help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Daniel, P. B.
Right arrow Articles by Habener, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Daniel, P. B.
Right arrow Articles by Habener, J. F.
Endocrinology Vol. 139, No. 9 3721-3729
Copyright © 1998 by The Endocrine Society

ARTICLES

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

Philip B. Daniel and Joel F. Habener2

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CYCLE of spermatogenesis is divided into 14 stages defined by patterns of cellular associations within the tubule (1). During the process of development, which takes approximately 45 days, a committed germ cell passes through 31/2 cycles, driven by the alternating influences of cAMP and androgenic hormones (reviewed in Ref. 2). cAMP increases transcriptional activation by several closely related bZIP transcription factors [cAMP response element-binding protein (CREB), cAMP response element modulator (CREM), and activating transcription factor-1 (ATF-1)] via phosphorylation by cAMP-dependent protein kinase A (reviewed in Ref. 3).

CREB and CREM are known to be expressed in a variety of isoforms, many of which are inhibitory to cAMP-induced transcription. CREB is predominantly a positive modulator of cAMP-responsive genes. However, in the testis, alternative exon splicing results in the expression of repressor CREB isoforms (4, 5). Several alternatively spliced exons in the CREB messenger RNA (mRNA) that contain in-frame stop codons have been described in mouse, rat, and human CREB genes (Fig. 1Go). The W, Z, and Y (or {gamma}) exons are testis-specific and are most strongly detected in germ cells (4, 6, 7). No function has been ascribed to the aminoterminally truncated proteins translated from the natural start site in exon B and terminating in exons Y or W. However, when exon W is spliced into CREB mRNA, a process of translational reinitiation within exon W and/or exon H allows the CREB-W transcript to generate two repressor bZIP proteins (I-CREBs) that inhibit CREB activator functions (4, 5).



View larger version (10K):
[in this window]
[in a new window]
  		Figure 1. Exonic structure of the rat CREB gene. The rat CREB gene consists of at least eleven exons (boxes). The coding regions are denoted by shading. The unshaded boxes denote the 5' and 3' untranslated exons. Exons W and Y (crossed boxes) introduce stop codons into the CREB reading frame. Exon D (lightly shaded) does not interfere with translation. Potential translation starts (small horizontal arrows) are located in exon B for CREB (c), and exons W and H for I-CREB long (l) and short (s). Arrows located on top of boxes denote oligonucleotides used for RT-PCR. The angled arrow on left of figure indicates transcription start site.

 
Earlier studies, using in situ hybridization (6) and immunocytochemical techniques (5), provided evidence suggesting that the expression of CREB-W mRNA is regulated during spermatogenesis (6). Here, mRNA from staged tubule segments is analyzed by RT-PCR to provide evidence that the expression levels of CREB, CREB-W, and CREB-Y mRNAs are regulated over the 12-day cycle of spermatogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Oligonucleotides
Primer pairs were designed to distinguish transcripts including or lacking exons Y and W. Primers were directed to exons flanking the alternatively spliced exons (see Fig. 1Go). For the detection of CREB-Y, the forward primer was based in exon C, in preference to the infrequently spliced exon D.

Oligonucleotides used as primers in the RT-PCR reactions were: Exons C-E primer pair: forward = 5'-CACTGTAACCTTAGTGCAGC-3'; reverse = 5'-AGGATTTCCCTTCGTTTTTGG-3'; Exon C probe: 5'-GCAGACAGTCCAGGTCCATGGGGTCATCC-3'; Exon D probe: 5'-TCTTCCTGTAAGGACTTAAAAAGAC-3'; Exon Y probe: 5'-GAGAACAGA-GATGTACTGTTTGCT-3'; Exons G-H primer pair: forward = 5'-GCAGACATTAACCATGACC-3'; reverse = 5'-CCTGTTCTTCAT- TAGACG-3'

Exon H probe: 5'-GACGAACCTCTCTCTTTCG-3'; Exon W probe/reverse primer: 5'-CAAAAAATTGTAAAGCAGG-3'.

FSH receptor (FSHR) primers: forward = 5'-GGAATCTGTGGAAGTTTTCG-3'; reverse = 5'-ATGGCCTGCTCTTCAGAAGG-3'; FSH receptor probe: 5'-CAAGGACAAAGGTCCATTCC-3'.

Adenine phosphoribosyltransferase primers: forward = 5'-TCCGA ATCTGAGTTGCAGC-3'; reverse = 3'-CTGCACACATGGTTCCTCC-3'.

