CYCLICAL ALTERNATIVE EXON SPLICING OF TRANSCRIPTION FACTOR CYCLIC ADENOSINE-MONOPHOSPHATE RESPONSE ELEMENT-BINDING PROTEIN (CREB) MESSENGER-RIBONUCLEIC-ACID DURING RAT SPERMATOGENESIS

During spermatogenesis, the levels of cAMP in seminiferous tubules und ergo stage-dependent cyclical fluctuations. We show that changes in cA MP levels are accompanied by alternative exon splicing of the RNA enco ding the cAMP-responsive transcription factor CREB (cAMP response elem ent-binding protein), expressed in both the Sertoli and germ cells. Ex ons Y and W are expressed exclusively in the testis, and they introduc e stop codons into the normal protein coding frame of CREB. The splici ng in of W was shown earlier to activate the internal translation of t wo alternative products of the CREB messenger RNA (mRNA) containing th e DNA-binding domain (I-CREBs). The I-CREBs act as potent inhibitors o f activator isoforms of CREB. The functions of the alternatively splic ed exon Y are unknown. To investigate whether the splicing of exons W and Y is regulated during spermatogenesis, seminiferous tubules, isola ted from adult rats, were dissected into segments representing differe nt stages of the spermatogenic cycle and were analyzed by RT-PCR. The analyses of pooled-tubule segments revealed stage-dependent splicing o f both exons W and Y in the CREB transcripts. Single tubules were diss ected into smaller segments for greater staging accuracy and were anal yzed by RT-PCR for CREB mRNAs containing either exons W or Y, as well as for FSH receptor mRNA. This analysis confirmed that a marked, cycle -dependent variation in CREB mRNA levels was occurring. Maximal splici ng of exons W and Y occurs independently at different stages of the sp ermatogenic cycle, stages II-VI and IX, respectively. The distinct spe rmatogenic cycle-dependent regulation of the splicing of exons W and Y provides further evidence in support of a functional relevance for CR EB-W and Y mRNA isoforms in spermatogenesis.