Credit Seminar of above topic which has got all latest information upto 2018 & is very much helpful in Reproductive Physiology of Ruminats

1. CREDIT SEMINAR
ROLE OF FOLLICULAR FLUID FACTORS IN OVULATION IN
BOVINES
BY
NITISH A KULKARNI
18-M-AP-03
DIVISION OF ANIMAL PHYSIOLOGY, NDRI, KARNAL

2. OVERVIEW
FOLLICULAR FLUID- COMPOSITION
INTRODUCTION
ROLE OF BIOCHEMICAL COMPONENTS
INTRACELLULAR SIGNALLING CASCADES
GENE EXPRESSION IN BOVINE OVULATING FOLLICLE
CONCLUSION

3. INTRODUCTION
• Folliculogenesis is the process in which a recruited primordial follicle grows and
develops into a specialized Graafian follicle with the potential to either ovulate
its egg into the oviduct at estrus to be fertilized or to die by atresia
• Dominant follicle continues to grow and mature to the preovulatory stage while others
undergo atresia
• Successful ovulation requires developmentally competent oocytes which are released at
appropriate timing from the ovarian follicle
• Complex intercellular communication coordinates critical stages of oocyte maturation and
links this process with release from the follicle
• Ovulation is controlled through endocrine hormones, immune and metabolic signals,
intrafollicular paracrine factors from the theca, cumulus granulosa cells and oocyte itself

4. Precise mechanisms in Ovulation
 Increased Intra-follicular pressure
 Proteolytic enzyme activity on follicular wall
 Morphological changes in stigma favoring follicular rupture
 Perifollicular Ovarian smooth muscle contraction
 Vascular alteration in perifollicular vessels
 Changes in ovarian collagen bundles causes increased cumulus disintegration

5. ENDOCRINOLOGY OF FOLLICULOGENESIS &
OVULATION
(Rahe et al., 1980,Hillier et al., 1994, Kullick et al., 1999)

6. FOLLICULAR FLUID
• It is a source for nutrients and also in the physiological, biochemical and metabolic aspects
of the nuclear and cytoplasmic maturation of the oocyte and the process of ovulation
• Follicular fluid is an indicator of the secretory activities and metabolism of follicular cells
• Oocyte and granulosa cells in the ovary are bathed in a fraction of extracellular fluid called
Follicular Fluid, which accumulates in the antrum of ovarian follicles
• Biochemical composition of follicular fluid may influence Oocyte maturation, Quality,
Ovulation, Transport of the oocyte to the oviduct, preparation of the follicle for
subsequent corpus luteum formation and its functioning
(Tripathi et al., 2015, Gérard et al., 2002, Sato et al., 2000, Hafez, 2000)
• Follicular fluid originates mainly from the peripheral plasma by transduction across the
follicle basement lamina and accumulates in the antrum

7. Metabolic and ionic concentrations of follicular fluid in cattle, Values are
in Mean ± SE
Metabolic and ionic
constituents SMALL(< 6 mm)
FOLLICLE
MEDIUM(6-8mm) LARGE(>8mm)
Glucose (mg/dl) 40.92 ± 1.02 47.81 ± 2.14 54.44 ± 2.32
Total proteins (g/dl) 6.49 ± 1.34 6.78 ± 1.45 7.01 ± 1.39
Triglyceride
(mg/dl)
33.33 ± 2.53 30.69 ± 2.23 30.24 ± 1.41
Cholesterol (mg/dl) 29.81 ± 2.51 25.65 ± 2.07 22.48 ± 2.23
Lactate (mM) 14.4 ± 0.35 9.4 ± 0.35 5.6 ± 0.37
Sodium (mM) 142.5 ± 0.34 142.4 ± 0.63 141.0 ± 1.14
Potassium (mM) 10.1 ± 0.21 7.9 ± 0.28 6.0 ± 0.23
Chloride (mM) 105.0 ± 0.50 104.0 ± 0.60 102.9 ± 0.76
Urea (mg/dl) 15.62 ± 1.08 16.81 ± 0.82 16.93 ± 1.25
• Developmental potential of oocyte largely depends on these components
(Tripathi et al., 2015)