Plasmids
Rat CREB complementary DNA (cDNA) and CREB-W cDNA in pCRII vector (Invitrogen, Carlsbad, CA) were provided by Dr. W. H. Walker. For the experiment described in Fig. 3CGo, the plasmids were linearized by digestion with XhoI restriction enzyme before dilution and PCR analysis.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 3. Control studies to verify the accuracy and quantitation of the RT-PCR procedure used to amplify mRNAs on stage-specific segments of rat seminiferous tubules. A, Optimization of cycle number and its effect on ratio of exon W+ products to exon W- products. In four separate experiments, a PCR reaction with CREB G-H primers was run for 30 cycles with aliquots withdrawn at indicated points. The numbers below each lane are the ratios of band intensities (adjusted for DNA content) between CREB-W and exon W- CREB products, quantified by densitometry and averaged over four experiments. B, Assessment of the linear range of quantitation of the RT-PCR procedure. A sample of the CREB G-H PCR product from a single tubule segment cDNA sample (stages VI-VII) was diluted to one half, one quarter, and one eighth by serial dilution and was analyzed as described. Two separate experiments are shown. C, Assessment of the ratio accuracy of the RT-PCR procedure. CREB and CREB-W cDNA clones in pCRII (Invitrogen) were apportioned in ratios of 1:1 through 1:10, keeping a constant mass of template (10 pg) per reaction. Ratios were quantified as described. The experiment was performed once.

 
Microdissection procedure
Seminiferous tubules were isolated from the testes of adult (60-day-old) Sprague-Dawley rats under transillumination microscopy. Intact tubules encompassing complete 14-stage cycles were selected and divided into consecutive segments (2), with stages estimated visually during dissection. Segments were stored at -70 C until RNA extraction was performed.

For the experiments shown in Figs. 4Go and 5Go, each of the three sample sets was produced from five seminiferous tubules. Tubules were divided into eight segments, representing stages IX-XI, XII, XIII-XIV, I, II-IV, V-VI, VII, and VIII. Segments were pooled according to their stage, and RNA was extracted in 200 ml of Trizol (Gibco BRL, Gaithersburg, MD), following the manufacturer’s protocol. To assist recovery during isopropanol precipitation, 20 µg linear polyacrylamide was added (8). Pellets obtained by centrifugation were washed in 70% ethanol, then resuspended in 20 µl DEPC-treated H20. RNA was quantified spectrophotometrically, and 2 µg was used for cDNA synthesis.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 4. Determinations of the ratios of CREB mRNAs with and without alternatively spliced exon W in rat seminiferous tubules during one cycle of spermatogenesis. A, Intact seminiferous tubules, covering a complete 14-stage cycle (12.5 days), were dissected into segments representing eight groupings of stages. The segments from 5 tubules were combined for each sample set, and a total of three sample sets were analyzed by RT-PCR with the G-H primer pair. Bands with (W+) and without (W-) the W exon are indicated. B, PCR results were analyzed densitometrically. The ratio of exon W+ to exon W- products was calculated and averaged over the three sample sets.

 


View larger version (29K):
[in this window]
[in a new window]
  		Figure 5. Determinations of the ratios of CREB mRNAs with and without alternatively spliced exon Y in rat seminiferous tubules during one cycle of spermatogenesis. A, Intact seminiferous tubules, covering a complete 14-stage cycle (12.5 days), were dissected into segments representing 8 groupings of stages. The segments from 5 tubules were combined for each sample set, and a total of three sample sets were analyzed by RT-PCR with the C-E primer pair. Bands with (Y+) and without (Y-) the W exon are indicated. B, PCR results were analyzed densitometrically. The ratio of exon Y+ to exon Y- products was calculated and averaged over the three sample sets. The D-exon-containing products were not quantified.

 
For experiments in Fig. 6Go, a single tubule, including two complete cycles, was divided into consecutive 3-mm segments. More accurate assignment of stages was achieved by microscopic examination of squashes of 0.5-mm portions from the distal end of each segment (9). Results in Fig. 6Go suggest an accuracy of ±1 stage. The remaining segment was extracted in 100 ml Trizol and processed as described above, except that RNA was not quantified, and all recovered RNA was used for cDNA synthesis.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 6. Identification of amounts and relative ratios of CREB mRNAs with alternatively spliced exons W and Y and FSHR mRNA in single-rat seminiferous tubules during two cycles of spermatogenesis. A, A single, intact seminiferous tubule containing two complete waves was dissected into consecutive 3-mm segments, as described in Materials and Methods. A 0.5-mm fragment from each segment was squashed and examined microscopically to assign stages for each segment. The individual segments were then analyzed by RT-PCR using primers for CREB G-H, CREB C-E, FSHR, and APRT in separate reactions. The identity of FSHR product is confirmed by hybridization to an oligonucleotide probe. B, Ratios of exon Y+/exon Y- (closed circles) and exon W+/exon W- (open circles) were calculated from densitometric analysis of CREB C-E and CREB G-H PCR results. The experiment was repeated twice. C, Quantities of CREB (closed circles) and FSHR (open circles) derived from densitometric analysis of CREB C-E and FSHR PCR results.

