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Cyclical Alternative Exon Splicing of Transcription Factor Cyclic Adenosine Monophosphate Response Element-Binding Protein (CREB) Messenger Ribonucleic Acid during Rat Spermatogenesis¹

Laboratory of Molecular Endocrinology, Massachusetts General Hospital and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Joel F. Habener, M.D., Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street, WEL320, Boston, Massachusetts 02114. E-mail: habenerj{at}a1.mgh.harvard.edu

During spermatogenesis, the levels of cAMP in seminiferous tubules undergo stage-dependent cyclical fluctuations. We show that changes in cAMP levels are accompanied by alternative exon splicing of the RNA encoding the cAMP-responsive transcription factor CREB (cAMP response element-binding protein), expressed in both the Sertoli and germ cells. Exons Y and W are expressed exclusively in the testis, and they introduce stop codons into the normal protein coding frame of CREB. The splicing in of W was shown earlier to activate the internal translation of two alternative products of the CREB messenger RNA (mRNA) containing the DNA-binding domain (I-CREBs). The I-CREBs act as potent inhibitors of activator isoforms of CREB. The functions of the alternatively spliced exon Y are unknown. To investigate whether the splicing of exons W and Y is regulated during spermatogenesis, seminiferous tubules, isolated from adult rats, were dissected into segments representing different stages of the spermatogenic cycle and were analyzed by RT-PCR. The analyses of pooled-tubule segments revealed stage-dependent splicing of both exons W and Y in the CREB transcripts. Single tubules were dissected into smaller segments for greater staging accuracy and were analyzed 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 splicing of exons W and Y occurs independently at different stages of the spermatogenic cycle, stages II-VI and IX, respectively. The distinct spermatogenic cycle-dependent regulation of the splicing of exons W and Y provides further evidence in support of a functional relevance for CREB-W and Y mRNA isoforms in spermatogenesis.

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