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THE DEVELOPMENTAL EXPRESSION PATTERN OF SMAD2 AND SMAD3 IN RAT TESTES.
Xu, Jian1, Oakley, Jennifer1, McGee, Elizabeth1, 1
ABSTRACT- Smads are a family of downstream mediators for the family of TGF growth factors. Smad2 and Smad3 are both associated with both TGF and activin signaling. In the present study, we have determined the expression pattern of these two important signaling molecules during the development of the rat testes. Testes were collected from 5-,10-,15-, 20-, 25-, 30- and 60-day-old rats, and fresh frozen or embedded in OCT and frozen. For western analysis, 15ug of protein lysate from each sample was subject to denaturing polyacrylamide gel electrophoresis and transfer to PVDF membrane. The membranes were probed with either Smad2 or Smad3 antibody. RNAse protection assay was performed with a-32P-CTP labeled antisense probes for Smad2 and Smad3. Cyclophilin was used as an internal control. Immunohistochemistry was performed with Smad2 and Smad3 antibodies in combination with a biotin-fluorescein avidin detection system. Western analysis showed that both Smad2 and Smad3 proteins were expressed from day 5 to day 60. Both Smad2 and Smad3 protein expression levels were highest from day 15 to day 30. Smad2 and Smad3 mRNA transcripts were detected at all time points. However for both Smads, expression increased on day 10 and remained elevated until day 30. Immunohistochemistry demonstrated that both Smad2 and Smad3 proteins were present in the cytoplasm of Sertoli cells and Leydig cells throughout development. Neither Smad2 or Smad3 protein was detected in spermatogonia, but staining for both proteins was prominent in meiotic germ cells. The pattern of Smad expression during testicular maturation in the rat is consistent with a role for TGF and activin in regulating Sertoli and Leydig cell functions during this time. The expression pattern of the Smads in germ cells suggests that TGF, activin or a related growth factor also play a role in regulating spermatogenesis through the Smad signaling transduction pathway.
KEY WORDS: Smad, spermatogenesis, activin, TGF
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