 
RT-PCR
RNA, in 10 µl H2O, was combined with 0.5 µg oligo nucleotide dT(16) and heated to 65 C for 10 min, then cooled on ice. RT buffer, deoxynucleotide triphosphates (50 µM each), dithiothreitol (5 mM), Superscript II (Gibco BRL) enzyme (50 U), and H2O were added for a total vol of 40 µl (pooled-tubule segments) or 20 µl (single-tubule segments), and reactions were incubated at 42 C for 40 min. For each set of samples, one additional segment (or pool of segments) was prepared as described, but without reverse transcriptase, as a control for reagent purity and contamination by genomic DNA.

PCR was performed in 50-µl reactions using 2 µl of the cDNA preparations. Reactions contained 20 pmol each of forward and reverse primers, 0.2 mM each of deoxynucleotide triphosphates, and 2.5 U of thermostable taq polymerase (TaKaRa Biomedical Inc., Berkeley, CA). Cycling conditions for both CREB primer pairs, and adenine phosphoribosyltransferase (APRT) primers, were 94 C for 20 sec, 55 C for 30 sec, and 72 C for 30 sec, for 25 cycles unless otherwise noted. For FSH receptor PCR conditions were modified as follows: 58 C annealing, temperature and extension time of 2 min, for 30 cycles. PCR products were resolved in 1.5% agarose gels containing 0.25 µg/ml ethidium bromide. Gels were scanned with a model 575 fluorimager (Molecular Dynamics, Sunnyvale, CA).

Densitometry on fluorimager-scanned gels was performed using Quantity One software (PDI, New York, NY). Signals from extra exon-containing products (CREB-W, CREB-Y, and CREB-WY) were corrected for additional DNA content relative to the shorter CREB products. (CREB-W: 0.809 in G-H PCR, 0.924 in C-H PCR; CREB-Y: 0.631 in C-E PCR, 0.875 in C-H PCR; CREB-WY: 0.816 in C-H PCR).

Southern analysis of PCR products
PCR products were transferred to MagnaGraph membrane (Micro Separations Inc., Westboro, MA) by capillary transfer and hybridized with {gamma}32P-ATP-labeled oligonucleotide probes in a solution of 5 x saline-sodium citrate, 1% SDS, 10 x Denhardt’s, and 100 µg/ml denatured sal-mon-sperm DNA for 3 h at 37 C. Blots were washed to a maximum stringency of 0.5 x saline-sodium citrate at 52 C. For rehybridization, probe was removed by washing blots in 0.5 m NaOH at room temperature for 1 h.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mRNA ratios of CREB variants are modulated in a stage-specific manner
Splice variants of the rat CREB gene are derived from the inclusion of exons D, Y, or W. Exons D and Y lie between exons C and E, and exon W between exons G and H (Fig. 1Go). PCR performed with primers based in these flanking exons produces bands representative of splicing events. Segments (2.5 mm) of a rat seminiferous tubule have been analyzed by RT-PCR and Southern blotting (Fig. 2Go). Hybridization results are consistent with molecular weight estimations for both G-H and C-E products (see Fig. 1Go).



View larger version (50K):
[in this window]
[in a new window]
  		Figure 2. Identification of exons W, Y, and D by RT-PCR of mRNA prepared from rat seminiferous tubules. A, The CREB RT-PCR products generated by the G-H primer pair of 271 bp (CREB, without exon W) and 335 bp (CREB-W). The template is pooled tubule segment cDNA from stages IV-V, VI, and VII. Identity of the products was confirmed by Southern blot hybridization with oligonucleotide probes for exons H and W. B, The C-E primer pair produces two major bands of 190 bp (CREB, without exon Y or D) and 301 bp (CREB-Y). Other products are two weaker bands of 232 bp (CREB-D) and 343 bp (CREB-YD) and a faint unidentified band between CREB-Y and CREB-D. Identity of the bands was confirmed by Southern blot hybridization with oligonucleotide probes for exons C, Y, and D.

