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Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, and 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: jhabener{at}partners.org
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In the testis, immunoreactive PACAP is highly abundant within germ cells (2, 11). Receptor-positive PACAP-responsive cell types in the testis include germ (12), Sertoli (13), and Leydig (10) cells. Much of the PACAP present in the testis is produced by the postmeiotic round spermatids (14), where the prohormone, pro-PACAP, is processed primarily to the isopeptide PACAP-38 (1, 2) compared with the alternative isopeptide PACAP-27. Several different PACAP messenger RNAs (mRNAs) exist. The major PACAP mRNA species in the rat testis is 1.5 kb shorter than the PACAP mRNA expressed in the hypothalamus (15). The testis PACAP mRNA also contains a novel first exon not found in the hypothalamic mRNA.
Here we characterize the arrangement of transcribed exons within the 5'-end of the PACAP gene and provide evidence for the existence of a single PACAP gene that contains multiples promoters, including a testis-specific promoter located 5' to the novel first exon. We show by transfection expression studies in isolated rat germ cells in vitro and in transgenic mice in vivo that expression of the PACAP gene is controlled by a tissue-specific promoter, primarily active in round spermatids. Examination of the developmental stage(s) of expression of PACAP mRNA during the spermatogenic cycle was carried out by RT-PCR analyses of segments of rat seminiferous tubules. PACAP mRNA is expressed at high levels at stages I–VII, with earliest expression at the time of meiosis (stage XIV).
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Materials and Methods |
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PCR products were prepared for TA cloning (Invitrogen, Carlsbad, CA) by phenol/chloroform extraction and ethanol precipitation, followed by incubation with Taq polymerase at 72 C for 10 min in the presence of 1 mM deoxy (d)-ATP.
Primer extension
Poly(A)+ mRNA was purified as described
above. Primer extension was performed using 1–3 µg
poly(A)+ mRNA, and 100 pmol oligonucleotide
primer end labeled with polynucleotide kinase and
[
-32P]ATP. Primer and template were mixed
and heated to 65 C for 10 min. RT was performed in a 10-µl reaction
including 250 µM dNTPs, and 100 U Superscript II reverse
transcriptase (Life Technologies, Inc., Gaithersburg,
MD).
PCR from genomic DNA
Amplification from rat genomic DNA was carried out using the
TaqPlus long PCR system (Stratagene, La Jolla, CA). The
13.5-kb fragment between the testis-specific and the first coding exon
of PACAP was amplified from rat genomic DNA in high salt buffer, using
a "touchdown" PCR protocol of 10-sec denaturation (94 C) followed
by 10-min annealing and extension. The annealing/extension temperature
was set at 72 C for the first 5 cycles, 70 C for the next 5 cycles, and
68 C for 25 cycles. The primers were PCPTF32 and PCPPR600. The product
was subcloned into pBluescript SK+
(Stratagene) by digesting with BamHI to give 2
fragments of approximately 3.5 and 10 kb containing the 3'-end of the
testis-specific exon and the 5'-end of the PACAP encoding sequence,
respectively.
RNA isolation and RT
Whole cell RNA was extracted with Trizol reagent (Life Technologies, Inc.) in accordance with the manufacturer’s
specifications.
RNA, in 10 µl H2O, was combined with 0.5 µg oligo(deoxythymidine)16 and heated to 65 C for 10 min, then cooled on ice. RT buffer, dNTPs (50 µM each), dithiothreitol (5 mM), Superscript II (Life Technologies, Inc.) enzyme (100 U), and H2O were added for a total volume of 40 µl (or 20 µl for single tubule segments), and reactions were incubated at 42 C for 40 min. For each set of samples one additional sample was prepared as described, except without reverse transcriptase (RT-) to control for reagent purity and nonspecific amplification from genomic DNA.
PCR and Southern blot hybridization
All PCR reactions were performed in 50-µl reactions using 2
µl templates. Reactions contained 20 pmol each of forward and reverse
primers, 0.2 mM each of dNTPs, and 2.5 U thermostable
Taq polymerase (TaKaRa Biomedical, Inc., Berkeley, CA).
