MICROSCOPY RESEARCH AND TECHNIQUE 32~459-497 (1995)
Spermatogenesis in Nonmammalian Vertebrates
Fearing Research Laboratory, Brigham and Women's Hospital, Haruard Medical School, Boston, Massachusetts 02115
Anamniotes, Spermatocysts, Amniotes
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.
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.
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).
In these nonmammalian vertebrates, the unit of
spermatogenesis is a spermatocyst, usually referred to
as a cyst (von La Valette, St. George, 1876). Incipient
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
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.
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
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
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
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
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.
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.
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).
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
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.
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
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.
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-
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-
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
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.
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-
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
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
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
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
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
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
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
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
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
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
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
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;
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
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.
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
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-
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.
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
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
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
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).
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
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-
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
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
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-
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
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
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.,
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
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.
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
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
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
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,
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
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
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-
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
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.
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
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.
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
SPERMATOGENESIS IN NONMAMMALIAN VERTEBRATES
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
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).
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
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-
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