Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

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 My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by El-Gehani, F.
Right arrow Articles by Huhtaniemi, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by El-Gehani, F.
Right arrow Articles by Huhtaniemi, I.
Agricola
Right arrow Articles by El-Gehani, F.
Right arrow Articles by Huhtaniemi, I.
Biology of Reproduction 63, 1482-1489 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Evidence That Pituitary Adenylate Cyclase-Activating Polypeptide Is a Potent Regulator of Fetal Rat Testicular Steroidogenesis1

Faraj El-Gehania, Manuel Tena-Semperea, and Ilpo Huhtaniemi2,a

a Department of Physiology, University of Turku, 20520 Turku, Finland

ABSTRACT

Testicular steroidogenesis in the fetal rat is activated before the onset of pituitary gonadotropin secretion. We studied here whether the pituitary adenylate cyclase-activating polypeptide (PACAP) could regulate this early Leydig cell activity. Effects of the two PACAP forms, 27 and 38, were studied on cAMP and testosterone production of dispersed Leydig cells of embryonic Day (E) 18.5. Furthermore, PACAP and PACAP type I receptor mRNA expression were measured by reverse transcription-polymerase chain reaction (RT-PCR), and testicular PACAP concentations by RIA. The two peptides were highly potent stimulators of fetal testes. Doses as low as 10-18 mol/L of PACAP-27 and 10-17–10-16 mol/L of PACAP-38 significantly stimulated cAMP and testosterone production, with magnitude comparable to that evoked by hCG. These effects were specific for fetal Leydig cells, because PACAP-responsive control cells, including murine Sertoli and granulosa cell lines, only responded to concentrations >=10-12 mol/L. By RT-PCR, PACAP and its type I receptor mRNAs were expressed in fetal testis as early as E15.5. By Northern hybridization, PACAP mRNA was first detectable on Day 30 postpartum and increased thereafter. Both forms of PACAP peptides were clearly detectable in E17.5 testes, with decreasing levels thereafter. In conclusion, the steroidogenesis of fetal rat Leydig cells responds to very low concentrations of PACAP, which may be an important physiological regulator of this activity before the onset of pituitary LH secretion.

cAMP, developmental biology, Leydig cells, testes, testosterone

INTRODUCTION

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been isolated from ovine hypothalamic tissues on the basis of its ability to stimulate adenylate cyclase in cultured rat anterior pituitary cells [1]. Pituitary adenylate cyclase-activating polypeptide exists in two forms: a longer form of 38 amino acids (PACAP-38) and a shorter one (PACAP-27) containing the 27 N-terminal amino acids of PACAP-38. Pituitary adenylate cyclase-activating polypeptide belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family of peptides [2].

Pituitary adenylate cyclase-activating polypeptide functions as a neurotransmitter, neuromodulator, and neurotrophic factor [2–4] in the central nervous system, and it is also widely distributed in endocrine organs [4–15]. Pituitary adenylate cyclase-activating polypeptide and its type I receptor were found in abundance in the adrenal medulla [4], where it may act as a potent noncholinergic secretagogue for catecholamines. Similarly, PACAP appears to play a regulatory role in insulin secretion [5]. Pituitary adenylate cyclase-activating polypeptide is considered a key player in the regulation of reproduction due to its effects at all levels of the hypothalamic-pituitary-gonadal axis. Pituitary adenylate cyclase-activating polypeptide acts in the hypothalamus to modulate the release of hormones such as LH and prolactin, probably by regulating the release of other hypophysiotropic factors such as GnRH, growth hormone-releasing hormone, and dopamine [6–8]. Additionally, PACAP has direct effects on pituitary gonadotroph cells by stimulating their LH release, as has been demonstrated in male rats in vivo [9]. Likewise, it stimulates the release of LH, FSH, and the common {alpha}-subunit in monolayer cultures of rat pituitary cells [10, 11]. Pituitary adenylate cyclase-activating polypeptide has been detected in the male and female urogenital system [12–15]. In the testis it might act as a paracrine or autocrine regulator of Sertoli and germ cells with a specific function during early spermiogenesis [13, 14], whereas in the female it may regulate the ovarian steroidogenic activity [15].

Both forms of PACAP bind to the same receptor, which exists as two major subtypes [8]. The type I PACAP receptors exhibit 100–1000 higher affinity for PACAP-27 and PACAP-38 than for VIP. The second type of receptor has the same affinity for PACAP-38, PACAP-27, and VIP. The latter receptor type can be subdivided into the classic VIP-binding site (also termed VIP1 receptor) and the helodermin-preferring sites (also known as VIP2 receptor) [8].

In fetal life, PACAP and PACAP receptors are expressed in the fetal brain as early as embryonic Day (E) 14.5 [16]. Pituitary adenylate cyclase-activating polypeptide may act as a trophic factor in the brain, involved in control of multiplication, differentiation, and/or migration of cells, based on its potent effect on cell proliferation, neurite outgrowth, and protein synthesis [17–19]. However, no reports on possible endocrine effects of PACAP during fetal life have been published.

Here, we have elaborated our preliminary finding that PACAP has a specific stimulatory effect on fetal testicular steroidogenesis [20]. To this end, we studied the dose-response effects of PACAP-27 and PACAP-38 on cAMP and testosterone production of dispersed fetal Leydig cells of E18.5 and followed this effect with advancing age of animals. We also explored expression of the PACAP and PACAP type I receptor genes in fetal testes. Finally, the testicular concentrations of both forms of PACAP were measured at different fetal ages.