PCR amplification of cDNA in the experiments shown in Fig. 2 was
carried out as follows: 10-sec denaturation at 94 C, 20-sec annealing
at 58 C, and 1-min extension at 72 C. Either 30 or 35 cycles were
employed depending upon the template. The PCR experiments shown in Fig. 5 were carried out with the following primers and conditions: for
PACAP, PCPF568, and PCPR1120: 10-sec denaturation at 94 C, 20-sec
annealing at 58 C, and 1-min extension at 72 C; for FSH receptor
(FSH-R), FRF11, and FRR2196: 10-sec denaturation at 94 C, 20-sec
annealing at 58 C, and 2-min extension at 72 C; and for adenine
phosphoribosyl transferase (APRT), APRTF, and APRTR: 10-sec
denaturation at 94 C, 20-sec annealing at 55 C, and 1-min extension at
72 C.
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-32P]ATP-labeled oligonucleotide probes was
performed in a solution of 5 x SSC (standard saline citrate), 1%
SDS, 10 x Denhardt’s solution, and 100 µg/ml denatured salmon
sperm DNA for 3 h at 37 C. Blots were washed to a maximum
stringency of 0.5 x SSC at 52 C. For rehybridization, the labeled
probe was removed by washing blots in 0.5 M NaOH at room
temperature for 1 h, then rinsing in 2 x SSC.
Microdissection and RT-PCR analysis of seminiferous tubules
Seminiferous tubules were isolated from the testes of adult
(60-day-old) Sprague Dawley rats and dissected under transillumination
microscopy by the method of Kangasniemi et al. (17). A
single tubule representing two complete cycles was divided into
consecutive 2.5-mm segments. Accurate assignment of stages was achieved
by microscopic examination of squashes of a 0.5-mm portion from the end
of each segment. Stages were assigned according to the scheme of
Leblond and Clermont (18). The accuracy of staging was ±1 stage. The
remaining segment was extracted in 100 µl Trizol, and pelleted RNA
was converted to cDNA by RT in 20-µl reactions, as described above.
PCR amplification was performed using 2-µl samples. All PCR reactions
(PACAP, FSH-R, and APRT) were performed for 30 cycles.
Ligation-mediated PCR from genomic DNA
Rat genomic DNA libraries were obtained as part of the Promoter
Finder kit (CLONTECH Laboratories, Inc.). In the first
round of PCR, reaction products were amplified using adapter primer 1
and PCPTR 86. Conditions were 94 C for 2 sec, 72 C for 3 min for 7
cycles, followed by 94 C for 2 sec and 67 C for 3 min for 32 cycles.
Products from the first round reactions were further amplified with
adapter primer 2 and PCPTR58. Second round amplification conditions
were 94 C for 2 sec, 60 C for 20 sec, and 72 C for 3 min for 30 cycles.
ExTaq polymerase (TaKaRa) was used in both rounds of PCR amplification.
Products were prepared for TA cloning as described above.
Transfection of isolated rat testicular germ cells
PACAP testis-specific promoter-luciferase reporter plasmids were
constructed by subcloning promoter fragments PPT1900, PPT820, and
PPT303 [along with 23 bp of the PACAP 5'-untranslated region
(5'-UTR)] into the promoterless pGL3-Basic plasmid (Promega Corp., Madison, WI). Testes obtained from adult Sprague Dawley
rats were decapsulated, and the remaining core tissue was sequentially
digested with collagenase and trypsin (0.5 mg/ml each) in enriched
Kreb-Ringer buffer (19). Single cell suspensions were prepared by
pipette-mediated disruption and sieving through 100-µm mesh cell
strainers (Falcon, Franklin Lakes, NJ). Mixed populations of testis
cells were cultured for 2 h in DMEM (Life Technologies, Inc.) with 10% FBS and antibiotics. Nonadherent germ cells were
collected by centrifugation of the culture medium at 250 x
g for 2 min. Cells were washed once with DMEM, resuspended
in OptiMEM medium (Life Technologies, Inc.), and
distributed into 12-well plates at a density of 2 x
105 cells/well in 400 µl OptiMEM. Cells were
transfected with reporter plasmids (1 µg/well) using the GeneFECTOR
lipid transfection system (Venn-Nova, Pompano Beach, FL) in amounts of
5 µl/well. The cells were exposed to the transfection conditions for
2 h, after which 500 µl DMEM with 20% FCS and antibiotics were
added to each well. Cells were harvested by centrifugation 18 h
later, and reporter gene expression was determined using a luciferase
assay reagent system (Promega Corp.).
