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Department of Medicine, Tulane University School of Medicine (M.L., A.A.), New Orleans, Louisiana 70112; U.S.-Japan Biomedical Research Laboratories, Tulane University Hebert Center (M.L., A.A.), Belle Chasse, Louisiana 70037; and Protein Chemistry Center, Loeb Health Research Institute, Ottawa Hospital Medical School (M.M.), Ottawa, Ontario, Canada K1Y 4K9
Address all correspondence and requests for reprints to: Dr. Min Li, U.S.-Japan Biomedical Research Laboratories, Tulane University Hebert Center, 3705 Main Street, Belle Chasse, Louisiana 70037-3001. E-mail: minlee{at}mailhost.tcs.tulane.edu
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A smaller amount of PACAP-LI was also found in the ovary (3). It has been reported in previous studies that PACAP is transiently expressed in the granulosa cells of the developing follicles of the rat ovary (6, 7, 8), and that its expression is stimulated by the pituitary gonadotropins (9, 10). It has also been demonstrated that treatment with PACAP38 suppresses apoptosis of follicular cells in a dose-dependent manner. Moreover, the LH-induced suppression of apoptosis of follicular cells was partially blocked by cotreatment with a PACAP antagonist, suggesting that the antiapoptotic effect of LH is mediated by endogenous PACAP38 (11).
Both PACAP38 and PACAP27 are derived from the same 176-amino acid precursor, from which also originates a structurally related peptide, PACAP-related peptide (12). The primary structure of the PACAP precursor is similar among mammals; the precursors in humans, sheep, rats, and mice contain PACAP38, PACAP27, and PACAP-related peptide (12, 13, 14, 15). We demonstrated that processing of the PACAP precursor in mammalian somatomammotropic GH4C1 cells transfected with PACAP precursor complementary DNA (cDNA) took place only when the cells were cotransfected with the expression vector for prohormone convertase PC1, PC2, or PC4 (16, 17). These endoproteases are subtilisin-like proteases belonging to the mammalian Kex2 family (18, 19, 20). In both the rat and mouse, PC1 and PC2 are expressed in neuroendocrine cells (18, 21), whereas PC4 is expressed in the testis (20, 22). Neither PC1 nor PC2 is expressed in the gonads. A recent study using an in vitro enzymic assay with recombinant convertase PC4 confirmed that PACAP is indeed a physiological substrate for PC4 (23), suggesting that PC4 is the best candidate enzyme for processing pro-PACAP to generate PACAP38 and PACAP27 in the gonads (23).
An in situ hybridization study revealed that PC4 transcripts are expressed in both pachytene spermatocytes and round spermatids (22). Thus, PC4 transcripts are expressed in the testicular germ cells just preceding or at approximately the same time when PACAP immunoreactivity appears (4). One current study showed that the messenger RNA (mRNA) for PC4 is also expressed in the ovary. PC4 transcripts were expressed in ovarian macrophages, and its absence in PC4 knockout animals rendered these cells hyperactive, leading to retardation of folliculogenesis (Mbikay, M., et al., unpublished observations).
Mbikay et al. (24) reported that the fertility of homozygous male PC4-null mice was severely impaired despite the absence of evident spermatogenic abnormalities, and the ability of spermatozoa from PC4-null mice to fertilize ova in vitro was considerably diminished (24). Moreover, the eggs fertilized by these spermatozoa failed to grow to the blastocyst stage, resulting in early embryonic death (24).
Our question is whether impaired fertility in PC4-deficient mutant mice is mediated by the absence of a regulatory gonadal peptide(s) whose processing is regulated by PC4. As the PACAP precursor is considered the best candidate for the physiological substrate for PC4 (16, 23), we decided to initially study whether the PC4-null mice lack mature PACAP in testes and ovaries. If we find neither PACAP38 nor PACAP27 expressed in the gonads of the PC4-deficient animals, these animals would become a powerful model in which we may study the physiological actions of testicular and ovarian PACAP.
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Materials and Methods |
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Tissue extraction for reverse phase HPLC (RP-HPLC) and RIA
The testes and ovaries were collected from 2- to 5-month-old
Pcsk-/- and
Pcsk+/+ mice under anesthesia (24).