MATERIALS AND METHODS

Animals and Experimental Design

Adult (2- to 3-mo-old) rats of the Sprague-Dawley strain (produced in our own vivarium) were housed under controlled photoperiod (14L:10D) and fed with commercial diet and water ad libitum. Females were caged with males overnight and checked the following morning for sperm in vaginal smear. The day after the night of mating was designated Day 0.5 of gestation, the day of birth was designated postnatal Day 1. Pups were housed with mothers in maternity cages until killed at 7, 13, 15, 16, or 20 days of age. Mothers were killed by decapitation under light CO2 anesthesia at daily intervals between E15.5 and E21.5. The fetuses were excised, pinned on a silicon rubber mat, the abdominal wall was opened, and the testes were removed, weighed, snap-frozen in liquid nitrogen, and stored at -70°C until used for analysis of PACAP content or mRNA. Alternatively, the testes were excised under sterile conditions and placed into ice-cold medium (Dulbecco modified Eagle medium/F12 [1:1], with 0.365 g/L L-glutamine) (Life Technologies, GIBCO BRL, Glasgow, Scotland), plus 0.1% BSA (Sigma Chemical Co., St Louis, MO), 4.5 g/L glucose, 20 mmol/L Hepes, and 0.1 g/L gentamicin (Biological Industries, Bet HaEmek, Israel) to be used for incubations and cultures (see below). All animal experiments were approved by the Turku University Committee on Ethics of Animal Experimentation.

Cultures of Dispersed Leydig Cells

The medium containing the fetal testes was changed into 15 ml of culture medium containing 0.4% collagenase type II (Sigma), and DNAse I (105 Kunitz units/L, Sigma), and the testes were incubated for 30 min at 37°C in a shaking water bath. Thereafter, the testes were dispersed mechanically by aspirating the tissue suspension through a pipette at 5-min intervals, continuing incubation until the tissue was completely disintegrated. The cells were then centrifuged at 200 x g for 5 min at 4°C, and the supernatant was discarded. The cells were finally washed twice with 50 ml of medium, resuspended in fresh medium, divided equally into a 24-well culture plate (Nunc, Roskilde, Denmark), and allowed to recover and stabilize for 24 h at 37°C in an atmosphere of 5% CO2 in air. The purity of the Leydig cells was assessed by cytochemical 3ß-hydroxysteroid dehydrogenase (3ß-HSD) reaction, and 30–40% of 3ß-HSD positive cells were found in the fetal testicular cell suspensions [20]. Thereafter, the medium was removed, and 1.0 ml of fresh medium containing 0.2 mmol/L 1-methyl-3-isobutylxanthine (MIX, Aldrich Chemie, Steinheim, Germany) and 10-19 to 10-6 mol/L of PACAP-27 or PACAP-38 (Peninsula Labs., Inc., Belmont, CA), or 30 µg/L of highly purified hCG (CR-121, 11 500 IU/mg, NIH, Bethesda, MD) were added and the cells incubated for 4 h. Thereafter, the medium was collected, (diluted 1:1 with 2 mmol/L theophylline [Sigma] in the case of cAMP measurement), heated at 100°C for 5 min, and stored at -20°C until analyzed (see below). The same steps were followed in dispersion and culture of Leydig cells of neonatal and immature rats after decapsulation of testes. All the experiments were run in triplicate or quadruplicate and repeated at least two times.

Culture of MSC-1 Cells

The MSC-1 cells are an immortalized Sertoli cell line derived from testicular tumor of a transgenic mouse carrying a fusion gene composed of the human anti-M;auullerian hormone transcripitional regulatory sequences linked to the coding sequence of simian virus 40 T-antigen [21]. One day before stimulation, the cells were plated into 24-well culture dishes (Greiner Labortechnik, Frickenhausen, Germany) at a density of 105 cells/well. The next day the medium was changed, and the cells were incubated in 0.5 ml culture medium (see above) in the presence and absence of 10-7–10-13 mol/L PACAP-27 or PACAP-38, with a final concentration of 0.2 mmol/L of MIX. The cells were incubated for 4 h at 37°C in an atmosphere of 5% CO2 in air, then the medium was collected, diluted 1:1 with 2 mmol/L theophylline, heated at 100°C for 5 min, and stored at -20°C until required for cAMP measurements (see below).

Isolation, Purification, and Culture of Adult Leydig Cells

For isolation and purification of adult Leydig cells, we used a previously described method [22]. In brief, the testes were decapsulated carefully and incubated in 10 ml of culture medium for 10 min at 34°C in an atmosphere of 5% CO2 in air in the presence of 0.3 mg/L collagenase type II (Sigma). After incubation, the tube (50 ml Falcon) was filled with medium and allowed to stand for 5 min at room temperature. The supernatant containing the dissociated interstitial cells was filtered through nylon gauze and centrifuged at 120 x g for 10 min. The resulting cell pellet was washed twice with medium and subjected to purification in a 50-ml continuous Percoll (Pharmacia, Uppsala, Sweden) gradient (density range 1.01–1.12 kg/L). After centrifugation (800 x g for 20 min at room temperature), the cell layer containing the purest Leydig cells (density ~1.05 kg/L) was collected, washed with medium, and incubated on 24-well plates (105 cells in 1.0 ml medium/well). The purity of the Leydig cells was assessed by the cytochemical 3ß-HSD reaction [23] after Percoll fractionation, and 75–85% of 3ß-HSD-positive cells were found. The purified Leydig cells were allowed to attach for 24 h in an atmosphere of 5% CO2 in air at 37°C, after which the medium was changed and the cells were stimulated as described above.