Transgenic animals
A PACAP testis-specific promoter fragment encompassing 820 bp of
sequence upstream of the transcription start site (determined by primer
extension and analysis) and 23 bases of 5'-UTR was subcloned into the
green fluorescence protein (GFP) reporter plasmid, pEGFP
(CLONTECH Laboratories, Inc.). A DNA fragment containing
the promoter, the reporter gene, and the 3'-RNA processing signals in
pEGFP was isolated by digestion with restriction enzymes
XhoI and SspI followed by electrophoresis and
purification from an agarose gel with the GlassMAX DNA isolation system
(Life Technologies, Inc.). The DNA was microinjected into
the pronuclei of fertilized mouse oocytes and transplanted into
pseudopregnant host female mice. Founder mice were genotyped, and those
bearing the PACAP promoter-GFP transgene were killed at 60 days of
age.
All animal protocols used in this study were subject to review and were approved by an animal ethics committee.
Single cell suspensions were prepared from the testis of some transgenic mice and sections of mouse testis were prepared from other transgenic mice and frozen in Tissue-Tek embedding compound (Sakura Finetek, Torrance, CA). GFP expression in cells and air-dried tissue sections was assessed by fluorescence microscopy. For immunocytochemistry, sections of frozen testis were fixed in 4% paraformaldehyde for 10 min and immersed in methanol at -20 C for 5 min. Sections were then blocked with 10% normal donkey serum before incubation with anti-GFP polyclonal antiserum (IgG fraction; 1:200 dilution; CLONTECH Laboratories, Inc.) overnight. Secondary antibody (Cy3-conjugated donkey antirabbit; 1:1500 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was applied for 1 h.
Digital overlays in Fig 4 were performed with Adobe Photoshop 5
software (Adobe Systems, Inc., San Jose, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsReferences |
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400 bp) was
obtained after 2 rounds of PCR amplification of DNA (not shown).
Sequence analysis of the cloned product obtained by the PCR
amplification indicates that the 5'-end of the cDNA contains an
additional 35 nucleotides in common with brain PACAP cDNA immediately
following the 13 bases with the dashed underline (Fig. 1A). Other minor sequence differences
found in the 5'-UTR are single nucleotide changes at positions 19 (T
instead of C) and 51 (A instead of G: positions 72 and 104,
respectively, in Ref. 15 ; Fig. 1A). These nucleotide changes were
confirmed by sequencing of several independently generated PCR
products.
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).
The sequence located immediately 5' to the first coding exon of the rat
PACAP cDNA was highly homologous to the corresponding region of the
mouse PACAP cDNA (Fig. 1B). It is important to note that in the mouse,
some PACAP transcripts initiate at a site in the gene preceding exon 2
by 282 bp. The equivalent rat sequence is therefore also indicated in
Fig. 1B as exon 1C/2. Comparing this sequence to the 5'-RACE sequence
(Fig. 1A) reveals that transcripts initiated from the testis-specific
first exon are processed using a 3'-splice site within the putative
exon 1B.
Analysis of genomic sequence from the 5'-end of the 13.5-kb intronic
region defines the 3'-terminal nucleotide of the testis-specific exon
as nucleotide 64 in Fig. 1A.
The previously described PACAP mRNA from rat brain (20) contains 366
nucleotides of 5'-UTR sequence that does not occur in the testis mRNA
described here or previously and has no similarity to any of the
alternative PACAP 5'-UTR sequences described for mouse (21). The
13.5-kb genomic DNA PCR product did not hybridize with two
oligonucleotide probes for this reported 5'-UTR sequence of PACAP from
rat brain (data not shown), indicating that it is not present in the
genomic DNA between the testis-specific first exon and the first coding
exon. Furthermore, this 366-nucleotide region is nearly identical
(365/366 nucleotides) to the reverse complement of the cDNA sequence
for rat ribosomal protein L15 (22) (nucleotides 310–666 in GenBank
X78167). Based on the PCR and nucleotide sequence data, a model for the
5'-region of the rat PACAP gene is presented (Fig. 1C).