 
Exon D, which is spliced-in in many tissues, does not shift the translational reading frame of CREB. The presence of exon D in a small fraction of the CREB mRNA population is confirmed by hybridization with an exon D-specific probe. Exon D is spliced into both natural CREB mRNA and CREB-Y mRNA (CREB mRNA containing exon Y), but these products are minor compared with CREB and CREB-Y themselves.

Control experiments were performed to determine whether the relative quantities of PCR product accurately reflect the relative levels of transcripts in CREB mRNA populations. The effect of cycle number on product ratios for whole testis cDNA was examined using the CREB G-H primer pair (Fig. 3AGo). When analyzed by densitometry (as outlined in Materials and Methods), the ratios of exon W-containing CREB PCR products (W+) to other CREB products (W-) remain relatively constant up to 30 cycles of amplification, confirming similar amplification kinetics of both cDNAs. Consequently, a protocol using 25 cycles was selected to remain within the linear range of amplification. The gel shows a representative result, and the values for the ratios of exon W+ to exon W- products (W+/W-) are the average of four experiments.

To ensure that the quantitative range of RT-PCR products analyzed in these experiments does not affect the accuracy of determination of product ratios by densitometric analysis, RT-PCR samples from single-tubule segments of stages VI-VII (amplified with CREB G-H primers) were serially diluted, and ratios were determined over the range of the dilution series (Fig. 3BGo). Product ratios (W+/W-) remained consistent at all dilutions. Note the presence of an extra band above the authentic CREB-W product. This band represents an anomalous product occasionally seen when amplifying CREB-W. The product can be detected in PCR experiments using CREB-W-containing plasmids as template (results not shown), and therefore, does not represent an additional splice variant. It is not included in the densitometric analyses.

The degree to which the observed product ratios reflect template concentrations was examined using plasmids containing CREB and CREB-W sequences. Using a constant total amount (10 pg) of both templates, the ratio of CREB-W to CREB was varied from 1 down to 0.1, and PCR analysis was carried out as described. Ratios of amplified products closely followed the input template ratios (Fig. 3CGo), indicating that a competitive amplification approach could accurately determine relative quantities of CREB isoforms.

This technique was then used to analyze cDNA from pooled-tubule segments. RT-PCR analysis for three independent preparations of five tubules each, using CREB G-H primers, was carried out to assess splicing of exon W (Fig. 4AGo), and CREB C-E primers were used to examine splicing of exon Y (Fig. 5AGo). Product ratios for CREB-W (W+/W-) and CREB-Y (Y+/Y-) were averaged over the three independent experiments and plotted against segment stage (Fig. 4BGo and 5BGo). A significant and reproducible variation in product ratios of CREB-W and CREB-Y mRNAs occurs over the course of the spermatogenic cycle (Figs. 4BGo and 5BGo, respectively). The results indicate that the timing of insertion for exon-W and exon-Y are significantly different: CREB-W reaches its maximum around stages V-VII, and CREB-Y peaks at stages IX-XI.

Alternative splicing of exons W and Y occurs at different temporal stages of the spermatogenic cycle
The suggestion that expression of CREB-W and CREB-Y may be regulated independently was further examined by RT-PCR analysis of consecutive segments from a single tubule. Whereas the pooled-tubule segments provided quantifiable amounts of RNA, single-tubule experiments offer a more detailed view of the changes in ratio between splice variant PCR products. The segments are smaller (3 mm) and are staged by microscopic examination, as described in Materials and Methods. Because the segments are consecutive and are taken from a single tubule, they are more representative of discrete stages of the spermatogenic cycle.

A single tubule, encompassing two complete cycles of spermatogenesis, was examined. The cDNA samples produced were used for multiple, separate PCR amplifications of CREB-Y, CREB-W, FSH receptor (FSHR), and APRT (Fig. 6AGo). Ratios for CREB-W and CREB-Y products, as fractions of products not containing the respective exons, were calculated as before (Fig. 6BGo). RT-PCR of the FSHR and APRT mRNAs were carried out as controls. Full-length (2.2 kb) FSHR was amplified as an indicator of stage-specific gene expression (10). The FSHR is expressed only in the Sertoli cells (11). Identity of the amplified band was confirmed by Southern blot hybridization using an oligonucleotide probe for the FSHR (Fig. 6AGo).