These mice received no hormonal treatments before the experiment. One
testis from each of two male mice (two testes) and two ovaries from
each of three female animals (six ovaries) of the same genotype were
pooled. Each specimen was added to 10 vol ice-cold distilled water. The
tissues were boiled for 5 min, cooled in ice, and combined with acetic
acid to a final concentration of 2.0 M containing 0.02%
ß-mercaptoethanol. They were then gently homogenized with a
Teflon-glass homogenizer and centrifuged (12,000 x g
for 30 min at 4 C). The resulting supernatants were prepurified on a
Sep-Pak Plus cartridge C18 column (Waters Corp., Milford, MA) and eluted with 50% acetonitrile
(CH3CN) containing 0.1% trifluoroacetic acid
(TFA). The eluate was concentrated to dryness in a Speed-Vac
concentrator (Savant Instruments, Inc., Holbrook, NY).
RP-HPLC analysis
The dried extracts of the tissues were dissolved in 10%
CH3CN-0.1% TFA. They were then subjected to
RP-HPLC on a TSK ODS-120T column (4.6 mm id x 25 cm; Tosohaas
Corp., Montgomeryville, PA) with a linear gradient system of
CH3CN-0.1% TFA from 10–30% over 10 min,
30–50% over 30 min, and finally 50–60% over 10 min. One microgram
each of synthetic PACAP38 and PACAP27 (American Peptide Co., Sunnyvale, CA) was also chromatographed in each experiment
as the reference standard. One-milliliter fractions were collected in
polypropylene tubes at 1-min intervals. The fractions were examined for
PACAP38, PACAP27, and total PACAP, including the precursor, by RIAs
using three different PACAP antisera with different recognition
sites.
RIAs for PACAP
Twenty-microliter aliquots of each HPLC fraction were examined
for PACAP by RIAs using rabbit antibodies with known recognition sites,
as described previously (3). Antiserum 89083–3 was
generated against synthetic PACAP38 in rabbits. This antiserum
recognizes PACAP38 and, to a lesser extent, PACAP27 and pro-PACAP.
Antiserum 88111–3 was generated against synthetic PACAP
(24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) and recognizes PACAP38 as well as peptides
containing the sequence of PACAP (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38), but not PACAP27
or its precursor. Antiserum 88123–3 was generated against synthetic
PACAP27 and recognizes PACAP27, but not PACAP38 or its precursor.
Ribonuclease (RNase) protection assay (RPAs)
RPAs were performed using a Direct Protect Lysate Ribonuclease
Protection Assay Kit (Ambion, Inc., Austin, TX). Briefly,
six male mice of each genotype of
Pcsk-/-,
Pcsk-/+, and Pcsk+/+ mice
were used as the donors of the testes. One testis from each animal was
extracted for RNAs in tissue lysates. Five female mice of each genotype
were used as the donors of the ovaries. Two ovaries from each animal
were pooled and extracted for RNAs in tissue lysates. pRB3Z3,
containing the 670-bp PstI/EcoRI fragment of rat
PACAP cDNA (14), was used to prepare
32P-labeled complementary RNA transcripts. The
plasmid DNA was linearized with HindIII and transcribed with
Redivue [
-32P]UTP (3000 Ci/mmol;
Amersham Pharmacia Biotech, Piscataway, NJ) using a
MAXIscript In Vitro Transcription T7 Kit (Ambion, Inc.).
To prepare the probe for ß-actin, pTRI-ß-actin-mouse (Ambion, Inc.), containing a 304-bp fragment of the mouse ß-actin gene,
was transcribed as described above. After the RNA transcription
reaction, the probes were treated with deoxyribonuclease I and purified
on a Sephadex G-50 NICK Spin Column (Amersham Pharmacia Biotech, Uppsala, Sweden). RNA from each sample in tissue
lysates was hybridized with approximately 2.2 x
105 cpm 32P-labeled PACAP
complementary RNA probe (6.7 x 108
cpm/µg) at 37 C overnight. The radiolabeled ß-actin probe was also
hybridized with the same amount of lysates from each sample. RNase
digestions were carried out at 37 C for 30 min using RNase T1. The
protected fragments were then precipitated and separated by size on a
5% polyacrylamide/8 M urea gel. The gel was
exposed at -80 C overnight to a Kodak BioMax MS
autoradiography film (Eastman Kodak Co., Rochester, NY)
without an intensifying screen. The levels of RNA were quantified using
a ScanMaker V scanner (MicroTek, Redondo Beach, CA) and the UN-SCAN-IT
gel analysis program (Silk Scientific, Orem, UT). The density of each
band for the PACAP or ß-actin transcripts was digitized and defined
in terms of pixels (PSL), which is the total pixel minus the
background. For each sample, the signal strength of the PACAP
transcript was normalized for the corresponding ß-actin signal.