Extraction of RNA

Total RNA was isolated from the adult and fetal tissues by the single step acid guanidinium thiocyanate-phenol-chloroform extraction method as described before [24].

Reverse Transcription-Polymerase Chain Reaction

Reverse transcription-polymerase chain reaction (RT-PCR) was used in order to screen the expression of PACAP and type I PACAP receptor mRNAs. For this purpose the following oligonucleotide primers were used: 1) PACAP mRNA: sense primer 5'-GTGACGCCTACGCCCTTTACTACC-3' (PACAPS) and antisense primer 5'-GCTATTCGGCGTCCTTTGTTTTT-3' (PACAPAS), corresponding to nucleotides 398–422 and 691–713 of the rat PACAP cDNA sequence, respectively [25]; 2) PACAP type I receptor mRNA: sense primer 5'-TTAACTTTGTCTTTTCATCGGC-3' (PACAP type I receptorS) and antisense primer 5'-TCCCTCTTGCTGACGTTCTC-3' (PACAP type I receptorAS) corresponding to nucleotides 952–976 and 1118–1133 of the rat PACAP type I receptor cDNA sequence, respectively [26]. The primers used for amplification of PACAP mRNA were designed to span over intron 4 of the PACAP gene, assuming that the human and rat PACAP genes are structurally similar [27], thus allowing the exclusion of potential contamination with genomic DNA. For PACAP type I receptors, the primers were selected to amplify an area where alternative splicing takes place, giving rise to different mRNA transcripts according to a previous report [28]. The RT and PCR reactions were performed sequentially in the same tube [29]. Fifty microliters of the RT-PCR mixture contained 1 nmol/L of each oligo primer, 200 µmol/L dNTPs, 1.5 mmol/L MgCl2, 20 U RNasin (Promega, Madison, WI), 12.5 U avian myeloblastosis virus reverse transcriptase, and 2.5 U Dynazyme-DNA polymerase (Finnzymes Oy, Espoo, Finland). The reaction was started at 50°C for 10 min (RT), followed by a period of 3 min at 97°C, and then run for 40 PCR cycles (96°C for 1.5 min, 57°C for 1.5 min, 72°C for 3 min), and final extension for 10 min at 72°C. For all reactions, liquid controls were run in parallel with RNA samples. These control samples yielded negative reactions.

Southern Hybridization Analysis

The cDNA fragments generated by RT-PCR were resolved on 1.2% agarose gels and transferred onto nylon membranes (Hybond-N, Amersham, Aylesbury, UK). The membranes were prehybridized for 2–4 h at 42°C in a total volume of 25 ml containing 5x SSPE (1x SSPE = 180 mmol/L NaCl, 10 mmol/L sodium phosphate, and 1 mmol/L EDTA, pH 7.7), 5x Denhardt solution, 0.5% (w/v) SDS, and calf thymus DNA (20 µg/L). Hybridization was performed at 42°C overnight in prehybridization solution after addition of the corresponding 32P-end-labeled antisense oligo probe: 1) for PACAP mRNA: 5'-GGCGAGGTTCTCGCCCATGCCCCTCGCCACCATGGA-3' corresponding to nucleotides 511–543 of the PACAP cDNA sequence [25]; 2) for PACAP type I receptor R mRNA 5'-GATGAGCAGTAGGGTGGAGCGGGCCAGCCG-3' corresponding to nucleotides 1044–1073 of the PACAP type I receptor cDNA sequence [26]. The blots were washed twice for 10 min in 2x SSPE-0.1% SDS at room temperature and once in 1x SSPE-0.1% SDS at 50°C for 30 min and then exposed to x-ray film (Kodak XAR-5, Eastman Kodak, Rochester, NY) for 2–48 h. The molecular sizes of the RT-PCR products were determined by comparison with molecular size markers run together with the DNA fragments.

Northern Hybridization Analysis

For Northern hybridization analyses, RNA samples (20 µg/lane) were resolved on 1.2% denaturing agarose gel and transferred onto Hybond-N+ nylon membranes (Amersham) by the capillary method [30]. The membranes were prehybridized for 4 h at 64°C in a solution containing 50% deionized formamide (Sigma), 3x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5x Denhardt solution, 0.1 g/L heat-denatured calf thymus DNA, 1% SDS, and 0.1 g/L yeast tRNA. For hybridization, a 32P-labeled complementary RNA (cRNA) probe for the rat PACAP was synthesized using a Riboprobe system II kit (Promega) and a fragment of PACAP cDNA (spanning nucleotides 1025–1410) subcloned into T vector pGEM-5f as template. Hybridization was performed for 18–20 h at 66°C in the same prehybridization solution after addition of the cRNA probe. After hybridization, the membranes were washed in 2x SSC and 0.1% SDS at room temperature for 15 min, and to remove nonspecific hybridization, treated with ribonuclease-A (3 mg/L in 2x SSC) for 15 min at room temperature, followed by two washes in 0.2x SSC and 0.1% SDS at 64°C for 30 min. The filters were exposed for 18 h to imaging plates using Fujix Bio-Image Analyzer Bas 5000 (Düsseldorf, Germany).