The FSHR RT-PCR reveals a striking down-regulation of FSHR mRNA around stages III-IV (Fig. 6CGo), a finding that is consistent with the results reported by Rannikko et al. (10) using a Northern blot analysis. Taking FSHR mRNA down-regulation as a stage-specific marker, staging accuracy seems to be ± 1 stage. The changes in FSHR gene expression are believed to be mediated by cAMP, which reaches its highest levels at stages XIV-V (2, 9). cAMP also rapidly represses FSHR mRNA levels in cultured Sertoli cells (12).

Amplification of APRT was carried out as a control for mRNA integrity (13). Levels of APRT product remain relatively constant over the course of two cycles, although there is some small (possibly stage-dependent) modulation. By comparison, there is a marked stage-dependent variation in levels of CREB mRNA. This variation is consistently found with both CREB G-H and CREB C-E primer pairs, with CREB PCR product elevated in stages I-VII. These findings are consistent with earlier results from in situ histohybridization of CREB mRNA in sections of rat testis (6, 14, 15).

The inclusion of exon W seems to reach a peak relative to CREB transcripts lacking exon W at stages IV-V, coincident with the peak in cAMP levels and the down-regulation of FSHR mRNA. Stages IV-V also coincide with the highest levels of CREB PCR product, consistent with up-regulation of CREB gene expression by cAMP (16, 17). The ratio of CREB-Y to exon Y+ transcripts remains low during peak cAMP levels; but as cAMP and overall CREB product fall in stages VIII-IX, the CREB-Y product declines less rapidly, resulting in a high ratio of exon Y+ to exon Y- mRNA in stages VII-X (Fig. 6BGo). The results of this experiment were confirmed with two other single-tubule cDNA sample sets (results not shown).

CREB exons Y and W are significantly (but not exclusively) cospliced
The marked difference in regulation between CREB-W and CREB-Y mRNAs could be caused by expression within different cell populations during spermatogenesis. To address this possibility, PCR was performed using primers specific for exon C (forward) and exon H (reverse). As with CREB C-E and CREB G-H PCR, the identity of products was confirmed by Southern blot hybridization with exon-specific oligonucleotides (Fig. 7AGo).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 7. Detection of mRNAs with cosplicing of both exons W and Y in rat seminiferous tubules during 1 cycle of spermatogenesis. A, The C-H primer pair produces 4 major bands of sizes 774 bp (CREB without exon W or Y: W+Y-), 838 bp (CREB-W: W+Y-), 885 bp (CREB-Y: W-Y+), and 949 bp (CREB-WY: W+Y+). The identity of the bands, amplified from pooled-tubule segment cDNA representing stages IV-V, VI, an VII, is confirmed by Southern hybridization with oligonucleotide probes for exons C, W, and Y. B, Intact seminiferous tubules covering a complete 14-stage cycle (12.5 days) were dissected into segments representing 8 groupings of stages. The segments from 5 tubules were combined and analyzed by RT-PCR with the C-H primer pair. The identity of bands, with respect to inclusion of exons W and Y, are indicated. C, PCR results were analyzed by densitometry. The ratios of exon-inclusive products (CREB-W, CREB-Y, and CREB-WY) to CREB were plotted.

 
For stage-dependent splicing studies, a single set of pooled-tubule cDNA was analyzed (Fig. 7BGo). The results show that CREB mRNA can contain exons Y and W, either singly or together. The pattern of W and Y inclusion seen with this primer combination conforms to that established with C-E and G-H primer pairs (Fig. 7CGo). Cosplicing of exons W and Y occurs in a significant proportion of CREB transcripts in stages VIII-XII. Although independently regulated, exons W and Y must, therefore, be cospliced in the same cell types.