Tissue-specific expression of PACAP mRNA isoforms
It has been reported previously that the size of the rat PACAP
mRNA in testis is 800 bp, approximately 1.5 kb shorter than the mRNA
detected in the hypothalamus. The difference in size is not accounted
for by the difference between the alternative first exons and probably
results in part from the use of alternative polyadenylation sites. It
is therefore possible that the testis-specific exon and accompanying
promoter may drive some expression of PACAP in other tissues. The
expression of the different transcripts was assessed in hypothalamus
and testis by RT-PCR using 5'-primers that amplify PACAP cDNA from the
coding region (F-2), the testis-specific exon (F-TS), or putative exons
1A and 1B (F-1A and F-1B; Fig 2). The
primer for exon 1B is based on a region upstream of the apparent splice
site for testis-specific transcripts; therefore, cDNA from
testis-specific mRNAs should not amplify with this primer in the primer
set. The 3'-primer in all cases was PCPR1120. Amplification was carried
out for either 30 or 35 cycles depending on the amount of product. The
identities of the PCR products were confirmed by Southern blotting and
hybridization with an oligonucleotide probe specific for the coding
region (PCPR743).
Using a forward primer based in the coding region (F-2), PACAP mRNA is
detected in both testis and hypothalamus, but more readily in testis
(30 cycles as opposed to 35; Fig. 2A). This finding is consistent with
the 3- to 4-fold greater abundance of PACAP mRNA in testis compared
with hypothalamus by Northern hybridization (15). A testis-specific
primer (F-TS) amplifies DNA only from testis cDNA. Even after 35 cycles
of amplification of hypothalamic cDNA, no product was detected. Primers
F-1A and F-1B amplify DNA products from both hypothalamus and testis.
Some alternative splicing is evident, particularly with the F-1A primer
products (Fig. 1B).
These results indicate that the novel testis-specific exon located 13.5 kb upstream of the PACAP-coding region is flanked by a tissue-specific promoter that drives the transcription of the majority of testicular PACAP mRNAs.
Isolation of the PACAP testis-specific promoter
Three independent clones containing 5'-PACAP promoter sequences
were generated from rat genomic DNA libraries using ligation-mediated
PCR. Gene-specific primers PCPTR86 and PCPTR58 were used in nested PCR
reactions. Using Southern hybridization with PCPTR30 as a probe,
products of approximately 1900, 840, and 330 bp were selected as
candidate sequences containing the PACAP testis promoter region.
Comparison of these DNAs yielded a consensus sequence for the proximal
823 bp of the promoter (Fig. 3A).
Analysis of the promoter with MatInspector (23) identified potential
TATA boxes and transcription factor-binding sites. Of particular
interest are binding sites for SOX5, a member of the SRY-related high
mobility group box family of DNA-binding factors, that is
coexpressed with PACAP in round spermatids (24) and has overlapping
binding specificity with testis-determining factor SRY.
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is 49 bp shorter at the 5'-end
than the clone previously described (15). A primer extension assay was
performed to estimate the position of the major transcriptional
initiation site. Poly(A)+ RNA from whole testis
was reverse transcribed using end-labeled PCPTR86 as a primer (Fig. 3B). By comparison to an accompanying sequencing reaction performed on
an unrelated template, it can be determined that the majority of PACAP
transcription originates 52 bp upstream of the 3'-end of PCPTR86. Using
the previously published PACAP testis 5'-UTR sequence and the novel
PACAP testis promoter sequence, the putative major transcription start
site was determined (arrow 2 in Fig. 3A). The major
transcription start site determined here is actually 32 bp downstream
from the 5'-end of the previously published testis-specific 5'-UTR
clone (15) (indicated as arrow 1 in Fig. 3A). The experiment
was repeated using mRNA extracted specifically from seminiferous tubule
segments expressing large amounts of PACAP (stages II–VIII) (14) with
identical results (data not shown).
PACAP testis-specific promoter analysis by transient transfection
of testis germ cells in vitro
PACAP promoter sequences that were isolated by ligation-mediated
PCR were subcloned into the pGL3 luciferase transcriptional reporter
plasmid. The three constructs containing varying lengths of the
testis-specific PACAP promoter were then transfected into rat testis
cell cultures comprised predominantly of germ cells. Luciferase
activity was assayed 18 h after transfection. All three promoter
constructs activate the transcription of the GFP reporter gene in the
transfected germ cells (Fig. 3, C and D).
Tissue-specific expression in transgenic mice
The expression of a GFP reporter gene (EGFP) controlled by an
843-bp segment of the PACAP testis-specific promoter (including 23 bp
of 5'-UTR) was assessed by fluorescence microscopy in a male transgenic
mouse.