Radioimmunoassays of Testosterone and cAMP

Testosterone was measured in the culture media by RIA as described before [31]. The assay sensitivity was 2 fmol/tube. The intra-assay coefficient of variation (CV) was below 6% and the interassay CV below 12%. Extracellular cAMP concentrations in the culture media were assayed by RIA as described before [32]. Succinyl-cAMP, radioiodinated with Na[125I]iodide (IMS 300, Amersham) in our laboratory, served as the tracer [33].

Pituitary Adenylate Cyclase-Activating Polypeptide Measurements

Testicular homogenates were comprised of pairs of testes from embryos at E17.5, E19.5, and E21.5. The frozen tissues were extracted by boiling in 0.9% NaCl for 10 min, followed by homogenization for 1 min and centrifugation. Each sediment was re-extracted in boiling 0.5 mol/L acetic acid for 15 min, followed by similar homogenization and centrifugation. The pooled supernatants were freeze-dried and redissolved in 1.0 ml of 0.5 mol/L sodium phosphate buffer, pH 7.5, for determination of PACAP-27 and PACAP-38 concentrations by RIA kits purchased from Peninsula Laboratories. The sensitivity of the PACAP assay was 10 pg/tube, and its intra-assay coefficient of variation was about 5%. All samples were analyzed in the same assay, to eliminate interassay variability.

Statistical Analysis

All values presented are means ± SEM. A Macintosh version of the superANOVA program (Abacus Concepts, Inc., Berkeley, CA) was used to perform one-factor ANOVA, followed by factorial tests and Fishers protected least significant difference posthoc tests. A P value less than 0.05 was chosen as the limit of statistical significance.

RESULTS

Stimulation of Dispersed Fetal, Neonatal, Immature, and Adult Rat Leydig Cells and MSC-1 Cells by PACAP-27 and PACAP-38

Both PACAP-38 and PACAP-27 stimulated cAMP and testosterone production by dispersed E18.5 fetal Leydig cells in culture in dose-dependent fashion (Fig. 1). Interestingly, both PACAP-38 and PACAP-27 were effective at extremely low concentrations. Doses as low as 10-18 mol/L of PACAP-27 and 10-17–10-16 mol/L of PACAP-38 were able to evoke significant stimulation of cAMP and testosterone production (P < 0.05). It is apparent from the cAMP dose-response curves that both forms of PACAP acted on two types of receptors, one of them at a low concentration of 10-18–10-12 mol/L, and after their saturation, another type of receptor was stimulated at higher concentrations of 10-12–10-7 mol/L (Fig. 1A). Cyclic AMP production at the lower PACAP concentrations was sufficient for maximal stimulation of steroidogenesis, because no further increase of testosterone was observed at the higher levels of the secondary cAMP increase.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 1. Dose-dependent stimulation of cAMP (A) and testosterone (B) production in dispersed fetal (E18.5) Leydig cells by PACAP-27 and PACAP-38. Fetal rat Leydig cells were dispersed as described in Materials and Methods, and incubated in the absence and presence of 10-19 to 10-7 mol/L PACAP-27 and PACAP-38. The bar indicates the testosterone production stimulated by hCG (30 µg/L). Each point represents the mean ± SEM of three different experiments (n = 6–10). The asterisks and the arrow indicate the beginning of significant differences from the basal (control) cAMP and testosterone production (*P < 0.05, **P < 0.001). Note that the Y-axis in B does not start from zero

As a control, we assessed the dose-response of cAMP to both forms of PACAP in MSC-1 cells. We found that both peptides were effective in these cells only at doses of 10-12–10-11 mol/L and higher (Fig. 2). These results are in good agreement with a previous report [34]. Similar findings were made when we stimulated the NT-1 cells (immortalized granulosa cell line derived from a mouse ovarian tumor) [35] (data not shown). When we followed the effect of PACAP-27 over different ages, it continued until the age when adult-type Leydig cells replace those of the fetal population around Day 20 postpartum [36] (Fig. 3). Accordingly, steroidogenesis of adult Leydig cells was not markedly stimulated by PACAP.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. The effects of PACAP-27 and PACAP-38 on cAMP production by MSC-1 cells. The cells were prepared as described in Materials and Methods, and incubated in the absence and presence of 10-13 to 10-7 mol/L PACAP-27 and PACAP-38. Each point represents the mean ± SEM of three different experiments (n = 6–9). The asterisks and the arrow indicate the beginning of significant differences from the basal (control) cAMP production (*P < 0.05). Note that the Y-axis does not start from zero



View larger version (61K):
[in this window]
[in a new window]
  		FIG. 3. The effects of PACAP-27 and hCG on cAMP and testosterone production in Leydig cells obtained from testes on Postnatal Days (D) 13, 16, 20, and in adults. The Leydig cells were dispersed, purified, and incubated as described in Materials and Methods. The incubation time was 4 h in the absence (C, {square}) or presence of 10-6 mol/L PACAP-27 (), and 30 µg /L hCG (). Each point represents the mean ± SEM of three different experiments (n = 6–9). The asterisks indicate significant differences from the basal (C) cAMP and testosterone production (***P < 0.001, ****P < 0.0001).