The inclusion of exons Y and W during meiosis and early spermiogenesis was examined using testis cDNA from rats between 20 and 35 days old, and amplified with the CREB C-H primer pair (Fig. 8AGo). Because of low yield, products from 20- and 25-day cDNA were concentrated 3-fold. Exons W and Y are detectable at low ratios in 20-day rats, increasing in relative abundance up to day 30 for exon Y, and day 35 for exon W (Fig. 8BGo).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 8. Occurrence of CREB mRNA isoforms during the onset of spermiogenesis. A, Testis cDNA from rats, 20–60 days old, analyzed by PCR using the C-H primer pair. PCR products in lanes representing RT-, 20 days, and 25 days were concentrated 3-fold to improve detection. The identity of bands, with respect to inclusion of exons W and Y, are indicated. B, PCR results were analyzed by densitometry. The rations reflect the sum of exon-inclusive products (i.e. W+Y+ together with either W+Y- or W-Y+) over the sum of exon-exclusive products (i.e. W-Y- together with either W-Y+ or W+Y-).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The approach of examining isolated contiguous segments of seminiferous tubules by RT-PCR is a direct and reliable method for the quantitative analysis of changes in specific gene expression during spermatogenesis. Here, we have used the RT-PCR method to investigate changes in the alternative splicing of CREB gene transcripts during the spermatogenic cycle. Our findings indicate that splice variants of CREB mRNA are regulated in a stage-dependent manner, rising and falling relative to levels of the exon W- or exon Y- transcript. The highest ratios for CREB-W and CREB-Y occur at stages II-V and VIII-XI, respectively, as summarized in Fig. 9Go. The possibility that CREB-W or CREB-Y transcripts may dominate within distinct populations of cells is suggested by previous results suggesting maximal amounts of CREB-W in round spermatids (6), and CREB-Y prevalent in mid- to late-pachytene spermatocytes (14). Alternatively, CREB-W and Y may be preferentially cospliced, as is the case for the W and Z exons of CREB in the human testis (4). This question was addressed with PCR primers based in exons C and H. This primer combination yields products both with exons Y or W, or both. Proportions vary stage-dependently in accordance with results from primer pairs targeting exons Y or W specifically. A significant, but not dominant, proportion of product contained both exons Y and W, indicating that W and Y are expressed in the same cell types but that their splicing events are not codependent.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 9. Diagram depicting the changes in the cyclical splicing of exons W and Y in CREB during the spermatogenic cycle, in conjunction with the reported cyclical fluctuations of cAMP levels in rat seminiferous tubules (2 ). Projected cAMP production, in relation to the peaks of CREB-W and CREB-Y splicing. Overall levels of CREB mRNA closely follow the cAMP and CREB-W lines.

 
The increase in expression of exon W correlates closely with the highest levels of cAMP production. In this respect, CREB-W expression may parallel the regulation of ICER, a bZIP repressor derived from the CREM gene, which is positively regulated by cAMP. ICER is not expressed in germ cells, but it is expressed in Sertoli cells. ICER is rapidly induced by cAMP, and it down-regulates its own promoter (18), as well as those of other genes regulated by cAMP (19). It is possible that the I-CREB repressor isoforms perform functions in germ cells parallel to those of the ICER repressor in Sertoli cells by modulating the cAMP responsiveness of key target genes.

The expression of exons W and Y is confined largely to the germ cells, but the exact type remains uncertain. In situ hybridization of tissue sections, and either Northern or RT-PCR analysis of purified cell populations, have detected CREB mRNA containing exons W and Y in both pachytene spermatocytes and round spermatids (6, 14, 17). RT-PCR analysis of testis from 20- to 35-day-old rats detects exons Y and W as early as 20 days, with proportions increasing significantly up to 30 days for exon Y, and 35 days for exon W. This is consistent with splicing-in of exons W and Y occurring in mid- to late-pachytene spermatocytes that first appear at approximately 20–25 days after birth, but suggests that round spermatids may also contribute significantly to CREB-Y and W mRNA production.

The CREB promoter is cAMP-responsive (16, 17, 20, 21), and the results presented here indicate a significant stage-dependent increase in CREB mRNA coincident with the time of known elevated cAMP levels in rat seminiferous tubules. In other experiments, a strong cAMP pulse, delivered by intratesticular injection of dibutyryl cAMP, caused down-regulation of CREB mRNA after 16 h (15). Taken together, these results suggest that the CREB gene is subject to both positive and negative regulation by cAMP.

Using antisera raised against the unique aminoterminal sequence of I-CREB(l), a product of exon W-containing mRNA, high levels of I-CREB(l) protein were detected at stages V-XIV in pachytene spermatocytes (5). Stage V is the approximate maximum for exon W splicing, whereas stages IX-XIV have the lowest levels. This pattern of I-CREB protein accumulation and persistence is consistent with the overall levels of CREB mRNA detected by RT-PCR. Thus, the presence of I-CREB protein through stages IX-XIV may account for the reduced levels of CREB PCR products in those stages.

The inclusion of exon Y in the CREB transcripts peaks in stages VIII-IX. This splicing-in of exon Y occurs as overall levels of the CREB transcripts are decreasing, so the highest levels of Y-containing mRNAs may actually occur earlier than stages VIII-IX. However, if exon Y-containing transcripts are generating I-CREB molecules, the efficacy of a repressor will be greatest when the ratio of CREB-Y to CREB is highest. The Y exon has not been investigated for its potential to generate I-CREBs, and the sequence of exon Y does not contain the hallmark translational initiation sequence found in exon W (5). Thus, similar to its regulation, the function of CREB-Y may be distinct from that of CREB-W.

