Differential Regulation of Cyclic Adenosine 3′,5′-Monophosphate (cAMP) Response Element-Binding Protein and cAMP Response Element Modulator Messenger Ribonucleic Acid Transcripts by Follicle-Stimulating Hormone and Androgen in the Adult Rat Testis

Abstract

Hormonal regulation of the expression of mRNA transcripts for cAMP response element-binding protein (CREB) and cAMP response element modulator (CREM) during spermatogenesis was studied in the adult rat testis. Northern analysis of CREB and CREM identified two mRNA transcripts for CREM (2.4 and 1.6 kb) and one transcript for CREB (2.0 kb). Analysis of mRNAs from isolated testicular cells by reverse transcriptase polymerase chain reaction (RT/PCR) showed that CREM mRNAs were expressed by the germ cells but not the Sertoli or interstitial cells, whereas CREB mRNA was located in germ cells, Sertoli cells, and interstitial cells. RNA was isolated and analyzed from the testes of 1) rats treated for 24 h with FSH, 2) rats in which androgen withdrawal had been induced by ethane dimethane sulphonate (EDS) treatment 6 days earlier (EDS-treated), 3) EDS-treated rats supplemented with testosterone (EDS + T), or 4) intratesticular administration or dibutyryl cAMP (dbcAMP) in the preceding 24 h. CREM mRNA transcript expression was found to be decreased after all of these treatments in samples from intact testis and from isolated cells. Expression of the CREB transcript was also decreased by EDS-induced androgen withdrawal, but not by FSH or EDS + T. In situ hybridization of paraffin-embedded testis sections probed with digoxigenin-labeled riboprobes confirmed the localization of CREB and CREM mRNA to the same cell types as found with RT/PCR. No stage-dependent expression of CREM mRNA transcripts could be observed. Hybridization of the CREB probe was highest around the base of stage VII-VIII tubules, and this was shown to be androgen-dependent. The data presented suggest that regulation of the expression of CRE-binding protein mRNAs in Sertoli and germ cells during spermatogenesis is dependent on both androgen and FSH. However, the effects of androgen or FSH on the regulation of CRE-binding protein mRNAs are different.

Hormonal regulation of male germ cell development

“…There is also evidence for a non-genomic action of androgens, in which testosterone elicits responses through secondary messengers such as cAMP and signaling pathways different from classical AR-mediated transcription (reviewed in Walker (2003)). Cellular responses following secondary messenger or signaling pathways in Sertoli cells include the phosphorylation of CREB and CRE modulator, and these transcription factors have also been shown to be regulated by FSH (West et al 1994, Scobey et al 2001. Despite common pathways being activated by FSH and testosterone, some differences exist, for example, testosterone does not up-regulate cAMP production in Sertoli cells (reviewed in Walker & Cheng (2005)).…”

Developmentally distinct in vivo effects of FSH on proliferation and apoptosis during testis maturation

“…References for those genes previously reported to be in the rat testis and/or regulated by FSH are in parentheses. Yes; (Walker et al 1994, West et al 1994, Monaco et al 1995 (Wolgemuth et al 1998, Buemer et al 2000, Strothmann et al 2004 Yes Androgen Binding Protein (ABP); sex hormone binding globulin…”

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

“…Among the methods described in the literature for the separation of rat germ cells, the combination of centrifugal elutriation and isopycnic density (Percoll ® ) has produced the highest degree of purity of spermatogenic cells from the adult rat testis (1). This method has been used by other investigators to study molecular aspects of rat germ cells (2,5) and therefore conclusions have been made about molecular and biochemical aspects of the cells, assuming that the purity of the cells was as high as described in the original study.…”