Characterization of anti-Mullerian Hormone in the Stallion and Mare: Changes During Testis Development and Determination of Serum Concentrations in Normal Mares, Pregnant Mares and Mares with Granulosa-cell Tumors

Anti-Mullerian hormone (AMH) is a dimeric glycoprotein, member of the transforming growth factor beta superfamily of growth and differentiation factors. During sexual differentiation, testosterone is responsible for Wolffian (mesonephric) duct development in males while AMH is required for Mullerian (paramesonephric) duct regression. AMH is produced from the time of differentiation of seminiferous tubules in the fetal testis until pubertal maturation and appears to regulate Leydig cell differentiation and testosterone secretion. In females, AMH is not expressed before the time of birth, guaranteeing normal differentiation of the female genital tract. After birth, AMH is expressed in granulosa cells of preantral and small antral follicles in the ovary and has been related to inhibition of recruitment of primordial follicles into the pool of growing follicles. In addition, circulating AMH concentrations have been used for monitoring granulosa-cell tumor (GCT) in women.;In chapter 1 and 2, changes in expression of anti-Mullerian hormone (AMH) and its receptor (AMHR2), androgen receptor (AR), cyclin-dependent kinase inhibitor (CDKN1B), connexin 43 (Cx43), 3beta hydroxysteroid dehydrogenase/Delta 5-Delta4- isomerase (3betaHSD), 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase cytochrome P450 (P450arom) in testes of normal stallions throughout puberty were characterized based upon immunohistochemistry (IHC) and real-time quantitative PCR (RT-qPCR). In chapter 3, the same markers were characterized in undescended testes of cryptorchid stallions and compared with testes of normal stallions. In chapter 4, a specific enzyme immunoassay from Immunotech-Beckman Coulter was validated for determination of serum AMH in the horse and for determination of concentrations of AMH in the blood of mares during the estrous cycle and pregnancy as well in mares with granulosa cell tumors.;During testis development in the stallion, AMH expression decreased with age and was accompanied by increased AR and CDKN1B expression in Sertoli cells coinciding with the final stages of Sertoli cell maturation and AMH regulation by testosterone. In addition, expression and distribution of Cx43 changed throughout development, suggesting a role of Cx43 in the Sertoli cell signaling and maturation, hormone secretion and blood-testis barrier formation. As expected, P450c17 and 3betaHSD immunolabeling and mRNA expression increased with age. 3betaHSD was also observed within the seminiferous tubules (presumptive Sertoli cells) of prepubertal testes and disappeared in the postpubertal testes. P450arom was absent or weakly expressed in immature Leydig cells of prepubertal testes and increased in the postpubertal and adult testis. In cryptorchid testes, AMH and AMHR2 immunoexpression was increased compared to normal testes. The persistence of high AMH expression accompanied by low AR expression in Sertoli cells of cryptorchid testes indicates failure of maturation of Sertoli cells and/or lack of testosterone suppression. The steroidogenic enzymes P450arom, P450c17 and 3betaHSD showed lower immunolabeling in the cryptorchid testes, which is also consistent with an underdeveloped testis. In addition, Cx43 expression is decreased in the cryptorchid testis, indicating disruption in intercellular communication.;In chapter 4, ovariectomized mares showed AMH concentrations at or below the limit of detection for the assay. AMH concentrations were higher in cycling and pregnant mares when compared to ovariectomized mares and did not change significantly throughout estrous cycle or pregnancy. Mares with granulosa-cell tumor showed an increase in AMH concentrations, when compared to normal cycling mares, pregnant mares and ovariectomized mares. A decrease of approximately 50% in AMH concentration was observed after 48 hours of tumor removal. These data indicate that AMH is expressed in equine GCTs and that serum levels are a useful biomarker for their diagnosis.