FIG. 1.

Downregulation of MIS expression by TNF-α in the testis. (A) MIS gene regulation by TNF-α in the organ-cultured testis. Testes from 7-day-old male mice were organ cultured in the presence or absence of 20 ng of TNF-α/ml and collected at the indicated time points for the preparation of total RNA. Testes from two to three mice were used for each time point. At the top of the panel, a Northern blot analysis of total RNAs from organ-cultured testes with ³²P-labeled mouse MIS cDNA as a probe is shown. The expression of GAPDH was used as an internal control. At the bottom of the panel, MIS mRNA signal quantified by using a phosphorimager and normalized by determining the GAPDH mRNA level in each sample is shown. Data are representative of three similar experiments. (B) MIS expression during the development of TNF-α knockout testis. Total RNAs from TNF-α wild-type and mutant testes were prepared at different developmental days. Testes from two to three mice were combined to prepare total RNAs for day 7 to day 26 samples. A Northern blot analysis of the total RNAs from testes with ³²P-labeled mouse MIS cDNA as a probe is shown in the top panel. 28S rRNA was used as an internal control. In the bottom panel, the MIS mRNA signal was quantified and normalized by using the 28S rRNA level in each sample. Two independent experiments were performed, and error bars represent the standard error of the mean (SEM). (C) Downregulation of MIS expression in TNF-α knockout testis by TNF-α treatment. Northern blot analysis was performed with total RNAs from testes of three 32-day-old TNF-α knockout mice, which were injected with or without 50 μg of TNF-α/kg. (D) Expression of TNF-α in meiotic germ cells. The immunohistology of mouse adult testis was evaluated with anti-TNF-α antibody. The negative control was processed with exclusion of the primary antibody. Strong signals in pachytene spermatocytes and elongated spermatids are indicated by arrows.