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The objective of the current study was to determine the role of transforming growth factor beta (TGFβ) during seminiferous cord formation and embryonic testis development. The expression pattern of mRNA for TGFβ isoforms was evaluated during testis development through a quantitative reverse transcription polymerase chain reaction (QRT-PCR) procedure. Expression of mRNA for TGFβ1 was highest at postnatal day 0 (P0) and P10. In contrast, TGFβ2 was high at embryonic day 15 (E15), declined at E16, and showed a transient increase at P0 through P3 of testis development. Interestingly, expression of mRNA for TGFβ3 was high during embryonic development and then declined after P3. Immunohistochemical localization of TGFβ1 and TGFβ2 demonstrated expression in Sertoli cells at E14 and in the seminiferous cords at P0. Selective interstitial cells expressed high concentrations of TGFβ1 and TGFβ2 in P0 testis. TGFβ3 was expressed in selective cells at the junction of the E14 testis and mesonephros. The cells expressing TGFβ3 in the testis appeared to be preperitubular cells that resided around the seminiferous cords. TGFβ3 was localized to gonocytes in P0 testis. TGFβ1 was found to have no influence on seminiferous cord formation in embryonic organ cultures of E13 testis. In contrast, growth of both E13 and E14 embryonic organ cultures was inhibited by TGFβ1 and resulted in reduced testis size (40% of controls) with fewer cords present. A P0 testis cell culture and thymidine incorporation assay were used to directly examine the effects of recombinant TGFβ1. TGFβ1 alone had no influence on thymidine incorporation in P0 testis cell cultures when compared to controls. Interestingly, TGFβ1 inhibited epidermal growth factor (EGF), and 10% calf serum stimulated P0 testis cell growth but not FSH-stimulated growth. Therefore, TGFβ1 appears to inhibit testis growth in both the embryonic and early postnatal periods. The hormonal regulation of TGFβ expression was measured using P0 testis cell cultures and a QRTPCR procedure for each TGFβ isoform. High concentrations of EGF stimulated expression of mRNA for TGFβ1 after 24 h but suppressed expression of TGFβ3. In contrast, there was no effect of FSH on TGFβ isoform expression. In summary, TGFβ regulates embryonic and P0 testis growth through inhibiting the actions of positive growth factors such as EGF. In addition, EGF but not FSH appears to regulate TGFβ isoform expression. Combined observations from the present study demonstrate that TGFβ isoforms are differentially expressed and appear to be regulators of testis growth during the embryonic and early postnatal periods.