Statistical analysis
The mean of PACAP mRNA levels in six testes from six animals
(one testis from each mouse) of each genotype was calculated. For the
ovarian PACAP mRNA, the mean of five replicates (two ovaries from each
of five animals pooled) for each genotype was determined. The results
were analyzed with one-way ANOVA, followed by the Turkey-Kramer
multiple comparisons test, using InStat 2.03 (GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered
statistically significant.
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and 2.
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, upper panel). No evident
immunoreactivity corresponding to the PACAP precursor, which usually
elutes before PACAP38, was detected. When the same HPLC fractions were
examined by RIA using antiserum 88111–3, specific for PACAP38, only
one peak that was coeluted with PACAP38 (fraction 19) was
detected. Similarly, when the fractions were assayed with antiserum
88123–3, specific for PACAP27, only one immunoreactive peak that was
coeluted with PACAP27 was observed (Fig. 1, B and C, upper
panels). However, RIA of the HPLC fractions for the testicular
extract from Pcsk-/- mice
(homozygous PC4-null) demonstrated only a small peak of PACAP-LI that
was detected with antiserum 89083. The peak was eluted just before
PACAP38, corresponding to PACAP precursor (16, 17). As
mentioned above, no such peak corresponding to the PACAP precursor was
found in the fractions for the testes from the wild-type mice (Fig. 1A, upper panel). On the other hand, no peak corresponding to
PACAP38 (Fig. 1, A and B, lower panels) or PACAP 27 (Fig. 1C, lower panel) was eluted for the testicular extracts from
PC4-deficient mice.
Similar elution patterns of PACAP-LI were observed for the
extracts of the ovaries from female mice (Fig. 2). A large PACAP38-LI
and small PACAP27-LI peaks were eluted for the ovarian extracts from
the Pcsk+/+ mice (Fig. 2, upper
panels). Limited, but distinct, PACAP immunoreactivity detected by
antiserum 89083–3 was also eluted just before PACAP38 for the ovaries
from these wild-type animals, probably representing the PACAP
precursor. In addition, low-level immunoreactivity detected by
antiserum 89083 was found in the very early fractions and in late
fractions. With antiserum 88111–3, only one peak corresponding to
PACAP38 was eluted for the ovarian extracts from wild-type mice.
Likewise, with antiserum 88123–3, only a small, but distinct, peak was
coeluted with PACAP27. However, none of the peaks corresponding to
PACAP38 or PACAP27 was found for the ovarian fractions from PC4-null
mice. Moreover, there was no peak corresponding to the PACAP precursor
for the ovarian extracts from
Pcsk-/- mice (Fig. 2, lower panels).
Expression of PACAP transcripts in testis and ovary of
Pcsk-/-, Pcsk-/+,
and Pcsk+/+ mice
The levels of PACAP transcripts were measured using direct
solution hybridization in crude lysates followed by nuclease
protection, which is more sensitive than Northern blot analysis
(25). There was no loss of sample due to omission of the
RNA isolation and precipitation step for purification. The levels of
PACAP mRNA in both the testis and ovary of
Pcsk-/- mice were
remarkably higher than those in the testis and ovary of the
Pcsk-/+ and Pcsk+/+ mice
(Figs. 3A and 4A). The mRNA levels in the
testis and ovary from Pcsk-/+ mice did not differ
from those in the wild-type animals.
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and 4B). This finding
suggests significant up-regulation of the PACAP precursor mRNA in
the Pcsk-/- mutant
mice. |
Discussion |
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In Pcsk-/- mice, the
levels of transcript for PACAP precursor were considerably elevated
compared with those in Pcsk-/+ and
Pcsk+/+ mice. As in the ovary (Fig. 4), expression of PACAP mRNA in the
testis is controlled by pituitary gonadotropins. Hypophysectomy
transiently reduced PACAP mRNA levels in the rat testis 1 week after
removal of the pituitary, but the levels significantly increased 2
weeks afterward, with a concomitant decrease in radioimmunoassayable
PACAP levels (25). The replacement treatments of
hypophysectomized rats with gonadotropins restored testicular PACAP-LI
and normalized PACAP mRNA (25). Increased levels of PACAP
mRNA in testis and ovary from PC4-deficient mice may indicate enhanced
transcription resulting from the absence of bioactive PACAP, which may
form a negative feedback loop of PACAP on PACAP mRNA expression. PACAP
in the gonads might negatively regulate the promoter of transcription
of its own gene.
Analysis of testicular extracts from wild-type mice by RP-HPLC followed by RIAs showed that no evident immunoreactivity detected by antiserum 89083–3 was eluted before PACAP38. The precursor is eluted immediately before PACAP38, as was found in our previous studies with GH4C1 cells transfected with the PACAP expression vector (16, 17). In contrast, the testicular extracts from homozygous PACAP-null mice showed a small, but distinct, peak detected by antiserum 89083–3, which was eluted immediately before PACAP38. This finding suggests that the precursor has not accumulated to a level detectable by the RIA in the testis of PC4-deficient mice, suggesting an efficient conversion of the precursor to mature PACAP in the testis. On the other hand, the precursor remains unprocessed in the absence of PC4 in the homozygous PC4-null mice. It should be noted, however, that antiserum 89083–3 recognizes the precursor as well as mature PACAP, but the extent of recognition of the precursor may not necessarily be comparable for PACAP38 and PACAP27. Thus, the size of the immunoreactive peak for the PACAP precursor may not necessarily reflect the accurate amount of precursor present in the extract.
In Pcsk-/- mice, an analysis of ovarian extracts using RP-HPLC and RIA did not show a clear-cut immunoreactive peak corresponding to the PACAP precursor. Some immunoreactivity detected by antiserum 89083 eluted immediately before PACAP38, which appears to be PACAP precursor-LI in the ovarian extracts from the wild-type mice, but not from homozygous PC4-null mice. This unexpected finding cannot be explained by the present study. Furthermore, the RP-HPLC profile with antiserum 89083 for the ovarian extracts from both wild-type and PC4-deficient mice showed small, but numerous, immunoreactivities eluted in early and late fractions. It is possible that the PACAP precursor in the ovarian tissues was degraded by other enzymes into numerous immunoreactive fragments.
Alternatively, the mRNA for the PACAP precursor in the testis is approximately 1.5 kb shorter than that cloned from the rat hypothalamus (26). During spermatogenesis, a testisspecific promoter in germ cells regulates the expression of the PACAP gene (27). The testicular PACAP transcript has a truncated 3'-noncoding region and a unique 5'-end region that are not observed in the brain PACAP transcript, a result of alternative splicing and posttranscriptional modification (polyadenylation) of the prepro-PACAP mRNA (28). These observations imply that the conformation of the testicular PACAP precursor could be different from that of the brain PACAP precursor, and that antibody 89083–3 could not specifically or strongly recognize the truncated PACAP precursor in the testis and ovary. This may account for the clear-cut demonstration of PACAP precursor-LI in the extracts of GH4C1 cells transfected with the human brain PACAP cDNA, but not in testicular and ovarian extracts.
In the rat testis, the concentration of PACAP-LI is comparable to that in the brain (3), and its expression appears to be stage specifically regulated during spermatogenesis (4). Immunohistochemistry showed that the specific PACAP-LI was found primarily in spermatids during the cap and acrosome phases, but not in spermatogonia, primary spermatocytes, mature spermatids, testicular or epididymal spermatozoa, or Sertoli or Leydig cells (4). These findings indicate that PACAP is transiently expressed in germ cells during spermatogenesis. Expression of PACAP in testis is also developmentally regulated, as PACAP is first expressed when rats are about 22–23 days old, when FSH secretion begins to increase (29). Prohormone convertase PC4 mRNA, detected by Northern blot analysis in an ontogenic study, was found restrictively in testicular germ cells after the 20th postnatal day in mice (20) and between days 19–22 in postnatal rats (22) when spermatogenesis was about to begin. It is interesting that PACAP mRNA in the testis was also first detected on postnatal days 22–23 (30). In situ hybridization histochemistry detected PC4 gene expression in pachytene spermatocytes and round spermatids, but not in elongating mature spermatids (22). PACAP-LI was also detected in the spermatids in the cap and acrosome phases (4). Therefore, expression of PC4 mRNA appears to occur in the testicular germ cells that express PACAP and is expressed at the same time as or slightly preceding the expression of PACAP-LI. The PC4-null mice, with apparently normal spermatogenesis, exhibited severely impaired fertility in vivo (24). The fertilizing ability of spermatozoa in vitro was also severely impaired. Oocytes fertilized by the spermatozoa from PC4-null mice did not develop to the blastocyst stage, causing early embryonic death (24). The motility of PC4-null spermatozoa was reduced, as indicated by the decreased percentage of hyperactivate spermatozoa (24). Accordingly, although spermatogenesis in PC4-deficient mice appeared to be normal morphologically, the functional capacity of the spermatozoa was impaired. These findings imply that the germ cells in PC4-null mice lack some key molecules that are required for mature function of spermatozoa. As PACAP is expressed in the spermatids at the cap and acrosome phases, testicular PACAP might be involved in the regulatory mechanisms of transcription of the key molecule(s) required for functional maturation of spermatozoa.
