The expression and localization of inhibin isotypes in mouse testis during postnatal development

Abstract

Inhibin, which is important for normal gonadal function, acts on the pituitary gonadotropins to suppress folliclestimulating hormone (FSH) secretion. The level and cellular localization of the inhibin isotypes, alpha, betaA and betaB, in the testis of mice were examined during postnatal development in order to determine if inhibin expression is related to testicular maturation. Mouse testes were sampled on postnatal days (PNDs) 1, 3, 6, 18, 48 and 120, and analyzed by Western blotting and immunofluorescence. Western blot analysis showed very low levels of inhibin alpha, betaA and betaB expression in the testes at days 1 to 6 after birth. The levels then increased gradually from PND 18 to 48-120, and there were significant peaks at PND 48. Inhibin alpha, betaA and betaB were detected in testicular cells during postnatal development using immunohistochemistry. The immunoreactivity of inhibin alpha was rarely observed in testicular cells during PND 1 to 6, or in the cytoplasmic process of Sertoli cells surrounding the germ cells and interstitial cells during PND 18 to 120. Inhibin betaA and betaB immunoreactivity was rarely observed in the testis from PND 1 to 6. On the other hand, it was observed in some spermatogonial cells, as well as in the interstitial space between PND 48 and PND 120. We conclude that the expression of inhibin isotypes increases progressively in the testis of mice with increasing postnatal age, suggesting that inhibin is associated with a negative feedback signal for FSH in testicular maturation.

Fig. 1

Light micrographs of the mouse testes at postnatal day (PND) 1 (A), PND 18 (B), and PND 48 (C). The arrows in A indicate gonocytes in undifferentiated seminiferous epithelium. The asterisk in C indicates the defined lumens of the tubules including mature sperm cells. H&E stain. Scale bars = 40 µm.

Fig. 2

Expression of inhibin isotypes α, β_A and β_B in mouse testis increased progressively with postnatal age. Photographs: Representative photographs of Western blots for inhibin isotypes α, β_A and β_B and beta-actin (A). Arrowheads indicate the positions of the inhibin isotypes (40~47 kDa) and beta-actin (45 kDa). Minor bands at various molecular weights were detected on the immunoblots for the inhibin isotypes α, β_A and β_B. Bar graph: The results of densitometric data analysis (mean ± SE, n = 3 mice/group). The relative expression levels of the inhibin isotypes were calculated after normalization to the beta-actin band from three different samples. The value for the testis at postnatal day (PND) 1 was arbitrarily defined as 1 (B, C and D, graphs). ^*p < 0.05, ^**p < 0.01 vs. PND 1-6.

Fig. 3

Immunofluorescent localization of inhibin α, β_A, and β_B isotypes in mouse testis at postnatal days 48. (A and B) Double-immunofluorescent staining in the same section showed the co-localization of inhibin α with vimentin in cell bodies of Sertoli cells (arrowheads), the cytoplasmic process of Sertoli cells (arrows) and in interstitial spaces (asterisks). (C) Immunofluorescent localization of the inhibin β_A subunit was observed in the cell membrane of some spermatogenic cells (arrows) as well as in the interstitial cells (asterisk). (D) Immunofluorescent localization of the inhibin β_B subunit was observed mainly in cell membranes of interstitial cells (asterisk) as well as in some spermatogonia (arrows). Scale bars = 30 µm.