Expression of anti-Müllerian hormone, cyclin-dependent kinase inhibitor (CDKN1B), androgen receptor, and connexin 43 in equine testes during puberty.

Sertoli cells are essential in development of a functional testis. During puberty, Sertoli cell maturation can be characterized by a number of markers, including anti-Müllerian hormone (AMH) and its receptor (AMHR2), androgen receptor (AR), cyclin-dependent kinase inhibitor (CDKN1B), and connexin 43 (Cx43). In the present study, immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) were used to characterize changes in expression of AMH, AMHR2, AR, CDKN1B, and Cx43 in prepubertal, postpubertal, and adult equine testes. During puberty, AMH expression decreased, and expression of AR as well as CDKN1B increased in Sertoli cells coinciding with the period of Sertoli cell maturation, arrest of cell proliferation, and presumptive AMH regulation by testosterone. Expression of AMHR2 appeared to decrease in Sertoli cells and increase in Leydig cells during pubertal maturation of the equine testis. In addition, expression and distribution of Cx43 changed during puberty in the stallion, suggesting a role for Cx43 in Sertoli cell signaling and maturation, hormone secretion, and blood-testis barrier formation. We concluded that Sertoli cell maturation during puberty in the stallion was accompanied by a reduced expression of AMH and its receptor, arrest of cell proliferation, increased expression of AR, and organization of gap-junctional communication.

Biological and clinical significance of anti-Müllerian hormone determination in blood serum of the mare.

Anti-Müllerian hormone (AMH), a member of the transforming growth factor ß superfamily of growth and differentiation factors, is expressed in granulosa cells of preantral and small antral ovarian follicles. In humans, AMH appeared to regulate recruitment and growth of small ovarian follicles. Furthermore, circulating AMH concentrations were elevated in women with granulosa-cell tumors (GCT). In the horse, GCTs are the most common tumor of the ovary, and a variety of endocrine assays have been used to diagnose presumptive GCTs. The objectives of the present study were to validate a heterologous enzyme immunoassay for determination of serum AMH in the horse, and to determine concentrations of AMH in the blood of mares during the estrous cycle, pregnancy, and in mares with granulosa-cell tumors. Mares with normal estrous cycles (n = 6) and pregnant mares (n = 6) had blood samples collected throughout one interovulatory period and monthly throughout gestation, respectively. Mares diagnosed with GCT had blood samples taken before (n = 11) and after ovariectomy (n = 5). Tumors were sectioned and fixed for immunohistochemistry and snap frozen for immunoblot analyses and RT-qPCR. In normal cyclic mares and in pregnant mares, there was no effect of cycle stage or month of gestation on serum AMH concentrations. In GCT mares, serum concentrations of AMH (1901.4 ± 1144.6 ng/mL) were higher than those in cyclic (0.96 ± 0.08 ng/mL) or pregnant (0.72 ± 0.05 ng/mL) mares and decreased after tumor removal. Both AMH and AMH receptor (AMHR2) immunolabeling and expression were detected by immunohistochemistry in the tumor and cyst fluid obtained from mares with GCTs. Therefore, we concluded that AMH was a useful biomarker for detection of granulosa-cell tumors in mares.

Expression of steroidogenic enzymes during equine testicular development.

In the mammalian testis, Leydig cells are primarily responsible for steroidogenesis. In adult stallions, the major endocrine products of Leydig cells include testosterone and estrogens. 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase (3ßHSD) and 17α-hydroxylase/17,20-lyase (P450c17) are two key steroidogenic enzymes that regulate testosterone synthesis. Androgens produced by P450c17 serve as substrate for estrogen synthesis. The aim of this study was to investigate localization of the steroidogenic enzymes P450c17, 3ßHSD, and P450arom and to determine changes in expression during development in the prepubertal, postpubertal, and adult equine testis based upon immunohistochemistry (IHC) and real-time quantitative PCR. Based on IHC, 3ßHSD immunolabeling was observed within seminiferous tubules of prepubertal testes and decreased after puberty. On the other hand, immunolabeling of 3ßHSD was very weak or absent in immature Leydig cells of prepubertal testes and increased after puberty. HSD3B1 (3ßHSD gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal testes (P=0.0041). P450c17 immunolabeling was observed in small clusters of immature Leydig cells in prepubertal testes and increased after puberty. CYP17 (P450c17 gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.030) and postpubertal testes (P=0.0318). A weak P450arom immunolabel was observed in immature Leydig cells of prepubertal testes and increased after puberty. Similarly, CYP19 (P450arom gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal (P=0.0001) testes. In conclusion, Leydig cells are the primary cell type responsible for androgen and estrogen production in the equine testis.

Iris colour and the influence of local anaesthetics on pre-corneal tear film stability.

Using non-invasive methods we report here that the stability of the pre-corneal tear film is lower in the brown eye than in the blue eye. The average stability in the blue eye is 15.8 sec (SD +/- 5.8) and in the brown eye it is 12.3 sec (SD +/- 2.9). On average, instillation of topical anaesthetics, benoxinate hydrochloride (0.4%) or amethacaine hydrochloride (0.5%), depress the stability of the pre-corneal tear film in blue eyes but not in brown eyes.