Germ cells have been shown to exhibit stage-dependent expression of different transcription factors, such as the cyclic AMP-responsive element modulator/cAMP response element-binding protein multigene family, which includes proteins functioning as the activators and inhibitors of gene transcription after the phosphorylation that follows the triggering of intracellular signaling pathways (31, 32). Such a reaction is reportedly regulated by mitogen-activated protein (MAP) kinases (MAPKs) (33). Our preliminary study showed that PACAP interacts with its specific receptor in the soluble cytosolic proteins of rat testis and activates extracellular signal-regulated kinase (ERK1/2) of MAP kinases at a lower concentration of 10-12 M, but not adenylate cyclase (34). If testicular PACAP plays a pivotal role in activation of MAPKs at a critical period during germ cell maturation, its absence may result in impaired fertility in male animals. It has also been reported that ERK1/2 activation is required for sperm capacitation (35, 36).
As in the testis, PACAP38 and PACAP27 are absent in the ovary of PC4-null mice. Although gonadotropins are the most important hormones to regulate ovarian physiology, several studies have demonstrated that PACAP also influences important ovarian functions (9, 10, 11, 37, 38, 39, 40), in part by mediating the actions of gonadotropins (11). In the rat, PACAP positively affects ovarian steroidogenesis, oocyte maturation, and plasminogen activator production (9, 10, 11, 38, 39, 41). Most recently, Fahrenkrug and his associates (38) reported that immunoneutralization of endogenous PACAP significantly reduced acute accumulation of progesterone and impaired subsequent luteinization induced by gonadotropins, suggesting an important auto- or paracrine role for ovarian PACAP.
PACAP-LI was detected by RIA in whole tissue extracts in the ovaries of humans, rats, and mice (3, 6, 41, 42, 43). Both immunohistochemistry and in situ hybridization showed intense PACAP-LI and PACAP mRNA in the majority of granulosa cells in the preovulatory follicles, but no transcripts for PACAP were observed in the primordial or mature follicles (6, 7, 8). The expression of PACAP in the ovary is stimulated by either LH or FSH and occurs during the periovulatory period (9, 10). PACAP suppresses the apoptosis of granulosa cells in cultured follicles. Although LH suppresses the apoptosis of granulosa cells, its effect is partially blocked by cotreatment with a PACAP receptor antagonist (11). In the ovary of PC4-null mice, folliculogenesis was significantly delayed compared with that in wild-type mice (Mbikay, M., et al., unpublished observations). These findings showed that blockage of the action of endogenous PACAP causes an impairment of the ovarian function similar to that found in PC4-deficient mice.
Although the physiological role of ovarian PACAP has been gradually unveiled by recent studies, the role of testicular PACAP in spermatogenesis or maturation of spermatozoa remains entirely unknown. The present study indicated that PC4 is the sole processing enzyme for the PACAP precursor in the testis and ovary. Because no appropriate in vitro culture system exists for investigating spermatogenesis in germ cells, it is difficult to examine the direct effect of PACAP on the process of spermatogenesis in vitro. In vivo studies have determined that the presence of the blood-testis barrier hampers entry of systemically administered PACAP into the seminiferous tubules, making it difficult to study the physiological action of PACAP in the testis. As PC4-null mice exhibit severely impaired fertility both in vivo and in vitro and a lack of bioactive PACAP in the testis, we speculate that the impaired fertility in those mice may result from absence of PACAP expression in the germ cells at a critical period during spermatogenesis. PC4 knockout (Pcsk-/-) mice with an absence of PACAP in the testicular germ cells will undoubtedly be used as a unique and effectual mechanism in studying the physiological role of testicular PACAP.
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Acknowledgments |
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Footnotes |
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Received April 20, 2000.
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