The phenomenon of alternative internal translation is not limited to CREB mRNA. Recently, a repressor isoform of CREM was described in endometrial stromal cells that also arises from a reinitiation of translation within a transcript in which the splicing of an additional exon prematurely terminates translation (22).

The generality of this mechanism of alternative exon splicing, coupled with the tissue-specific and now stage-dependent regulation of CREB-W and Y exons, suggests that they may be important for controlling CREB-mediated gene transcription.


   Acknowledgments
 
We thank J. Seufert and M. Thomas for helpful discussions, and T. Budde for help in the preparation of the manuscript.


   Footnotes
 
1 This study was supported, in part, by United States Public Health Service Grant DK-25532. Back

2 An investigator with the Howard Hughes Medical Institute. Back

Received January 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. LeBlond CP, Clermont Y 1952 Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci 55:548–570
  2. Parvinen M 1982 Regulation of the seminiferous epithelium. Endocr Rev 3:404–417[Abstract/Free Full Text]
  3. Habener JF, Miller CP, Vallejo M 1995 Cyclic AMP-dependent regulation of gene transcription by cAMP response element-binding protein and cAMP response element modulator. Vitam Horm 51:1–57[Medline]
  4. Girardet C, Walker WH, Habener JF 1996 An alternatively spliced polycistronic mRNA encoding cyclic adenosine 3',5'-monophosphate (cAMP)-responsive transcription factor CREB (cAMP response element-binding protein) in human testis extinguishes expression of an internally translated inhibitor CREB isoform. Mol Endocrinol 10:879–891[Abstract/Free Full Text]
  5. Walker WH, Girardet C, Habener JF 1996 Alternative exon splicing controls a translational switch from activator to repressor isoforms of transcription factor CREB during spermatogenesis. J Biol Chem 271:20145–20158[Abstract/Free Full Text]
  6. Waeber G, Meyer TE, LeSieur M, Hermann HL, Gérard N, Habener JF 1991 Developmental stage-specific expression of cyclic adenosine 3',5'- monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing [published erratum appears in Mol Endocrinol 7:1501]. Mol Endocrinol 5:1418–1430[Abstract/Free Full Text]
  7. Ruppert S, Cole TJ, Boshart M, Schmid E, Schutz G 1992 Multiple mRNA isoforms of the transcription activator protein CREB: generation by alternative splicing and specific expression in primary spermatocytes. EMBO J 11:1503–1512[Medline]
  8. Gaillard C, Strauss F 1990 Ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res 18:378[Free Full Text]
  9. Kangasniemi M, Kaipia A, Mali P, Toppari J, Huhtaniemi I, Parvinen M 1990 Modulation of basal and FSH-dependent cyclic AMP production in rat seminiferous tubules staged by an improved transillumination technique. Anat Rec 227:62–76[CrossRef][Medline]
  10. Rannikko A, Penttila TL, Zhang FP, Toppari J, Parvinen M, Huhtaniemi I 1996 Stage-specific expression of the FSH receptor gene in the prepubertal and adult rat seminiferous epithelium. J Endocrinol 151:29–35[Abstract/Free Full Text]
  11. Heckert LL, Griswold MD 1991 Expression of follicle-stimulating hormone receptor mRNA in rat testes and Sertoli cells. Mol Endocrinol 5:670–677[Abstract/Free Full Text]
  12. Themmen AP, Blok LJ, Post M, Baarends WM, Hoogerbrugge JW, Parmentier M, Vassart G, Grootegoed JA 1991 Follitropin receptor down-regulation involves a cAMP-dependent post-transcriptional decrease of receptor mRNA expression. Mol Cell Endocrinol 78:7–13[CrossRef][Medline]
  13. Dush MK, Briggs MR, Royce ME, Schaff DA, Khan SA, Tischfield JA, Stambrook PJ 1988 Identification of DNA sequences required for mouse APRT gene expression. Nucleic Acids Res 16:8509–8524[Abstract/Free Full Text]
  14. Waeber G, Habener JF 1992 Novel testis germ cell-specific transcript of the CREB gene contains an alternatively spliced exon with multiple in-frame stop codons. Endocrinology 131:2010–2015[Abstract/Free Full Text]
  15. West AP, Sharpe RM, Saunders PTK 1994 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. Biol Reprod 50:869–881[Abstract]
  16. Meyer TE, Waeber G, Lin J, Beckmann W, Habener JF 1993 The promoter of the gene encoding 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription. Endocrinology 132:770–780[Abstract/Free Full Text]
  17. Walker WH, Fucci L, Habener JF 1995 Expression of the gene encoding transcription factor cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB): regulation by follicle-stimulating hormone-induced cAMP signaling in primary rat Sertoli cells. Endocrinology 136:3534–3545[Abstract]
  18. Molina CA, Foulkes NS, Lalli E, Sassone-Corsi P 1993 Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell 75:875–886[CrossRef][Medline]
  19. Monaco L, Foulkes NS, Sassone-Corsi P 1995 Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor. Proc Natl Acad Sci USA 92:10673–10677[Abstract/Free Full Text]
  20. Widnell KL, Chen J-S, Iredale PA, Walker WH, Duman RS, Habener, JF, Nestler EJ 1996 Transcriptional regulation of CREB (cyclic AMP response element-binding protein) expression in CATH.a cells. J Neurochem 66:1770–1773[Medline]
  21. Walker WH, Daniel PB, Habener JF Inducible cAMP early repressor ICER down-regulation of CREB gene expression in Sertoli cells. Mol Cell Endocrinol, in press
  22. Gellersen B, Kempf R, Telgmann R 1997 Human endometrial stromal cells express novel isoforms of the transcriptional modulator CREM and up-regulate ICER in the course of decidualization. Mol Endocrinol 11:97–113[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 ReproductionHome page
X. Huang, J. Zhang, L. Lu, L. Yin, M. Xu, Y. Wang, Z. Zhou, and J. Sha
Cloning and expression of a novel CREB mRNA splice variant in human testis
Reproduction, December 1, 2004; 128(6): 775 - 782.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
H. F. S. Huang, M.-T. Li, S. Wang, G. Wang, and J. E. Ottenweller
Spinal Cord Contusion Impairs Sperm Motility in the Rat Without Disrupting Spermatogenesis
J Androl, May 1, 2003; 24(3): 371 - 380.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
E. J. Abraham, C. A. Leech, J. C. Lin, H. Zulewski, and J. F. Habener
Insulinotropic Hormone Glucagon-Like Peptide-1 Differentiation of Human Pancreatic Islet-Derived Progenitor Cells into Insulin-Producing Cells
Endocrinology, August 1, 2002; 143(8): 3152 - 3161.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. A. Hussain, C. P. Miller, and J. F. Habener
Brn-4 Transcription Factor Expression Targeted to the Early Developing Mouse Pancreas Induces Ectopic Glucagon Gene Expression in Insulin-producing beta Cells
J. Biol. Chem., May 3, 2002; 277(18): 16028 - 16032.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
S. A. Shell, C. Fix, D. Olejniczak, N. Gram-Humphrey, and W. H. Walker
Regulation of Cyclic Adenosine 3',5'-Monophosphate Response Element Binding Protein (CREB) Expression by Sp1 in the Mammalian Testis
Biol Reprod, March 1, 2002; 66(3): 659 - 666.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
H. Lou and R. F. Gagel
Alternative Ribonucleic Acid Processing in Endocrine Systems
Endocr. Rev., April 1, 2001; 22(2): 205 - 225.
[Abstract] [Full Text]

