Immunocytochemical localization of cytochrome P450 aromatase in the testis of prepubertal, pubertal, and postpubertal horses

Abstract

The large amount of testicular estrogens produced by the stallion is unique compared to the amounts found in other domestic species. Although the cellular locale of the cytochrome P450 aromatase (P450_arom) enzyme that converts C₁₉ androgens to C₁₈ estrogens has been identified in the Leydig cell of adult equine testis, the location in the immature equine testis is not known. The goal of this work was to localize the enzyme in colts and stallions during sexual development. Testes were obtained from prepubertal (n=7), pubertal (n=6), and postpubertal (n=8) colts and stallions during both the breeding and non-breeding seasons. Tissue was fixed and prepared for immunocytochemistry (ICC), carried out with an antiserum against human placental P450_arom. In prepubertal colts, there was distinct immunopositive staining of a similar degree within both the Leydig cell and the seminiferous tubule. Horses in the pubertal group had strong Leydig cell immunopositive staining and a slight degree of positive staining within the seminiferous tubules. Postpubertal stallions exhibited definitive immunopositive staining within Leydig cells but not within the seminiferous tubules. Therefore, P450_arom is present within the Leydig cell throughout sexual development. In contrast, the presence of P450_arom within the seminiferous tubule based upon ICC appeared to be gone by adulthood, suggesting that an age-dependent shift in the locale of this enzyme as the stallion matures.

Introduction

Stallions are different from most other domestic species in the large amount of estrogens that are produced by the testis. Baggett et al. [1] were the first to demonstrate that the stallion testis was capable of producing estrogens from androgens. The pig is another species that exhibits elevated estrogens; however, blood plasma levels of conjugated estrogens in the stallion are over 60 times higher than those in the boar [2], [3].

The cytochrome P450 aromatase (P450_arom) enzyme is responsible for the conversion of C₁₉ androgens to C₁₈ estrogens. This enzyme has been immunolocalized to the Leydig cells of many adult mammals, including stallions [4], [5], rams [6], brown and black bears [7], [8], bank voles [9], rats [10], [11], mice [12], and men [13], [14]. Far fewer studies have investigated the locale of P450_arom in the testes of prepubertal and pubertal animals. The prepubertal boar [15] and ram [6] both exhibited positive immunostaining for the enzyme in Leydig cells only. In the immature rat, both the Sertoli cell [11] and the Leydig cell [10] were found to contain immunoreactive P450_arom. As the young rat matures, enzyme activity increased in Leydig cells and decreased in Sertoli cells, suggesting an age-related shift in the cellular location of the aromatase enzyme [16], [17].

Previous immunocytochemistry (ICC) work in the postpubertal stallion by Eisenhauer et al. [4] demonstrated that the aromatase enzyme was only localized within the Leydig cell. Recently, our laboratory has observed that estradiol production by Leydig cell, Sertoli cell, and seminiferous tubule cultures of prepubertal, pubertal, and postpubertal colts and stallions suggested that the aromatase enzyme is present in both Leydig cells and Sertoli cells and that the distribution of P450_arom within these cells may be age-dependent [18], [19], [20].

To determine the locale of the aromatase enzyme during puberty, ICC was conducted on testicular tissue from prepubertal, pubertal, and postpubertal colts and stallions using an antiserum against the human placental enzyme.