Pituitary Adenylate Cyclase-Activating Polypeptide and PACAP Receptor Gene Expression

The RT-PCR and Southern hybridization showed that PACAP and PACAP type I receptor mRNAs are expressed in the fetal rat testis as early as E15.5. Using the PACAP cDNA primers, only one amplicon of the expected size was observed (Fig. 4A), while three bands were observed in the case of PACAP type I receptors, corresponding to the expected sizes of the known splice variants of this receptor mRNA, namely the regular PACAP receptors cDNA, the PACAP-HIP and/or PACAP-HOP receptor variants and the PACAP-HIP-HOP receptor variant [26, 37] (Fig. 4B).



View larger version (63K):
[in this window]
[in a new window]
  		FIG. 4. Southern hybridization analysis of RT-PCR products of PACAP (A) and PACAP type I receptor (B) transcripts in the fetal rat testes of E15.5–E17.5, 1 day postpartum (1 D), fetal brain (FB), adult brain (AB), and liquids alone as a negative control. The migration of the expected cDNA products are depicted on the right sides of the panels.

Northern hybridization analysis of total testicular RNA from pre- and postnatal rats of different ages using a PACAP cRNA probe showed no signal in fetal and neonatal testes. There was a faint signal in 30-day-old testes, and the level of expression increased clearly thereafter (Fig. 5). The testicular transcript was about 0.8 kilobases (kb) in size, while it was 3.0 kb in the brain sample, in agreement with a previous report [25].



View larger version (81K):
[in this window]
[in a new window]
  		FIG. 5. Northern hybridization analysis of rat testicular PACAP mRNA during fetal and postnatal development. An aliquot of 20 µg total testicular RNA from different ages was loaded and resolved on a denaturing 1.2% agarose gel, then transferred onto nylon membrane, and hybridized with a cRNA probe for PACAP. A single transcript of approximately 0.8 kb was observed in the testes and about 3.0 kb in the brain but not in liver (negative control). The positions of the 28S and 18S rRNAs are indicated. The bottom panel shows the amounts of the 18S rRNAs in the same lanes by ethidium bromide staining.

Pituitary Adenylate Cyclase-Activating Polypeptide Measurements

Testicular PACAP-27 content was 12.9 ± 0.3 ng/g on E17.5, and it decreased to 7.6 ± 0.7 ng/g on E19.5, and further to 5.0 ± 0.04 ng/g on E21.5. The respective concentrations of PACAP-38 were 7.0 ± 0.2 ng/g on E17.5, 3.6 ± 0.08 ng/g on E19.5, and 3.03 ± 0.1 ng/g on E21.5 (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Rat testicular PACAP concentration at different ages

DISCUSSION

In the rat, fetal testicular steroidogenesis is activated on E15.5, whereas significant levels of pituitary LH appear in fetal circulation on E18.5 [20]. Therefore, fetal testicular steroidogenesis must start independently of LH, pointing out that some nongonadotropic autocrine, paracrine, or endocrine factor/s likely turn on and maintain this early activity. Our previous findings demonstrate that PACAP and VIP are likely candidates for such endo/paracrine stimuli of the fetal testis [20, 38]. In accordance, the results of the present study suggest that PACAP is a highly potent regulator of fetal testicular steroidogenesis, apparently engaging type I PACAP receptors.

Both PACAP forms stimulated cAMP and testosterone production of E18.5 fetal Leydig cells at concentrations as low as 10-17–10-18 mol/L. Because dispersed fetal testicular cells were used in the PACAP stimulations, the possibility that part of cAMP originates from other cells than those of Leydig cannot be excluded. However, the high sensitivity of the testosterone response indicates that the Leydig cells are the site of the very sensitive response to PACAP. This peptide is at least five orders of magnitude more potent than VIP in stimulating the fetal testicular steroidogenesis [38]. As far as we know, this is the first report on a paracrine factor with such high potency. The results suggest that PACAP acts on two types of receptors to stimulate the cAMP production by fetal Leydig cells. At concentrations 10-18–10-12 mol/L, it first stimulates the type I PACAP receptors that exhibit 1000-fold higher affinity for PACAP over VIP. When these receptors have become fully saturated it acts through VIP2 receptors that are expressed in the fetal testis as early as E15.5 [38]. It seems likely that the fetal testis contains a special form of PACAP type I receptors with high affinity for PACAP. Different variants of PACAP type I receptors have been proposed, as five different mRNA splice variants were identified, resulting from alternative splicing of two cassettes, named HIP and HOP [37]. These variants differ in the third intracellular loop and may have different abilities to activate adenylate cyclase or inositol triphosphate production. Radioligand binding and functional studies suggested the presence of type IA receptors that have equally high affinity for PACAP-27 and PACAP-38, and type IB receptors that prefer PACAP-38 over PACAP-27 [4, 8]. Some other studies suggested that the PACAP type I receptor, and its variants so far cloned correspond to type IA [8]. In most of the tissues so far studied [28, 37], the predominant mRNA variants are the normal receptor and the HOP1 variant. Although the RT-PCR technique used here is not quantitative, the predominant mRNA form in fetal testis had a size corresponding to the HIP and/or HOP1 variant (with the primers used, we could not distinguish between the HIP and HOP forms), followed by the normal receptor. The large HIP-HOP form was only faintly detectable. However, further binding studies are needed to characterize the possible unique receptor subtypes of the fetal testis.

