Luteolysis

mechanism of luteolysis in bovines with reference to recent concepts

1. Endocrine and Paracrine control of luteolysis in bovines MAJOR CREDIT SEMINAR Dr. Santhosh Shinde Roll no- P 1673 Ph. D scholar

2. Historical perspective Corpus luteum. Where it comes from? What it secretes? What is its function? How it is regulated? Luteolysis Why should the corpus luteum capitulate? Signal for luteolysis Control of luteolysis a) Endocrine control b) Paracrine control Overview

3. Historical perspective Coiter (1573)- Presence of cavities filled with yellow solid on ovary Regnier de Graaf (1641–1673)- First accurately described the structure Marcello Malpighi (1628–1694) – Named structure as Corpora (bodies) lutea (yellow) Prenant (1898) - Corpus luteum acts as a gland internal secretion . Magnus (1901)- Luteal extract has biologically active substance maintain pregnancy Butenaudt , Hartman and Slotta (1934)- Isolation of the pure crystalline hormone Slotta (1934)- Named the compound progesterone and suggested a structural formula Butenandt and Westphal (1934)- Compound was synthesized Leo Loeb (1923)- Influence of uterus on regression of the corpus luteum McCrackenit (1972)- Identified PGF2 α as - luteolysis hormone

4. Formation of corpus luteum Preovulatory surge of LH - major signal for CL development LH induces , transformation of ovulated follicle cells into the CL- Luteinization (Richards, 1987)

5. LHR Preovulatory LH surge LH induced Luteinization Ovulation Theca interna cell- Small luteal cell Granulosa cells- Large luteal cells ( Miyanoto , 2009)

6. Cell types found in the CL Cell type Percentage (%) Volume (%) Large luteal cells 4 25 Small luteal cells 19 18 Endothelial cells 53 17 Fibrocytes 17 11 Vascular endothelial cells - 50% of all cells in bovine CL (Miyamoto and Shirasuna , 2009) LLC secrete 10 times more progesterone than SLC (Fitz, 1982; Koose , 1971)

7. Luteal development From Day 4 to 16 of estrous cycle, 2- fold increase in size of LLC ( 15×10 6 /corpus luteum ), 2- fold increase in number of fibroblasts (from 21 to 50×10 6 ), 5- fold increase in number of SLC (from 10 to 50 ×10 6 ) 6.5- fold increase in number of endothelial cells (from18 to 120 ×10 6 ) ( Farin , 1986: Jablonka-shariff , 1993) Factors regulating proliferation of SLC and fibroblasts - Fibroblast growth factors - insulin growth factors - Growth hormone - LH ( Redmer , 1996: Juengel , 1995)

8. Cont… Proliferation of endothelial cells neo- vascularization (Reynolds,1992) Vascular endothelial growth factor ( VEGF )- mitogen Capillary lumina - 22% of the total volume of the corpus luteum ( Dharmarajan , 1985) Rate of blood flow ( 6–10 ml/g/min ) to the corpus luteum Consume 2-6 times more oxygen per unit weight than does the liver, kidney, or heart (Swann and bruce , 1986)

9. Regulation of luteal function Matured CL secrete progesterone – Maintenance of pregnancy Hormones that support the growth and/or function of the corpus luteum are termed luteotropic hormones (Acosta et al., 2002) Hormones includes, LH, GH, prolactin , IGF-I, PGE2, and PGI2 (Gordon et al., 2000) How does the progesterone secretion maintained ?

10. Molecular Mechanism of LH on Luteal Cell Receptor PKA Nucleus DNA Histones Protein Synthesis (Enzymes) mRNA Plasma Membrane ( cAMP second messenger system) Cholesterol Gs Adenylate Cyclase C R cAMP LH ATP cAMP (+ PO 4 ) R-ER Protein Synthesis Mitochondria Cholesterol Pregnenolone S-ER Steroid Synthesis Progesterone

11. Acute effects of GH and IGF-I on progesterone secretion Direct effect – Activation tyrosine kinase JAK2 Indirect effect - expression of IGF-I - but how does it secrete pregesterone is unknown ? (Parmer et al., 1991; Talawera , 1992) (Davis et al., 1996; Lucy,1993)

12. Effects of prostaglandins on progesterone secretion Prostaglandins E and I - normal luteal function Prostaglandins E and I - higher amounts in the early luteal phase (Mitchell et al.,1991) Binding sites for PGI2 reside on both small and LLC - Elevation of cAMP levels (Huggins et al., 1983; Fitz, 1984) High- affinity receptors for PGE2 LLC (Fitz et al., 1982) Bovine luteal tissue addition of PGE2- levels of cAMP and presumably activated PKA (Miyashita et al., 1998)

13. Luteolysis Luteolysis is defined as lysis or structural demise of the corpus luteum. Two events occur during luteolysis, a. Functional luteolysis- loss of capacity to synthesize and secrete progesterone b. Structural luteolysis- loss of the cells 16-17 day of estrous cycle- concentrations of mRNA encoding 3 β-HSD and StAR decreased ( Mcguire et al., 1999) secretion of progesterone - luteal blood flow and steroidogenic capacity of luteal cell ( Shirasuna et al., 2004) Why mammals have developed mechanisms to curtail luteal function?