GFP fluorescence was detectable in seminiferous tubules of the
transgenic mouse, but not in those of a nontransgenic littermate (Fig. 4A, panels 1 and 2). No fluorescence was
detected in squash preparations of cerebellum, hypothalamus, pituitary,
lung, kidney, spleen, liver, pancreas, skeletal muscle, or heart from
the transgenic mouse compared with nontransgenic mouse tissues (data
not shown). By this criterion, activity of the 843-bp segment of the
PACAP promoter appears to be specific to the testis.
In freshly cut sections prepared from frozen testis, a diffuse
fluorescence was visible toward the middle of transgenic tubules that
was absent in nontransgenic testis (Fig. 4A, panels 3 and 4). In
vivo expression visualized in recently dissected seminiferous
tubule segments (Fig. 4A, panels 5 and 6) appears localized near the
tubule lumen.
Localization of the fluorescence signal observed with an antiserum
specific for GFP indicates significant signal associated with small
cells toward the lumen of the tubule, probably round spermatids (Fig. 4B, left panel is normal mouse, right panel is
transgenic mouse). However, there was also significant signal in some
larger cells further from the lumen, possibly indicating expression in
pachytene spermatocytes.
Fluorescence microscopy of single cells from a transgenic testis showed
that GFP expression is found predominantly in cells less than 12 µm
in diameter, most likely round spermatids. Approximately 50% of the
smaller cells were positive for GFP (data not shown), consistent with
segregation of the transgenic allele at meiosis. A small minority of
larger cells (15–20 µm in diameter) also expressed GFP, possibly
indicating transgenic promoter activity in pachytene spermatocytes
(data not shown). GFP protein persisted into late spermiogenesis, as
shown in panel 2 of Fig. 4C, where an elongated spermatid is shown
shedding its fluorescent cytoplasm (arrowed).
Developmental stage-dependent PACAP expression in rat seminiferous
tubules
The expression of PACAP in the seminiferous tubules of rats has
been shown to be developmental stage dependent, as assessed by Northern
blot analysis of pooled tubule segments representing specific temporal
developmental stages in the spermatogenic cycle (14).
Here we used RT-PCR to assay for PACAP mRNA in RNA prepared from
consecutive 2.5-mm segments taken from a single rat seminiferous
tubule, representing two full cycles of spermatogenesis. Expression of
PACAP mRNA, as assessed by RT-PCR using the primer pair PCPF568 and
PCPR1120, shows that levels of PACAP mRNA undergo a marked
stage-dependent fluctuation, increasing greatly between stages
XIV–VIII, then declining to minimal levels by stage X (Fig 5). The cDNA for FSH-R was also amplified
using the primers FRF11 and FRR2194 and was confirmed by hybridization
with FRR747. As reported previously (25), the FSH-R signal undergoes a
marked stage-dependent fluctuation in levels. In our experiments, FSH-R
mRNA is sharply reduced in stages II–III in the first cycle and stages
IV–V in the second cycle. Rannikko et al. (25) observed
that the minimal levels of FSH-R mRNA are expressed in stage VI of the
rat spermatogenic cycle. Some of the disparity between our results and
those of Rannikko et al. may be due to the difficulties in
accurately staging the tubule segments. The quality and consistency of
cDNA were assessed by amplification of a ubiquitously expressed mRNA
APRT.
Conclusions
There is now convincing evidence to support the conclusion that
PACAP mRNA in both testis and hypothalamus is the product of the same
gene expressed from two distinct promoters. The majority of PACAP mRNA
in the testis contains a unique first exon not expressed in the
hypothalamus. This unique testis-specific exon is located 13.5 kb
upstream of the first coding exon of the PACAP gene.
The organization of the structure of the PACAP gene is similar to that of the related GH-releasing hormone (GHRH) gene (26), which also contains a testis-specific first exon and a testis-specific promoter located 10.7 kb upstream from the 5'-end of the transcription start site in the hypothalamus. Only a single testis-specific exon of the PACAP gene has been detected to date. In comparison, a second alternatively spliced GHRH exon is detected in testis mRNA transcripts. The second exon of the GHRH gene is located 3' of the testicular first exon and 5' of the placental first exon (26).
