FOLLICULAR FLUID FACTORS IN OVULATION- DR.NITISH KULKARNI

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

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 D ominant 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 O vulation 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 I t 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 O ocyte and granulosa cells in the ovary are bathed in a fraction of extracellular fluid called F ollicular Fluid , which accumulates in the antrum of ovarian follicles B iochemical 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 I nsulin-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. I mmune 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 M atrix M etalloproteinases ( 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 Th i s pat h w a y act i v at e s Er k a s d o Eg f r e c eptors i n r e s p onse t o Eg f - 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 s i g nal t r ans d uct i on mechanisms include HAS-2, TSG-6, PTX-3, COX-2, PGE2 receptor (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) I ntrafollicular treatment with an inhibitor of ERK1/2 pathway ( 50 µM PD0325901 ) abrogates ovulation in cattle( Walsh et al ., 2012) 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 C ell-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 M ost 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 C o-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, O ocyte maturation & Ovulation