Immunolocalization of AMH and cell quantification in seminiferous tubules of buffalo fetuses

The testicular development of buffalo fetuses has been studied by several authors (Abdel-Raouf et al, 1974; Kaur et al, 2011) and anti-Muleriano hormone (AMH), also known as Mullerian Inhibiting Substance (MIS) expressed by Sertoli cells plays an important role in sexual differentiation by inducing regression of the Mullerian ducts in the male fetus (Jost, 1953). AMH was identified in Sertoli cells of equine fetuses between 5 and 11 months of gestational age (Ball et al, 2008), however there are no studies in the literature with immunolocalization of AMH in buffalo fetuses. This work aims to make the morphometry of the seminiferous tubules (sexcords), to quantify the support cells (Pre-Sertoli) and gonocytes and make the immunolocalization of AMH in buffalo fetal testis of different ages. Testis of buffalo fetuses (n = 12) aged between 3 and 8 months (15-98cm CRL) (Abdel-Raoulf et al, 1974) were collected in slaughterhouse processed for conventional histology and stained with H.E for morphometry. The imunolocalization of the AMH was performed on histological sections deparaffinized using anti-AMH antibody 1:50 (SC 28912) according to manufacturer instructions. For morfometry and quantification were analyzed 100 tubules in each age. The average of tubular diameter was 38.06 (± 5.4), 46.27 (± 8:47), 44.11 (± 6.83), 32.56 (± 5.65) and 35.5 (± 4.26) µm; the average of supporting cell (pre-Sertoli) was 7.72 (± 1:53) 8.13 (± 1.68) 9.24 (± 3.10) 05.08 (± 1:56) and 8.86 (± 1.73) and the average of gonocytes was 0.82 ( ± 0.77) 0.94 (± 0.78); 1:24 (± 0.95), 0.70 (± 0.64) and 1.13 (± 0.86) to 3-4, 5, 6, 7 and 8 months, respectively, showing a a statistically significant difference between different age groups (p ≤ 0.05). Immunohistochemistry for AMH in testicular parenchyma sections showed intense labeling in the cytoplasm of pre-Sertoli cells within the sexual cords (seminiferous tubules in development) in all ages analyzed and immunostaining was not observed in gonocytes and interstitial tissue. Herein, the results showed that AMH is present in testis of buffalo fetuses between 3 and 8 months, however further studies are needed to clarify its role in testicular development.