Home page
 EndocrinologyHome page
J. k. Chen and L. L. Heckert
Dmrt1 Expression Is Regulated by Follicle-Stimulating Hormone and Phorbol Esters in Postnatal Sertoli Cells
Endocrinology, March 1, 2001; 142(3): 1167 - 1178.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
H. Zulewski, E. J. Abraham, M. J. Gerlach, P. B. Daniel, W. Moritz, B. Müller, M. Vallejo, M. K. Thomas, and J. F. Habener
Multipotential Nestin-Positive Stem Cells Isolated From Adult Pancreatic Islets Differentiate Ex Vivo Into Pancreatic Endocrine, Exocrine, and Hepatic Phenotypes
Diabetes, March 1, 2001; 50(3): 521 - 533.
[Abstract] [Full Text]

Home page
 EndocrinologyHome page
P. B. Daniel, L. Rohrbach, and J. F. Habener
Novel Cyclic Adenosine 3',5'-Monophosphate (cAMP) Response Element Modulator {theta} Isoforms Expressed by Two Newly Identified cAMP-Responsive Promoters Active in the Testis
Endocrinology, November 1, 2000; 141(11): 3923 - 3930.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Daniel, P. B.
Right arrow Articles by Habener, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Daniel, P. B.
Right arrow Articles by Habener, J. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals