ICR mice used in this experiment were obtained from the animal center at the Korea Research Institute of Bioscience and Biotechnology. Mice were housed in a room maintained under the following conditions: a temperature of 23 ± 2℃, a relative humidity of 50 ± 5%, with artificial lighting from 08:00 to 20:00 and 13-18 air changes per h. The mice were fed a standard animal diet. Three mice at postnatal days (PNDs) 1, 3, 6, 18, 48 and 120 were obtained from the same litters.

Mice were sacrificed and testes were immediately removed (n = 3). A sample of the testes was embedded in paraffin wax after routine fixation in 10% buffered formalin. Paraffin sections (5 µm thick) were used in all immunostaining experiments. The opposite testis was snap-frozen and stored for immunoblot analysis. All experiments were carried out in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (USA).

Rabbit polyclonal anti-inhibin α (H-134), β_A (H-120) and β_B (H-110) antibodies were obtained from Santa Cruz Biotechnology (USA). Mouse monoclonal anti-beta-actin and vimentin antibodies were purchased from Sigma (USA) and Neomarkers (USA), respectively.

Testes tissues were immersed quickly in buffer H (50 mM β-glycerophosphate, 1.5 mM EGTA, 0.1 mM Na₃VO₄, 1 mM DTT, 10 µg/ml aprotinin, 2 µg/ml pepstatin, 10 µg/ml leupeptin, 1 mM PMSF, pH 7.4), and sonicated for 10 sec. The homogenate was transferred to microtubes and centrifuged at 19,340 × g for 10 min. The supernatant was then harvested. For the immunoblot assay, the supernatant was loaded into individual lanes of 10% sodium dodecyl (lauryl) sulfate-polyacrylamide gels, electrophoresed and immunoblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, USA). The residual binding sites on the membrane were blocked by incubation with 5% nonfat milk in phosphate-buffered saline (PBS, pH 7.4) for 1 h. Subsequently, the membrane was incubated overnight at 4℃ with rabbit polyclonal anti-inhibin-α, β_A and β_B antibodies (1 : 1,000 dilution). After extensive washing and incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (1 : 20,000 dilution; Pierce, USA), signals were visualized using chemiluminescence (Super Signal West Pico; Pierce, USA). For normalization purposes, membranes were re-probed with antibodies against beta-actin (1 : 20,000 dilution; Sigma, USA). Several exposure times were used to obtain signals in the linear range. The bands were quantified using Scion Image Beta 4.0.2 for Windows XP software (Scion, USA). The data were analyzed using one-way ANOVA followed by a Student-Newman-Keuls post hoc test for multiple comparisons. In all cases, a p value < 0.05 was considered significant.

Paraffin-embedded sections of testes (5 µm) were deparaffinized, treated with a citrate buffer (0.01 M, pH 6.0) in a microwave for 20 min, and then treated with 0.3% hydrogen peroxide in methyl alcohol for 20 min to block endogenous peroxidase activity. After three washes with PBS, sections were incubated with 10% normal goat serum, and then incubated with rabbit monoclonal inhibin α, β_A and β_B (1 : 100 dilution) for 1 h at room temperature. The immunoreactivity was visualized using fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (1 : 50 dilution; Sigma, USA). Cell phenotypes of inhibin α, β_A and β_B expression were examined by double label immunofluorescence using cell-type-specific markers, including vimentin (1 : 500 dilution) for the Sertoli and interstitial cells. First, the paraffin sections were reacted with primary rabbit anti-inhibin α, β_A and β_B followed by FITC-labeled goat anti-rabbit IgG (1 : 50 dilution; Sigma, USA). Slides were then incubated with mouse vimentin followed by tetramethyl rhodamine isothiocyanate-labeled goat anti-mouse IgG (1 : 50 dilution; Sigma, USA).

The testis at PND 48-120 showed an increase in the height of the seminiferous epithelium and the defined lumens of the tubules including mature sperm cells (Fig. 1C), while the tubules at PND 1-18 were largely undifferentiated (Figs. 1A and B). As shown in Fig. 1C, there was an abundant population of interstitial cells in the testis at PND 48. The seminiferous tubules contained primary spermatocytes, spermatids and Sertoli cells at various stages. This suggests that sexual maturation in this experimental animal occurs between PND 18 and 48.

The protein levels of the inhibin α, β_A and β_B isotypes in the testes during postnatal development were analyzed semiquantitatively by Western blotting to determine the developmental changes in the inhibin isotypes.

As shown in Fig. 2, a low intensity signal for inhibin α expression was detected in the testis at days 1-6 after birth. The level gradually increased at PND 18 to 120, and there was a significant peak (approximately 2 fold, p < 0.01 vs. PND 1-6) at PND 48 (Figs. 2A and B). A low level of inhibin β_A expression was observed in the early phase of development (PND 1-6). The level increased and showed a significant peak (approximately 2 fold, p < 0.05 vs. PND 1 and 6) at day 48 after birth (Figs. 2A and C). A low intensity signal for inhibin β_B expression was detected in the testis at PND 1-6. The level increased at PND 48-120, and there were substantial levels at both PND 48 (approximately 1.5 fold, p < 0.01 vs. PND 1-18) and PND 120 (approximately 1.6 fold, p < 0.05 vs. PND 1-18) (Figs. 2A and D).

At PND 1-6, there was little immunoreactivity for inhibin α, β_A and β_B subunits in testicular cells (data not shown). Inhibin α expression (Fig. 3A) was observed in cytoplasmic processes of vimentin-positive Sertoli cells surrounding spermatogenic cells (Fig. 3B) at PND 18-120. Immunoreactivity for inhibin β_A was observed in the interstitial and spermatogenic cells (Fig. 3C) during PND 48-120. Inhibin β_B immunoreactivity was observed mainly in cell membranes of some spermatogonia in the seminiferous tubules as well as in the interstitial cells after PND 48 (Fig. 3D).