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

Baines H, Nwagwu MO, Hastie GR, Wiles RA, Mayhew TM, Ebling FJ - Reprod. Biol. Endocrinol. (2008)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. Helen_baines@hotmail.com

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.

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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).
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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).

Mentions: Western blots revealed a clear band of ERα-immunoreactivity in extracts of pituitary tissue from adult hpg mice at a molecular weight of approximately 64 KDa (Fig. 1, top). There was no significant difference in abundance comparing samples from hpg (n = 4) and wild type (n = 4) mice. ERα-immunoreactive bands were also observed at approximately 64 KDa in extracts of testis from both hpg and wild-type mice, plus a slightly lower molecular weight band which is likely to be a degradation artefact (Fig. 1, bottom). No statistical difference in abundance of the two bands between the genotypes was observed (Fig. 1). Immunohistochemistry revealed a clear nuclear localization of ERα in anterior pituitary cells in both hpg mice and wild-type litter mates (Fig. 2). ERα-immunoreactivity was prominent in the nuclei of both Leydig cells and peritubular myoid cells in testes from hpg mice (Fig. 2, left). Leydif cells in the age-matched wild-type testis also expressed ERα-immunoreactivity (Fig. 2, right), and some peritubular cells also expressed ERα (Fig. 2, right). In addition, in wild-type mice there ws some positive immunostaining of cells adjacent to the basal membrane with an irregular shaped nucleus which are almost certainly Sertoli cells (Fig. 2, right). The immunoreactive signal was absent when the primary antiserum was replaced with rabbit IgG (Fig. 2, bottom left).


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

Baines H, Nwagwu MO, Hastie GR, Wiles RA, Mayhew TM, Ebling FJ - Reprod. Biol. Endocrinol. (2008)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254628&req=5

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).
Mentions: Western blots revealed a clear band of ERα-immunoreactivity in extracts of pituitary tissue from adult hpg mice at a molecular weight of approximately 64 KDa (Fig. 1, top). There was no significant difference in abundance comparing samples from hpg (n = 4) and wild type (n = 4) mice. ERα-immunoreactive bands were also observed at approximately 64 KDa in extracts of testis from both hpg and wild-type mice, plus a slightly lower molecular weight band which is likely to be a degradation artefact (Fig. 1, bottom). No statistical difference in abundance of the two bands between the genotypes was observed (Fig. 1). Immunohistochemistry revealed a clear nuclear localization of ERα in anterior pituitary cells in both hpg mice and wild-type litter mates (Fig. 2). ERα-immunoreactivity was prominent in the nuclei of both Leydig cells and peritubular myoid cells in testes from hpg mice (Fig. 2, left). Leydif cells in the age-matched wild-type testis also expressed ERα-immunoreactivity (Fig. 2, right), and some peritubular cells also expressed ERα (Fig. 2, right). In addition, in wild-type mice there ws some positive immunostaining of cells adjacent to the basal membrane with an irregular shaped nucleus which are almost certainly Sertoli cells (Fig. 2, right). The immunoreactive signal was absent when the primary antiserum was replaced with rabbit IgG (Fig. 2, bottom left).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. Helen_baines@hotmail.com

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.

Show MeSH
Related in: MedlinePlus