14. A New Beginning Who is responsible for this decrease in CL function? Inhibitory effect of Progesterone on FSH Secretion will be nullified

15. The Uterus Regulates the Life Span of the Corpus Luteum Luteal Phase Luteal Phase Hysterectomy 1 10 20 1 10 20 Intact Animal Hysterectomized Animal Progesterone Progesterone The substance responsible – Found to be PGF2 α ( McCrackenit , 1972)

16. What is the signal that initiates the release of PGF2a ? Uterus Ovary CL PGF2 a Anterior Pituitary Posterior Pituitary Oxytocin McCracken Model of Oxytocin control on CL Regression Estradiol from the developing preovulatory follicle triggers the release of hypophysial oxytocin (McCracken et al., 1981)

17. Countercurrent mechanism of luteolysis (McMillan, 1972) Ovarian Artery This mechanism is specific to certain species; How does PGF2a cause luteolysis ?

18. PGF 2α - Acute increase (from 30 min to 2 h) in blood flow at the periphery of the CL (Acosta et al., 2002) Additional evidence - blood flow to CL increased with each PGFM pulse - spontaneous luteolysis in the heifer ( Ginther and beg, 2012) Nitric oxide (NO) and Apelin (APJ) pathway - Potent vasorelaxant Administration of the NO donor (SNAP) into CL - Mimicked the actions of PGF 2α ( Shirasuna et al., 2008) NOS inhibitor (L-NAME) - suppressed acute increase in luteal blood flow Need of initial increase in luteal blood flow? Blood flow and vascular changes

19. Endothelin-1 The EDN1 – 21 a.a peptide produced by endothelial cells First isolated from porcine vascular endothelial cells (Yanagisawa et al., 1988) Cause luteolysis by, Reduce blood flow during early luteolysis - Arteriole constriction ( Granstrom and Kindahl , 1986) Inhibits the steroidogenic activity (Fraser et al., 1997) Antiluteolytic property of PGE2 -- attenuate the vasoconstrictive actions of endothelin-1 (Irvin, 1999) Luteolysis accelerators within the CL (Paracrine control)

20. Angiotensin II Ang II is a strong vasoconstrictive peptide (Hayashi and Miyamoto 1999; Speth et al., 1999) Ang II - inhibit LH-stimulated P4 release in bovine luteal cells ( Stirling et al., 1990) Spontaneous luteolysis in the cow- PGF2α and Ang II also have positive feedback during luteolysis ( Shirasuna et al., 2004)

21. Luteal PGF2a- PGF2a from peripheral sources - Synthesis of luteal PGF2a ( Ellendorff , 1998) PGF2a autoregulate its synthesis - stimulating the liberation of arachidonic acid by hydrolysis of membrane phospholipids (Watanabe et al., 1995)

22. Intracellular Signaling PGF2a acts by binding to specific receptors – LLC day 13 to 20- Binding affinity of the PGF2a to receptor increases 203 time ( Rao et al., 1979) Gordon et al., 2000)

23. Activation of PKC in LLC - post translational modification of cellular proteins - cholesterol availability - maintenance of the extracellular matrix - activation of proteins involved in apoptosis (Pate and Keyes, 2001)

24. Two types of intercellular communications Gap junction- exchange of molecules of less than about 1 kDa (calcium ions, cAMP and IP3) between adjacent cells (Yamasaki and Naus , 1996) Gap junctions are formed by connexin ( Cx ) proteins such as Cx43 ( Grazul-Bilska et al., 1997) Cadherin family- vascular endothelial cell cadherin (VE- cadherin ), epithelial cadherin (E- cadherin ) Cell adhesion and gap junction - necessary for transferring the luteolytic signal between luteal cells and endothelial cells ( Grazul-Bilska et al., 1997; Borowczyk et al., 2006) Involvement of cell adhesion systems within the CL

25. Immune-Mediated Events Immune cells infiltrate the CL during luteolysis - chemotaxic factor (Murdoch, 1987) Role of Macrophages during early stages of luteolysis: 1) phagocytosis of degenerative luteal cells, 2) cytokine mediated inhibition of steroidogenesis 3) Stimulation of PGF2a secretion by the corpus luteum. 4) degradation of the extracellular matrix (Parker, 1991) Infiltration of leukocytes - increased local production of cytokines (particularly IL-1, TNF- a,and IFN- γ )- stimulate luteal PGF2a synthesis ( Vallet et al., 1993)

26. Apoptosis - active, energy-dependent process by which nonessential populations of cells delete themselves from a tissue (Kerr, 1992) Biochemical and morphological changes- characteristics of apoptotic cells - Nuclear fragmentation, - Appearance of membrane-bound vesicles, - Ladder like fragmentation of genomic DNA and - Changes in gene expression (Bas, caspase ) (Sawyer, 1991) PGF2a - promotes apoptosis in cells of corpus luteum Cell fragments, or apoptotic bodies- targets for Phagocytosis ( Gemell et al.,1986) Apoptosis

27. Concept of luteolysis in cow (Miyamoto and Shirasuna,2009)

28. Conclusions Formation and regression of the CL involve a wide variety complex multifaceted biological processes Cow- PGF2α induced increase in luteal blood flow is one of the earliest signals of the luteolytic cascade Endocrine mediated luteolytic cascade- Endometrial PGF 2α and luteal OXT Luteolysis accelerators within the CL are responsible for Paracrine control of luteolysis Gap and adherence junctions composed of cell adhesion molecules (CAMs) involved transport of luteolytic Immune mediated cells are involved in luteolysis- inflammatory process

29. Thank you