In order to show the specificity of this extraordinarily potent effect of PACAP on fetal Leydig cells, we studied the dose-response relationship for both forms of PACAP in adult mouse Sertoli and granulosa tumor cell lines. We found, as did others [15, 34], that both PACAP forms were effective in the pico- to nanomolar range but inactive at lower concentrations.

When we followed the effect of PACAP on testicular steroidogenesis through the neonatal and immature ages, the stimulatory effect disappeared between 16 and 20 days postpartum, in accordance with the time when fetal-type Leydig cells are replaced by those of the adult growth phase [36]. These results are in agreement with our previous report in which we noted that PACAP has no effect on BLT-1 cell (immortalized adult mouse Leydig cell line) cAMP and testosterone production [20] and with the results by Romanelli et al. [39] demonstrating the absence of PACAP effects on basal testosterone production by the adult Leydig cells. They are in contrast to another recent report showing a stimulatory effect of PACAP on testosterone production by adult rat Leydig cells [40]. However, the PACAP binding sites within the adult testis have been localized in the seminiferous tubules and not in the Leydig cells [4, 41], and by immunocytochemistry PACAP immunoreactivity has been found in spermatogonia and primary spermatocytes but not in mature spermatides, spermatozoa [42], Sertoli cells, or Leydig cells [13]. These findings support the idea that during adult life, PACAP may function as a paracrine or autocrine regulator of germ cell function, being involved in the regulation of spermatogenesis but not of steroidogenesis.

The RT-PCR data demonstrated that PACAP can be produced locally to act as a paracrine or autocrine factor within the fetal testis, although part of it can also be of extratesticular origin. Although we could demonstrate PACAP gene expression in the fetal testis as early as E15.5, its level of expression remained low during the fetal and neonatal ages. In accordance, our attempts to identify the cellular localization of expression of PACAP and its receptor by in situ hybridization were unsuccessful (results not shown). This was not unexpected, because PACAP was needed at extremely low concentrations to stimulate fetal testicular steroidogenesis. The level of PACAP gene expression increases clearly after 30 days postpartum, hinting to a role in the regulation of spermatogenesis and sexual maturation.

The endogenous fetal testicular PACAP concentrations are sufficient to stimulate fetal testicular steroidogenesis. This means that both PACAPs are available in physiologically effective concentrations in the fetal rat testes at the early ages when testicular steroidogenesis is highly active despite the absence of LH in fetal circulation [20]. In contrast to adult tissues where PACAP is mainly represented by PACAP-38 and only 10% of the total immunoreactivity formed by PACAP-27 [12], the fetal testes contain a higher concentration of PACAP-27 than PACAP-38. However, PACAP-38 and PACAP-27 are generated from the same gene [4], but the physiological significance of this difference and whether PACAP-27 is derived directly from PACAP-38 or processed independently from the precursor remains to be studied.

In conclusion, our results suggest that PACAP, possibly produced within the fetal testis, is, at extremely low concentrations, a potent stimulus of fetal testicular steroidogenesis. The magnitude of steroidogenic stimulation by PACAP is comparable to that evoked by hCG, and the PACAP effect is probably produced, at low concentrations, via type I PACAP receptors that are expressed in the fetal testis as early as E15.5. These results indicate that PACAP, possibly together with VIP [38] and other, as yet unidentified, nongonadotropic factors, may contribute to the early gonadotropin-independent stimulation of fetal Leydig cells.

ACKNOWLEDGMENTS

The authors are grateful to Prof. Rolf Håkanson (University of Lund, Sweden) for his valuable comments on the manuscript and for the PACAP measurements done in his laboratory. The skillful technical assistance of Ms. T. Laiho and Ms. B. Carlsson is highly acknowledged.

FOOTNOTES

First decision: 25 February 2000.

1 This study was supported in part by a grant from the General Secretariat of Education and Scientific Research, Libya (F.E.-G.), by a postdoctoral grant from DGICYT, Ministry of Science, Spain (M.T.-S.) and by grants from The Academy of Finland and The Sigrid Jusélius Foundation. Back

2 Correspondence: Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. FAX: 358 2 2502610; ilpo.huhtaniemi{at}utu.fi Back

Accepted: June 8, 2000.

Received: January 27, 2000.

