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

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 μg 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

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>Western blot analysis revealed ERα immunoreactive bands of appropriate molecular weight in extracts of testis and pituitary glands from hpg mice, and immunohistochemical studies confirmed ERα 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.</p> <p>Conclusion</p> <p>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 <it>hpg </it>mice is predominantly via the stimulation of pituitary FSH release.</p

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-1

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>s) or testis of hypogonadal mice (, left panels) or age-matched wild-type mice (+/+, right panels). Note that many anterior pituitary cells express nuclear ERα immunoreactivity in both and +/+ mice. ERα-ir is abundant in the Leydig cells (lc) and peritubular myoid cells (pt) in the testis of 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 testis. All photomicrographs were taken at the same magnification so the 20 μm scale bar in top left panel applies to all panel

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-0

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>tom) from hypogonadal () and age-matched wild-type (+/+) mice. Membranes were stained with MC-20 ERα antiserum (Santa Cruz)

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-7

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>s) or testis of hypogonadal mice (, left panels) or age-matched wild-type mice (+/+, right panels). Note that many anterior pituitary cells express nuclear ERα immunoreactivity in both and +/+ mice. ERα-ir is abundant in the Leydig cells (lc) and peritubular myoid cells (pt) in the testis of 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 testis. All photomicrographs were taken at the same magnification so the 20 μm scale bar in top left panel applies to all panel

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-6

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>tom) from hypogonadal () and age-matched wild-type (+/+) mice. Membranes were stained with MC-20 ERα antiserum (Santa Cruz)

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-5

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>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

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-4

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>s 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