Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse

Abstract

Background: The hypogonadal (hpg) mouse is widely used as an animal model with which to investigate the endocrine regulation of spermatogenesis. Chronic treatment of these GnRH-deficient mice with estradiol is known to induce testicular maturation and restore qualitatively normal spermatogenesis. The aim of the current studies was to investigate whether these effects of estradiol are direct effects in the testis, or indirect actions via paradoxical stimulation of FSH secretion from the pituitary gland.

Methods: Initially, Western blot and immunohistochemistry were used to analyse tissues from hpg mice to identify potential sites of action of estradiol. In the main study, hpg mice were treated for 50 days with either an estradiol implant or daily injections of recombinant human FSH, or a combination of both, to determine whether estradiol would have an additive or synergistic effect with FSH on testis development, as assessed by histological analysis and stereological quantification of Leydig, Sertoli and germ cell proliferation.

Results: Western blot analysis revealed ERalpha immunoreactive bands of appropriate molecular weight in extracts of testis and pituitary glands from hpg mice, and immunohistochemical studies confirmed ERalpha in nuclei of anterior pituitary cells and Leydig and peritubular cells in hpg mice. Histological and morphometric analyses revealed that estradiol treatment alone was as effective as FSH in promoting Sertoli cell production and proliferation of the seminiferous epithelium, resulting in the production of elongating spermatids. Combined estradiol and FSH treatment did not produce a greater effect than either treatment alone, though an increased dose of FSH significantly increased seminiferous tubule volume and testis weight and increase Sertoli cell numbers further within the same time frame. In contrast, estradiol caused substantial increases in the wet weight of the seminal vesicles, whereas FSH was without effect on this tissue, and did not augment the actions of estradiol.

Conclusion: As ERalpha receptor is abundantly expressed in the pituitary gland of hpg mice, and estradiol did not exert effects on testis development over and above those of FSH, we conclude that the action of estradiol on testis development in hpg mice is predominantly via the stimulation of pituitary FSH release.

Figure 1

Western blot analysis of ERα immunoreactivity. Examples of a Western blot analysis of extracts of pituitary gland (top) and testis (bottom) from hypogonadal (hpg) and age-matched wild-type (+/+) mice. Membranes were stained with MC-20 ERα antiserum (Santa Cruz).

Figure 2

Immunohistochemical localization of ERα in hpg mice. Examples of immunohistochemical localization of ERα in the pituitary gland (upper panels) or testis of hypogonadal mice (hpg, left panels) or age-matched wild-type mice (+/+, right panels). Note that many anterior pituitary cells express nuclear ERα immunoreactivity in both hpg and +/+ mice. ERα-ir is abundant in the Leydig cells (lc) and peritubular myoid cells (pt) in the testis of hpg mice, and is also present in some Sertoli cells (sc) in wild-type mice. Bottom left panel shows a control study in which the primary antiserum was omitted, the section is from a hpg testis. All photomicrographs were taken at the same magnification so the 20 μm scale bar in top left panel applies to all panels

Figure 3

Organ weights and serum FSH concentrations in hpg mice after treatment with estradiol and/or rhFSH. Wet weight of paired testes (top), epididymides (second panel) and seminal vesicles (middle panel), and serum concentrations of FSH of male hpg mice as determined in the human FSH ELISA (fourth panel) and the rat RIA (bottom). Groups of mice were treated for 50 days with subcutaneous implants containing either cholesterol (chol) or 2% estradiol (E), and also daily treatment with vehicle, 1 or 5 U of recombinant human FSH (Gonal-F™). Values are group mean ± SEM, group sizes as indicated. *P < 0.05, **P < 0.01 and ***P < 0.001 vs group treated with cholesterol implants and vehicle; ^@P < 0.01 vs all other groups.

Figure 4

Testis histology after treatment with estradiol and/or rhFSH. Representative examples of testicular histology for hpg mice receiving subcutaneous implants containing either cholesterol (chol) or 2% estradiol (E), and also daily treatment with vehicle, 1 or 5 U of recombinant human FSH (Gonal-F™) for 50 days. Scale bars represent 50 μm.

Figure 5

Leydig cells in hpg mice after treatment with estradiol and/or rhFSH. Representative examples of 3β-HSD immunostaining in hpg mice receiving subcutaneous implants containing either cholesterol (chol) or 2% estradiol (E), and also daily treatment with vehicle, 1 or 5 U of recombinant human FSH (Gonal-F™) for 50 days. Scale bars represent 50 μm.

Figure 6

Numbers of Leydig and Sertoli cells in hpg mice after treatment with estradiol and/or rhFSH. Estimates of numbers of Leydig cells (top) and Sertoli cells (bottom) in testes of hpg mice that had been treated for 50 days with subcutaneous implants containing either cholesterol (chol) or 2% estradiol (E), and also daily treatment with vehicle, 1 or 5 U of recombinant human FSH (Gonal-F™). Values are group mean ± SEM, group sizes as indicated. *P < 0.05 and ***P < 0.001 vs group treated with cholesterol implants and vehicle.