Analysis of the proximal 820 bp of the testis-specific promoter reveals the existence of motifs with strong homology to binding sites for SOX5, a member of the high mobility group class of DNA-binding proteins. SOX5 and PACAP are both expressed in round spermatids (24, 14). A related transcription factor, SOX6 or SOX-LZ (27, 28), is also expressed in postmeiotic spermatids, as is the sex determination factor, SRY (29). The three related factors overlap in binding specificity (24, 27). The 820-bp testis-specific promoter, with an additional 23 bp of 5'-UTR, is capable of directing tissue-specific expression of a GFP reporter gene in the testis of transgenic mice. The highest levels of transgene expression are seen in 50% of the smaller (<12-µm diameter) cells, and expression persists in elongated spermatids. This suggests that round spermatids are the major cell type expressing the transgene, a result in accordance with the reported pattern of PACAP expression in the testis (14).
We show that in transient transfections of cultured germ cells, both a short (303-bp) and longer (820- and 1900-bp) promoter fragments direct transcription of a reporter gene.
Stage-dependent expression of PACAP was demonstrated by RT-PCR analysis of a single rat seminiferous tubule representing two full 14-stage cycles. As previously reported (14), PACAP mRNA undergoes a marked temporal and stage-dependent expression. This study of FSH-R mRNA levels using RT-PCR and a related study using Northern blot analysis (25) of pooled tubule segment RNA are in agreement, particularly in the observation that the maximal expression of PACAP mRNA levels coincides with the minimum levels of FSH-R mRNA. FSH-R mRNA levels are strongly down-regulated by the messenger cAMP generated in response to the actions of PACAP and FSH on Sertoli cells (30). These observations suggest that PACAP may play an important role in the stage-dependent temporal fluctuations in cAMP levels in the rat seminiferous tubule.
The cycle of spermatogenesis in rat seminiferous tubules is visible as a progressive darkening of the tubules from the lightest region at stage IX through to the darkest region at stage VIII. The abrupt transition between stages VIII and IX occurs due to the release of mature spermatozoa into the lumen of the tubule (spermiation). Superimposed on the visible wave of spermatogenesis is a modulation in cAMP production that is partly attributed to changes in responsiveness to FSH (31). Probable factors affecting cAMP accumulation include levels of phosphodiesterases (32, 33) and FSH receptor (30). The modulation of cAMP levels coordinates gene expression in the different cell types of the testis. In Sertoli cells, cAMP drives the expression of factors important for early germ cell survival and development, such as stem cell factor (34, 35, 36).
The role, if any, of cAMP in spermiogenesis is less certain.
Previously, cAMP was believed to be important for the activity of the
transcription factor CREM
that accumulates in postmeiotic germ cells
(37). CREM is closely related in structure to the cAMP response
element-binding protein and is similarly capable of the
trans-activation of cAMP-dependent transcription (38, 39).
CREM interacts with the promoters of several genes that are
important for the maturation of spermatids (40, 41, 42). The absence of
CREM in CREM-null mice results in the developmental arrest and
apoptosis of round spermatids (43, 44). However, recent evidence
suggests that CREM does not require cAMP-dependent phosphorylation
by protein kinase A for transcriptional activity in round spermatids
due to the presence of an alternative coactivator protein (45, 46).
Both germ cells and Sertoli cells respond to the actions of PACAP
in vitro (13, 12), and the consequent cAMP production may
act as a critical modulator of the spermatogenic cycle, augmenting the
effect of FSH. Although PACAP is reported to be less effective in the
stimulation of cAMP production in Sertoli cells than is FSH (40% of
FSH maximum) (13), mRNA for type 2 vasoactive intestinal polypeptide
receptors has been reported in germ cells (47). As germ cells do not
express FSH-R, PACAP may act through type 2 vasoactive intestinal
polypeptide receptors to provide the primary stimulus for cAMP
production in germ cells.
Stage-dependent expression of PACAP provides the spermatogenic tubule with an endogenous cAMP generator, the effects of which may be sufficient to compensate for the loss of FSH-driven cAMP production in homozygous null mutations of the human (48) and murine (49) FSH-R genes, and the murine FSH ß-subunit (50). In these mutational models a loss of the FSH signal does not negate male fertility, although it does lead to reduced spermatogenesis in humans and smaller testes in mice.
It seems likely that PACAP is capable of substituting for FSH as a cAMP-inducing agent in spermatogenesis. Given the high level of PACAP expression in the testis and the variety of testis cell types that express PACAP receptors, it seems reasonable to propose that PACAP is an essential factor for maintaining adult spermatogenesis in rodents.
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Acknowledgments |
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Footnotes |
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2 Investigator with Howard Hughes Medical Institute.
Received August 10, 1999.
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