8. Classical hormones ROLE IN OOCYTE DEVELOPMENT, MATURATION & OVULATION
• Estradiol • Cytoplasmic maturation of oocytes
• Increase Synthesis of IGF-1 from Granulosa cells
• Promotes expression of LH receptors
• Progesterone • Critical for end stages of follicle development & ovulation
• Induces Prostaglandins (PG’s) E & F production
• LH • Supports thecal steroidogenesis, induces ovulation and corpus luteum
formation
• Stimulate meiotic maturation of oocyte
• FSH • Stimulation of primordial follicles, dominant follicle selection
• Stimulate cumulus expansion and to facilitate the production of the protease
plasmin
• Prostaglandin • PGF2 – Rupture of follicles
• PGE – Remodelling of follicular layers, terminating in CL formation
• Stimulates production of Plasminogen Activator
• Androgens • Stimulates early follicle development
(Tripathi et al.,2015)
Follicular fluid classical hormones

9. Non-classical hormones/factors ROLE IN OOCYTE DEVELOPMENT, MATURATION &
OVULATION
• Activin • Increases FSH secretion, promotes ovarian
follicular development
• Follistatin • Activin-binding protein that neutralizes activin
bioactivity
• Anti-Mullerian Hormone(AMH) • Inversely correlated with the maturation and
developmental potential of oocytes
• Bone morphogenetic protein-15
(BMP-15)
• Influence oocyte maturation, Quality and COC
expansion
• Positively correlated to FF estradiol levels in
individual follicles
• Insulin-like growth factors (IGFs)
• Transforming growth factor (TGF) family (Tripathi et al.,2015)
Follicular fluid non-classical factors/hormones

10. Growth factors
A. Insulin-like growth factors (IGFs)
 IGFs I and II which are very active & are potent Mitogens
 IGF have synergistic action with Gonadotropins
 Insulin, as well as binding to IGF receptors, has its own ovarian receptors and is
known to promote androgen production
 IGF Binding Proteins have inhibitory actions with IGF
(Wetterau et.al., 2002)
B. Transforming growth factor (TGF) family
 The transforming growth factor (TGF) family is also well represented in
the ovary as is epithelial growth factor (EGF)
 All play a passive role in the regulation of gonadotropin activity
within the follicles
(Wetterau et.al., 2002)

11. Immune response-related genes and their
functions in ovulation
{Takeda et al., 2003, Shimada et al., 2007, Herath et al., 2007}

12. Gene products that are essential for
ovulation
(Henriquez et al., 2017, Breen et al., 2013, R. Duggavathi et al., 2009, Sela-Abramovich et al., 2005, Park et al., 2004 Salvador et al., 2002)

13. INTRACELLULAR SIGNALING ACTIVATED BY LH
(Salvador et al., 2002, Sela-Abramovich et al., 2005, Breen et al., 2013, Henriquez et al., 2017, Park et al., 2004)

14. Enzymes such as Matrix Metalloproteinases (MMPs) and ADAMTS(A disintegrin & Metalloproteinases
with Thrombospondin-like motifs) proteases are involved (although not all are essential):
Remodeling of the extracellular matrix Rupturing of the follicular apex
Degradation of basement membrane
 Tissue inhibitors of MMPs (TIMPs) are also involved in the ovulatory process and the pattern of in vivo
expression suggests that decreased TIMPs levels and increased MMPs are involved in follicle rupture
 The extracellular matrix remodeling is completed by a process similar to the inflammatory process, with the
participation of macrophages, neutrophils, cytokines produced by leukocytes, platelet activating factor and free
radicals
 Induction of Ovulation with mediators including endothelin-2, interleukin-6 and cGMP-dependent
protein kinase- II
(Sayasith & Sirois, 2015)
(Stassi, AF, et.al., 2017)
(Richards, et.al., 2005)

15. • Consequently, cAMP levels in the oocyte fall
Signal transduction in cumulus cells
in the mouse periovulatory phase
• FSH binds FSH receptors on cumulus cells to activate AC, cAMP production and PKA
This pathway activates Erk as do Egf receptors in response to Egf-L, amphiregulin (AR), betacellulin (BC)
and epiregulin (ER) (Seger et al., 2001; Salvador et al., 2002; Choi et al.,2005)
• Transcription factors regulated by these kinase
cascades include AP-1 factors, Elk-1 and cAMP
response element-binding protein (CREB)
• In growing follicles, cAMP is translocated to the
oocyte via gap junctions until these are inactivated
through phosphorylation by Erk and cells separate
from the oocyte through cumulus expansion
(Russell et al., 2003b)
(Marsh, 1976; Richards, 1994)
(Russell & Robker, 2007)

