microscope level €or several species of urodeles, e.g., Desmognathus fuscus (Kingsbury, 1901); Taricha torosa (Miller and Robbins, 1954); Cynops pyrrohogaster pyrrohogaster (Tanaka and Iwasawa, 1979); N . maculosus (Pudney et al., 1983). In general, spermatogenesis proceeds in a similar fashion for most urodeles and resembles that occurring in teleosts. For instance, in N . maculosus, nascent cysts develop when a primary spermatogonium becomes associated with a Sertoli cell (Fig. 19B). Subsequent maturational divisions of spermatogonia results in an increase in size of the cysts (Fig. 19C). During spermatogenesis, cysts migrate toward the distal end of the seminiferous lobule. By the time this occurs, the cysts contain mature spermatozoa. Spermiation then occurs, exposing Sertoli cells with isogeneic bundles of
  24. 24. 482 J. PUDNEY spermatozoa embedded in their apical cytoplasm (Fig. 19E). Sertoli cells are either sloughed with the spermatozoa or undergo degeneration. A number of studies have reported on the fine structure of different aspects of spermatogenesis in several species of urodels, e.g., Pleurodeles wnltlii (Picheral, 1967, 1972a,b,c, 1979); D. fuscus (Lommen, 1970); Salamandra salamandra (Bergmann et al., 1982; Schindelmeiser et al., 1985); Ambystoma mexicanum (Keyhani and Lemanski, 1981; Miltner and Armstrong, 1983). Primary spermatogonia are large cells characterized by highly irregularly shaped nuclei and with the cytoplasm appearing relatively devoid of organelles except for a number of mitochondria, several small Golgi regions and an occasional lipid droplet (Fig. 20A). The secondary spermatogonia appear smaller with large spherical nuclei, numerous mitochondria, long cisternae of SER, several Golgi areas (Fig. 20B) and profiles of annulate lamellae (Fig. 21B). An interesting feature of these spermatogonia is that during the early stage of cyst formation, both develop abundant blunt projections a t their periphery that invaginate the surrounding Sertoli cytoplasm (Fig. 21A). It is assumed these represent anchoring devices that keep individual spermatogonia associated with their corresponding Sertoli cell during development of the cysts. At a later stage of cyst development the germ cells have a smooth surface (see Fig. 20B). It has been reported that as cysts mature the germ cells lose close physical contact with Sertoli cells (Pudney, 1993). Furthermore, at this time, germ cells develop filopodia and the intercellular space between Sertoli cells and germ cells widens (Fig. 22). It was suggested this occurred to allow movement and/or translocation of cells as the cysts become organized within the seminiferous lobule. Spermatocytes of S. salamandra have been reported to contain annulate lamellae and an occassional lipid droplet (Schindelmeiser et al., 1985). Furthermore, in this species, mitochondria were located at the periphery of spermatocytes and this disposition of these organelles was also observed in early spermatids. Proacrosomal granules were also reportedly detected in secondary spermatocytes. As yet, it would appear that junctional specializations have not been described between germ cells and Sertoli cells at any stage of spermatogenesis. The fine structure of urodelean spermatozoa has been best studied in P. waltlii (Picheral, 1967, 1972a,b,c, 1979). The spermatozoa possess an acrosoma1 cap that ends in either a sharp point or rounded knob and in some species develops a hooked barb, e.g., P. waltlii, Euproctus aspes. An elongated, conical subacrosomal space contains the perforatorium which consists of an axial rod that extends deep into the nucleus within an endonuclear canal. The nucleus has prominent ridges composed of nonchromatin material consisting of minute tubules packed together (Fig. 23A). Many species of urodeles display this ridge on the spermatozoal nuclei but which differs in composition ranging from a single bundle, e.g., P. waltlii to several, e.g., S. salamandra. The neck, or connecting piece appears as a long cylinder that fits into a deep fossa on the posterior pole of the nucleus, resulting in this region being mostly enclosed by a shell of chromatin. At the caudal end of the neck lie the two centiroles. The presence of a ring centriole (annulus) has been reported for P. waltlii (Picheral, 1972b) and D. fuscus (Lommen, 1970). The spermatozoa of urodeles are unusual since they develop an undulating membrane that extends almost the length of the flagellum (Fig. 23B). The flagellum consists of an axial fiber or rod (also called SUPporting filament) that extends from the neck region and the attached thin undulating membrane with the axoneme located a t the free edge along with a dense structure called the marginal filament (Fig. 2 3 0 . The midpiece contains the mitochondria which are associated only with the axial fiber and so do not surround the axoneme (Fig. 23D). In A. mexicanurn the mitochondria are smaller than in other species and so closely packed as to exhibit an almost crystalline arrangement (Keyhani and Lemanski, 1981). The flagellum of Hynobius nebulosus lacks mitochondria which are present in a protoplasmic bead located around the nucleus (Picheral, 1979). Anura Like urodeles, anuran spermatogenesis occurs in cysts located in seminiferous lobules (Fig. 24A). Initially cyst development progresses similarly to that occurring in urodeles and teleosts (Fig. 24B), e.g., Rana temporaria (Witschi, 1915); Rana esculenta (Lofts, 1964);Rana pipiens (Burgos, 1955; Rugh, 1939). There is a distinct difference, however, in the process of spermatogenesis that distinguishes these amphibia from urodeles. During spermiogenesis, as elongate spermatids become embedded in a single Sertoli cell, the formation of the spermatid flagellum signals rupture of the cyst. These open cysts now form the wall of the seminiferous lobule which is lined by highly differentiated Sertoli cells with bundles of spermatids deeply embedded in their apical cytoplasm (Fig. 24C). At this stage the seminiferous lobule resembles morphologically the germinal epithelium present in amniotes. During this period, at the periphery of the lobule, primary spermatogonia can be detected surrounded by Sertoli cells that are preparing to form cysts for the next round of spermatogenesis (Fig. 24A). Spermiation then ensues and in some species, e.g., R . esculenta only the apical portion of the Sertoli cell is shed along with the spermatozoa. The basal portion of the Sertoli cell, containing the nucleus, remains to develop the next generation of cysts. In these amphibian species, therefore, a permanent germinal epithelium can be said to be present. An unusual mode of spermatogenesis has been reported for Bombina uariegata (Obert, 1976).In this species, apparently germ cells develop in the absence of Sertoli cells and spermatozoa do not aggregate into bundles but appear as a mass of single cells. It was unknown whether this represents a primitive condition or is a peculiarity restricted to discoglossid amphibia. Electron microscope studies are available on spermatogenesis in several species of anuran amphibians, e.g., Bufo arenarum (Burgos and Fawcett, 1956; Cavicchia and Moviglia, 1982); Rana clamitans (Poir-
  25. 25. Fig. 20. Low power electron micrographs of N. maculosus testis showing (A) Primary spermatogonia (SP)with irregularly shaped nuclei which are poorly differentiated and possess a number of mitochondria and a n occasionally lipid droplet. Note development of nu- merous filopodia at periphery of the germ cell. B Secondary spermatogonia possess large spherical nuclei. Note that at this stage of development the cells exhibit a smooth outline with no formation of filopodia.

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