Neonatal androgenization of hypogonadal (hpg) male mice does not abolish estradiol-induced FSH production and spermatogenesis

Abstract

Background: Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency in hypothalamic gonadotropin-releasing hormone (GnRH) synthesis. Chronic treatment of male hpg mice with estradiol induces FSH synthesis and secretion, and causes testicular maturation and qualitatively normal spermatogenesis. As estradiol negative feedback normally inhibits FSH production in the male, this study tested whether this paradoxical response to estradiol in the male hpg mouse might be due to inadequate masculinisation or incomplete defeminization in the neonatal period. Previous studies have demonstrated that treatment of hpg mice with testosterone propionate in the immediate neonatal period is necessary to allow full reproductive behaviors to be expressed following suitable endocrine stimulation at adult ages.

Methods: Hpg mice were treated with 100 mug testosterone propionate or vehicle on postnatal day 2. At 35 days of age, subgroups of these mice were treated with silastic implants containing estradiol or cholesterol. Reproductive behavior was scored in tests with steroid-primed female mice, then testicular development was assessed histologically, and measures of pituitary FSH content made at 85 days of age.

Results: The neonatal testosterone propionate treatment successfully defeminized female litter mates, as revealed by impaired vaginal opening and deficiencies in lordosis behavior, and it allowed appropriate male reproductive behavior to be expressed in a proportion of the hpg males when tested at an adult age. However, neonatal androgen supplementation did not block or even reduce the subsequent actions of estradiol in increasing pituitary FSH content, nor did it affect the ability of estradiol to induce qualitatively normal spermatogenesis.

Conclusion: The ability of the hpg male to show a "female" neuroendocrine response to estradiol is not a result of inadequate androgenization during neonatal development, and thus the actions of estradiol revealed in this rodent model are not an artefact of incomplete sexual differentiation, but reflect a physiological role of estradiol occurring during a specific early temporal window of male reproductive development.

Figure 1

Experimental design. Male hpg mice were treated with either vehicle (VEH) or 100 μg testosterone propionate (TP) on postnatal day 2, then received a subcutaneous silastic capsule containing either cholesterol (chol) or 2% estradiol in cholesterol on day 35 of age.

Figure 2

Female littermates of experimental male mice received a subcutaneous injection of either vehicle (VEH) or 100 μg testosterone propionate (TP) on postnatal day 2. Panels indicate body weight (top), reproductive tract weight, anogenital distance, proportion showing vaginal opening by day 70 of age and proportion showing aggressive behavior toward stud males in tests of reproductive receptivity. Values are group mean ± SE (upper panels) or numbers of mice (lower panels).

Figure 3

Proportions of male hpg mice displaying mounting behavior when paired with a steroid-primed female. Mice were treated with vehicle (VEH) or testosterone propionate (TP) on postnatal day 2, then either a cholesterol (chol) or a 2% estradiol (E) subcutaneous implant on day 35, and tested after 75 days of age. Three wild-type C3H mice were also tested (+/+).

Figure 4

Body weight (top), paired testis weight, anogenital distance, and wet weight of epididymides and seminal vesicles (bottom)) of male hpg mice receiving vehicle (VEH) or 100 μg testosterone propionate (TP) on postnatal day 2, then either a cholesterol (chol) or a 2% estradiol (E) subcutaneous implant on day 35. Values are group mean ± SE. **P < 0.001 vs groups treated with cholesterol implants.

Figure 5

Pituitary FSH content (top) and testis testosterone content (bottom) in male hpg mice; receiving either vehicle (VEH) or testosterone propionate (TP) on postnatal day 2, then either a cholesterol (chol) or a 2% estradiol (E) subcutaneous implant on day 35. For comparison, pituitary FSH and testicular testosterone values derived from wild-type litter mates analysed in the same assay are indicated (+/+). Values are group mean ± SE. **P < 0.001 vs groups treated with cholesterol implants.

Figure 6

Representative examples of testicular histology from 85 day old hpg mice treated with vehicle (VEH) or testosterone propionate (TP) on postnatal day 2, then receiving either a cholesterol (chol) or a 2% estradiol (E) subcutaneous implant on day 35. Note that the most mature cells types in testes from mice treated with cholesterol (VEH+chol, TP+chol) are type A spermatogonia (SA) and primary spermatocytes (PS); there is little evidence of lumen formation, and Sertoli cells are frequently observed adjacent to the basal lamina and more centrally (St). In contrast, after estradiol treatment (VEH+E, TP+E) the seminiferous tubules are expanded and have developed a lumen (L), Sertoli cells are adjacent to the basal lamina and round and elongating spermatids (ES) are present. Treatment with testosterone propionate in the neonatal period did not affect the testicular response to estradiol in later life. Scale bars represent 30 μm.