16. • In a separate pathway, oocyte-secreted growth and differentiation factor- 9 (Gdf-9) and bone
morphogenetic protein-15 (BMP-15) activate integral receptor kinase activity on cumulus cells, which
results in phosphorylation of SMAD2/3, which translocate to the nucleus in dimers with SMAD4
(Yan et al., 2001)
• These transcription factors promote
expression of key cumulus genes
required for specification of the
cumulus- specific response to the
ovulatory surge
• Genes induced via these two signal
transduction mechanisms include HAS-
2, TSG-6, PTX-3, COX-2, PGE2receptor
(EP2) and regulator of GPCR-2 (RGS-2)
• PGE2 activates the EP2 receptor to mediate a signal transduction pathway similar to FSH, and RGS-
2 may control the activation of G-proteins
(Gaytan et al., 2003, Downs and Longo, 1983; Davis et
al., 1999; Ochsner et al., 2003b)
(Espey and Richards, 2002)

17. • The cow is an excellent model to study the impact of signaling pathways in
theca cells, which is difficult in the murine model (Young et al., 2010)
 Successful ovulation is dependent on the trigger elicited through the LH surge which
activates a cascade of signalling pathways, like ERK1/2 pathway (Siddappa et al., 2010)
 Intrafollicular treatment with an inhibitor of ERK1/2 pathway (50 µM PD0325901)
abrogates ovulation in cattle(Walsh et al., 2012)
o In bovine granulosa cells in vitro, treatment with forskolin and ERK1/2 inhibitor (U0126)
decreased ADAMTS1 expression (Sayasith et al., 2013)
(Nature-Scientific Reports, 2018)

18. Fig.1. Effect of intrafollicular administration of the
MEK inhibitor, PD0325901 on ovulation in cattle
Fig.2. Inhibition of ERK1/2 activity in granulosa cells of
ovulating follicles by an intrafollicular administration of
PD0325901
(Yasmin Schuermann, et al., 2018)

19. {BIOLOGY OF REPRODUCTION (2015)}

20. Extracellular vesicles
 Cell-secreted vesicles carrying biomolecules such as proteins,
mRNAs, miRNAs, metabolites and lipids (Choi et al., 2013)
As the follicle develops extracellular vesicles quantity and
content in the follicular fluid vary (Navakanitworakul et al., 2016) and
have different effect on cumulus expansion and oocyte gene
expression (Hung et al., 2015)
Facilitate the transfer mechanism of different macromolecules
(Di Pietro, 2016)

21. Micro RNAs (miRNA)
• MicroRNAs are small non-coding RNAs regulating gene
expression present in cells or in follicular fluid
• miR-130b is involved in increasing granulosa and cumulus cells
proliferation and in oocyte maturation (Sinha et al., 2017)
• miRNAs transiting in EVs in the follicular fluid modulate signaling
pathways, such as the PI3K-Akt pathway (Andrade et al., 2017b)

22. MicroRNA (miRNA) levels in the ovary during the
estrous cycle
(Derek et al., 2018, Donadeu et al., 2012, Velthut-Meikas etal., 2013, Shen et al., 2013)

23. Regulatory Function of Follicular Fluid with Inhibitory or
Stimulatory Responses
SUBSTANCES PHYSIOLOGICAL RESPONSES
INHIBITORS
Oocyte Maturation Inhibitor (OMI) Inhibits completion of oocyte meiosis
Luteinization Inhibitor Prevents Luteinization of Granulosa cells
FSH Receptor-binding Inhibitor Depress the binding of FSH to Granulosa cells
Inhibin Depress secretion of FSH
STIMULATOR
Luteinization Stimulator Stimulates luteinization of Granulosa cells
(Hafeez et al., 1973)

24. CONCLUSIONS
 Most mammalian mRNAs are targeted by multiple miRNAs, thus additional miRNAs that
play a regulatory role in the expression of key enzymes involved in the biosynthesis of E2
and P4 which eventually helps in Ovulation
 Information regarding ionic, metabolic and composition in follicular fluid will extend our
understanding regarding nutritional environments for Folliculogenesis & Ovulation
 Co-ordination and synchronization of endocrine, paracrine, immune and metabolic
signals from follicular fluid acting mainly through the cumulus compartment exert control
on oocyte maturation, developmental potential and ultimately ovulation
 Biochemical components of follicular fluid thus helps in Folliculogenesis, Oocyte
maturation & Ovulation