REFERENCES

  1. Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD, Coy DH. Isolation of a novel 38 residues hypothalamic peptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 1989; 164:567–574.[CrossRef][Medline]
  2. Arimura A. Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regul Pept 1992; 37:287–303.[Medline]
  3. Arimura A, Somogyvari-Vigh A, Weill C, Fiore RC, Tatsuno I, Bay V, Brenneman DE. PACAP functions as a neurotrophic factor. Ann NY Acad Sci 1994; 739:228–243.[Medline]
  4. Arimura A, Shioda S. Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors: neuroendocrine and endocrine interaction. Front Neuroendocrinol 1995; 16:53–88.[CrossRef][Medline]
  5. Yada T, Sakurada M, Ihida K, Nakata M, Murata F, Arimura A, Kikuchi M. Pituitary adenylate cyclase activating polypeptide is an extraordinarily potent intra-pancreatic regulator of insulin secretion from islet beta-cells. J Biol Chem 1994; 269:1290–1293.[Abstract/Free Full Text]
  6. Leonhardt S, Jarry H, Kreipe A, Werstler K, Wuttke W. Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates pituitary hormone release in male rats. Neuroendocrinol Lett 1992; 14:319–328.
  7. Jarry H, Leonhardt S, Schmidt WE, Creutzfeldt W, Wuttke W. Contrasting effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats. Life Sci 1992; 51:823–830.[CrossRef][Medline]
  8. Rawlings SR, Hezareh M. Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior pituitary gland. Endocr Rev 1996; 17:4–29.[Abstract/Free Full Text]
  9. Osuga Y, Mitsuhashi N, Mizuno M. In vivo effect of pituitary adenylate cyclase-activating polypeptide 38 (PACAP 38) on the secretion of luteinizing hormone (LH) in male rats. Endocrinol Jpn 1992; 39:153–156.[Medline]
  10. Hart GR, Gowing H, Burrin JM. Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats. J Endocrinol 1992; 134:33–41.[Abstract/Free Full Text]
  11. Culler MD, Paschall CS. pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates the gonadotropin-releasing activity of luteinizing hormone-releasing hormone. Endocrinology 1991; 129:2260–2262.[Abstract/Free Full Text]
  12. Arimura A, Somogyvari-Vigh A, Miyata A, Mizuno K, Coy D, Kitada C. Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes. Endocrinology 1991; 129:2787–2789.[Abstract/Free Full Text]
  13. Shioda S, Legradi G, Leung WC, Nakajo S, Nakaya K, Arimura A. Localization of pituitary adenylate cyclase-activating polypeptide and its messenger ribonucleic acid in the rat testis by light and electron microscopic immunocytochemistry and in situ hybridization. Endocrinology 1994; 135:818–825.[Abstract]
  14. Kononen J, Paavola M, Penttilä T-L, Parvinen M, Pelto-Huikko M. Stage specific expression of pituitary adenylate cyclase-activating polypeptide mRNA in the rat seminiferous tubules. Endocrinology 1994; 135:2291–2294.[Abstract]
  15. Heindel JJ, Sneeden J, Powell CJ, Davis B, Culler MD. A novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, regulates the function of rat granulosa cells in vitro. Biol Reprod 1996; 54:523–530.[Abstract]
  16. Tatsuno I, Somogyvari-Vigh A, Arimura A. Developmental changes of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the rat brain. Peptides 1994; 15:55–60.[CrossRef][Medline]
  17. Matsumoto H, Koyama C, Sawada T, Koike K, Hirota A, Arimura A, Inoue K. Pituitary folliculo-stellate-like cell line (TtT/GF) responds to novel hypophysiotropic peptide (pituitary adenylate cyclase-activating polypeptide), showing increased adenosine 3',5'-monophosphate and interleukin-6 and cell proliferation. Endocrinology 1993; 128:728–734.[Abstract/Free Full Text]
  18. Deutsch PJ, Schadlow VC, Barzilai N. 38-amino acid form of pituitary adenylate cyclase-activating peptide induces process outgrowth in human neuroblastoma cells. J Neurosci Res 1993; 35:312–320.[CrossRef][Medline]
  19. West AP, McKinnell C, Sharpe RM, Saunders PT. Pituitary adenylate cyclase-activating polypeptide can regulate testicular germ cell protein synthesis in vitro. J Endocrinol 1995; 144:215–223.[Abstract/Free Full Text]
  20. El-Gehani F, Zhang F-P, Pakarinen P, Rannikko A, Huhtaniemi I. Gonadotropin-independent regulation of the testicular steroidogenesis in the fetal rat. Biol Reprod 1998; 58:116–123.[Abstract/Free Full Text]
  21. Peschon JJ, Behringer RR, Cate RL, Harwood KA, Idzerda RL, Brinster RL, Palmiter RD. Directed expression of an oncogene to Sertoli cells in transgenic mice using mullerian inhibiting substance regulatory sequences. Mol Endocrinol 1992; 6:1403–1411.[Abstract/Free Full Text]
  22. Parvinen M, Nikula H, Huhtaniemi I. Influence of rat seminiferous tubules on Leydig cell testosterone production in vitro. Mol Cell Endocrinol 1984; 37:331–336.[CrossRef][Medline]
  23. Mendelson C, Dufau M, Catt K. Gonadotropin binding and stimulation of cAMP and testosterone production in isolated Leydig cells. J Biol Chem 1975; 250:8818–8823.[Abstract/Free Full Text]
  24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate phenol-chloroform extraction. Anal Biochem 1987; 162:156–159.[Medline]
  25. Hurley JD, Gardiner JV, Jones PM, Bloom SR. Cloning and molecular characterization of complementary deoxyribonucleic acid corresponding to a novel form of pituitary adenylate cyclase-activating polypeptide messenger ribonucleic acid in the rat testis. Endocrinology 1995; 136:550–557.[Abstract]
  26. Pisegna JR, Wank SA. Molecular cloning and functional expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor. Proc Natl Acad Sci U S A 1993; 90:6345–6349.[Abstract/Free Full Text]
  27. Hosoya M, Kimura C, Ogi K, Ohkubo S, Miyamoto Y, Kugoh H, Shimizu M, Onda H, Oshimura M, Arimura A, Fujino M. Structure of the human pituitary adenylate cyclase activating polypeptide (PACAP) gene. Biochim Biophys Acta 1992; 1129:199–206.[Medline]
  28. Vertongen P, Ciccarelli E, Woussen-Colle M-C, Neef P-D, Robberecht P, Cauvin A. Pituitary adenylate cyclase-activating polypeptide receptors of type I and II and glucagon-like peptide-I receptors are expressed in the rat medullary carcinoma of the thyroid cell line 6/23. Endocrinology 1994; 135:1537–1542.[Abstract]
  29. Zhang F-P, Hämäläinen T, Kaipia A, Pakarinen P, Huhtaniemi I. Ontogeny of luteinizing hormone receptor gene expression in the rat testis. Endocrinology 1994; 134:2206–2213.[Abstract/Free Full Text]
  30. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning—A Laboratory Manual, 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989.
  31. Huhtaniemi I, Nikula H, Rannikko S. Treatment of prostatic patients with a gonadotropin-releasing hormone angonist analog: acute and long term effects on endocrine functions of the testes. J Clin Endocrinol Metab 1985; 61:698–704.[Abstract/Free Full Text]
  32. Harper J, Brooker G. Femtomole sensitive radioimmunoassay of cyclic AMP and cyclic GMP after 2'-O-acetylation by acetic anhydride in aqueous solution. J Cyclic Nucleotide Res 1975; 1:207–218.[Medline]
  33. Brooker G, Harper J, Terasaki W, Moylan R. Radioimmunoassay of cyclic AMP and cyclic GMP. In: Brooker G, Greengard P, Robinson G (eds.), Advances in Cyclic Nucleotide Research. New York: Raven Press; 1979: 1–55.
  34. Heindel JJ, Powell CJ, Paschall CS, Arimura A, Culler MD. A novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, modulates Sertoli cell function in vitro. Biol Reprod 1992; 47:800–806.[Abstract]
  35. Kananen K, Markkula M, Rainio E, Su JG, Hsueh AJ, Huhtaniemi I. Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines. Mol Endocrinol 1995; 9:616–627.[Abstract/Free Full Text]
  36. Tapanainen J, Kuopio T, Pelliniemi L, Huhtaniemi I. Rat testicular endogenous steroids and number of Leydig cells between the fetal period and sexual maturity. Biol Reprod 1984; 31:1027–1035.[Abstract]
  37. Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH, Journot L. Differential signal transduction by five splice variants of the PACAP receptor. Nature 1993; 365:170–175.[CrossRef][Medline]
  38. El-Gehani F, Tena-Sampere M, Huhtaniemi I. Vasoactive intestinal peptide (VIP) is an important endocrine regulator of fetal rat testicular steroidogenesis. Endocrinology 1998; 139:1474–1480.[Abstract/Free Full Text]
  39. Romanelli F, Fillo S, Isidori A, Conte D. Pituitary adenylate-cyclase activating polypeptide regulates rat Leydig cell function in vitro. Neuropeptides 1997; 314:311–317.
  40. Rossato M, Nogara A, Gottardello F, Bordon P, Foresta C. Pituitary adenylate-cyclase activating polypeptide stimulates rat Leydig cells steroidogenesis through a novel transduction pathway. Endocrinology 1997; 138:3228–3235.[Abstract/Free Full Text]
  41. Shivers BD, Gärcs TJ, Gottschall PE, Arimura A. Two high affinity binding sites for pituitary adenylate-cyclase activating polypeptide have different tissue distributions. Endocrinology 1991; 128:3055–3065.[Abstract/Free Full Text]
  42. Hannibal J, Fahrenkrug J. Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) gene by rat spermatogenic cells. Regul Pept 1995; 51:111–115.[CrossRef]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
T. Takeda, Y. Matsumoto, T. Koga, J. Mutoh, Y. Nishimura, T. Shimazoe, Y. Ishii, T. Ishida, and H. Yamada
Maternal Exposure to Dioxin Disrupts Gonadotropin Production in Fetal Rats and Imprints Defects in Sexual Behavior
J. Pharmacol. Exp. Ther., June 1, 2009; 329(3): 1091 - 1099.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
C. Gyamera-Acheampong and M. Mbikay
Proprotein convertase subtilisin/kexin type 4 in mammalian fertility: a review
Hum. Reprod. Update, March 1, 2009; 15(2): 237 - 247.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. J. Kuhl, S. M. Ross, and K. W. Gaido
CCAAT/Enhancer Binding Protein {beta}, But Not Steroidogenic Factor-1, Modulates the Phthalate-Induced Dysregulation of Rat Fetal Testicular Steroidogenesis
Endocrinology, December 1, 2007; 148(12): 5851 - 5864.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
C A Wilson and D C Davies
The control of sexual differentiation of the reproductive system and brain
Reproduction, February 1, 2007; 133(2): 331 - 359.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Lacombe, V. Lelievre, C. E. Roselli, W. Salameh, Y.-h. Lue, G. Lawson, J.-M. Muller, J. A. Waschek, and E. Vilain
Delayed testicular aging in pituitary adenylate cyclase-activating peptide (PACAP) null mice
PNAS, March 7, 2006; 103(10): 3793 - 3798.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
F. El-Gehani, M. Tena-Sempere, H. Ruskoaho, and I. Huhtaniemi
Natriuretic Peptides Stimulate Steroidogenesis in the Fetal Rat Testis
Biol Reprod, August 1, 2001; 65(2): 595 - 600.
[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 My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by El-Gehani, F.
Right arrow Articles by Huhtaniemi, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by El-Gehani, F.
Right arrow Articles by Huhtaniemi, I.
Agricola
Right arrow Articles by El-Gehani, F.
Right arrow Articles by Huhtaniemi, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS