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Endocrinology Vol. 139, No. 4 1474-1480
Copyright © 1998 by The Endocrine Society

ARTICLES

Vasoactive Intestinal Peptide Is an Important Endocrine Regulatory Factor of Fetal Rat Testicular Steroidogenesis1

Faraj El-Gehani, Manuel Tena-Sempere and Ilpo Huhtaniemi

Department of Physiology, University of Turku, Turku, Finland

Address all correspondence and requests for reprints to: Prof. Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study elaborates our recent preliminary finding that vasoactive intestinal peptide (VIP) has a specific stimulatory effect on fetal rat Leydig cells. We examined the dose-response relationship for the effect of VIP on cAMP and testosterone production by dispersed fetal Leydig cells isolated from rat testes on embryonic day (E) 18.5. Further, we used RT-PCR to examine the expression of the VIP gene in fetal brain and testes and that of the VIP receptor genes in fetal testes and used RIA to measure VIP in testes and plasma during the fetal period. VIP stimulated fetal testicular cAMP production at a dose of 10-9 mol/liter, whereas a dose as low as 10-12 mol/liter stimulated testosterone production. This suggests that VIP at low doses may stimulate testosterone production using second messenger pathways other than cAMP. RT-PCR analysis could not reveal either VIP messenger RNA (mRNA) in fetal tissues or VIP1 receptor mRNA in the fetal or newborn testes, whereas VIP2 receptor mRNA was detected in fetal testes as early as E15.5. Northern hybridization analysis showed that the level of expression of VIP2 receptor mRNA is very low in fetal and neonatal testes and increases with age. The testicular VIP content was unmeasurable by our RIA method (i.e. <1 fmol/testis), whereas the circulating level of VIP was 82.9 ± 1.1 pmol/liter on E17.5 and decreased with advancing fetal age.

In conclusion, our results suggest that VIP from an extratesticular source, possibly from the maternal compartment, may regulate fetal testicular steroidogenesis through type 2 receptors as early as E15.5. These findings may be of physiological significance, because the onset of fetal testicular steroidogenesis occurs at an age (E15.5–19.5) before the onset of pituitary LH secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASOACTIVE intestinal peptide (VIP), a 28-amino acid molecule, was first isolated from porcine duodenum (1) and later identified in endocrine cells (2, 3, 4) and in the nervous system (5, 6, 7). In the nervous system it is thought to function as both a neurotransmitter and a modulator affecting cellular proliferation and survival, local metabolism, and vascular tone (8). Most studies of VIP have concentrated on its role as a central nervous system neurotransmitter and neuromodulator with neurotropic properties (9, 10, 11). However, VIP has recently been shown to regulate embryonal growth in rodents during the early postimplantation period (12). In the cultured whole mouse embryo on E9.5, VIP dramatically accelerated growth (13). Treatment with VIP stimulated mitoses and doubled DNA.

VIP acts through two types of receptors (VIP1 and VIP2); both of them have been cloned and shown to have very different amino acid sequences (8). Both are members of the seven-transmembrane domain, G protein-coupled receptor family, and VIP1 and VIP2 receptors have similar affinity to VIP, peptide histidine isoleucine (PHI), pituitary adenylate cyclase-activating polypeptide-38 (PACAP38), and PACAP27. VIP1 receptor messenger RNA (mRNA) is found in the lung, small intestine, thymus, and brain in the cerebral cortex and hippocampus. VIP2 receptor mRNA is present in the testis, stomach, pituitary, pancreatic islets, and hypothalamus (8). cAMP is the main second messenger for both VIP receptors (14, 15). However, VIP actions mediated through alternative signal transduction pathways have also been reported (16, 17, 18, 19, 20, 21).

We suggested recently that VIP stimulated fetal testicular steroidogenesis (22). In continuation of this work, we studied the relationship of the dose-response effect of VIP on cAMP and testosterone production by embryonic day 18.5 (E18.5) dispersed fetal rat Leydig cells and followed this effect over time. We also studied VIP gene expression in fetal tissues and characterized VIP receptors in fetal testis. Moreover, the testicular and circulating concentrations of VIP were measured at different fetal ages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
Adult (2- to 3-month-old) rats of the Sprague-Dawley strain (produced in our own vivarium) were housed under a controlled photoperiod (14 h of light, 10 h of darkness) and fed a commercial diet and water ad libitum. Females were caged with males overnight and checked the following morning for sperm in the 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 they were killed at 7, 13, 14, 16, 20, or 28 days of age. Mothers were killed by decapitation under light CO2 anesthesia between 0800–1200 h at daily intervals between E15.5–21.5. The fetuses were excised and pinned on a silicon rubber mat, and blood was taken by axillary puncture into heparinized syringes kept on ice and centrifuged. Plasma was stored at -20 C until analyzed. The abdominal wall was opened, and the testes were removed, snap-frozen in liquid nitrogen, and stored at -70 C until analyzed for VIP or RNA (see below). Alternatively, the testes were excised under sterile conditions and placed into ice-cold medium [DMEM-Ham’s F-12 (1:1), with 0.365 g/liter L-glutamine (Life Technologies, Glasgow, Scotland)] with 0.1% BSA (Sigma Chemical Co., St. Louis, MO), 4.5 g/liter, 20 mmol/liter HEPES, and 0.1 g/liter gentamicin (Biological Industries, Bet HaEmek, Israel) to be used for all incubations and cultures (see below).

Cultures of dispersed Leydig cells
The medium containing the fetal testes was changed into 15 ml of the culture medium containing 0.4% collagenase type II (Sigma) and deoxyribonuclease I (105 Kunitz units/liter; 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 the incubation until the tissue was disintegrated completely. The cells then were 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 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 (3ßHSD) reaction, and 30–40% of 3ßHSD-positive cells were found in the fetal testicular cell suspensions (22). Thereafter, the medium was removed, and 1.0 ml fresh medium containing 0.2 mmol/liter 1-methyl-3-isobutylxanthine (Aldrich Chemie, Steinheim, Germany) and 10-13–10-6 mol/liter VIP (Peninsula Laboratories, Belmont, CA) or 30 µg/liter highly purified hCG (CR-121; 11,500 IU/mg; NIH, Bethesda, MD) were added, and the cells were incubated for 4 h. Thereafter, the medium was collected [diluted 1:1 with 2 mmol/liter theophyline (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 the immature rats after decapsulation of the testes. All experiments were performed in quadruplicate and repeated three times.

Isolation, purification, and culture of adult Leydig cells
For isolation and purification of adult Leydig cells, we used a previously described method (23). In brief, the testes were decapsulated carefully and incubated in 10 ml culture medium for 10 min at 34 C in an atmosphere of 5% CO2 in air in the presence of 0.3 mg/ml collagenase type II (Sigma). After incubation, the tube (50 ml; Falcon, Oxnard, CA) 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 the medium and subjected to purification in a 50-ml continuous Percoll (Pharmacia, Uppsala, Sweden) gradient (density range, 1.01–1.12 kg/liter). After centrifugation (800 x g for 20 min at room temperature), the cell band containing the purest Leydig cells (density, ~1.05 kg/liter) was collected, washed with medium, and incubated in 24-well plates (105 cells in 1.0 ml medium/well). The purity of the Leydig cells was assessed by cytochemical 3ßHSD reaction (24) after the Percoll fractionation, and 75–85% 3ßHSD-positive cells were found. The purified Leydig cells were allowed to attach to 24-well culture plates for 24 h in an atmosphere of 5% CO2 in air, after which the medium was changed, and the cells were stimulated as described above.

RNA extraction
Total RNA was isolated from the adult and fetal tissues by the single step acid guanidinium thiocyanate-phenol-chloroform extraction method (25).

RT-PCR
RT-PCR was used to screen the expression of VIP as well as VIP1 and VIP2 receptor mRNAs. For this purpose, the following oligonucleotide primer pairs were used: 1) VIP mRNA: sense primer, 5'-GCCTGTTCAGGAAGCTGCACTG-3' (VIPS); and antisense primer, 5'-CTTCCGAGATGCTACTGCTGATTC-3' (VIPAS), corresponding to nucleotides 2–22 and 307–330 of the rat VIP complementary DNA (cDNA) sequence, respectively (26); 2) VIP1 receptor mRNA: sense primer, 5'-GCCTGTTCAGGAAGCTGCACTG-3' (VIP1 RS); and antisense primer, 5'-AGGTAGAGGCCCTCTACCAG-3' (VIP1 RAS), corresponding to nucleotides 556–577 and 762–781 of the rat VIP1 receptor cDNA sequence, respectively (15); and 3) VIP2 receptor mRNA: sense primer, 5'-GTCACAGTACAAGAGGCTCGC-3' (VIP2 RS); and antisense primer, 5'-CCCTCATACAGAGCTGACAGTG-3' (VIP2 RAS), corresponding to nucleotides 1025–1045 and 1389–1410 of the rat VIP2 receptor cDNA sequence, respectively (27). The primers used for determination of VIP mRNA were designed to amplify a cDNA fragment spanning introns B, C, and D according to the human VIP gene (28), allowing exclusion of potential genomic DNA contamination of the samples. For VIP1 and VIP2 receptors, as no report on gene structure was available, the primers were designed according to previous reports (15, 27). The RT and PCR reactions were performed sequentially in the same tube (29). Fifty microliters of the RT-PCR mixture contained 1 nmol/liter of each oligo primer, 200 mmol/liter deoxy (d)-NTPs, 1.5 mmol/liter MgCl2, 20 U RNasin (Promega), 12.5 U AMV-reverse transcriptase, and 2.5 U Dynazyme-DNA polymerase (Finnzymes Oy, Espoo, Finland). The reaction was started at 50 C for 10 min at room temperature, 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, and 72 C for 3 min), with 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 gel 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 5 x SSPE (1 x SSPE = 180 mmol/liter NaCl, 10 mmol/liter sodium phosphate, and 1 mmol/liter EDTA, pH 7.7), 5 x Denhardt’s solution, 0.5% (wt/vol) SDS, and calf thymus DNA (20 mg/liter). Hybridization was performed at 42 C overnight in the prehybridization solution after the addition of the corresponding 32P end-labeled antisense oligoprobe: 1) for the VIP mRNA, 5'-TGGTGAAAACTCCATCAGCATGCCTGGC-3', corresponding to nucleotides 240–213 of the VIP cDNA sequence (26); 2) for the VIP1 receptor mRNA, 5'-TTGAAGAGGGCCATGTCCTTGATGAAGACG-GC-3[primes], corresponding to nucleotides 663–632 of the VIP1 receptor cDNA sequence (15); and 3) for the VIP2 receptor mRNA, 5'-CAGGCACAGGCCTCTCCACCTTCTTTTCAG-3', corresponding to nucleotides 1250–1221 of the VIP2 receptor cDNA sequence (27). The blots were washed twice for 10 min each time in 2 x SSPE-0.1% SDS at room temperature and once in 1 x 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 analysis, RNA samples (20 µg/lane) were resolved on a 1.2% denaturing agarose gel and transferred onto Hybond-N+ nylon membranes (Amersham International, Aylesbury, UK) by the capillary method (30). The membranes were prehybridized for 4 h at 64 C in a solution containing 50% deionized formamide (Sigma), 3 x SSC, 5 x Denhardt’s solution, 0.1 g/liter heat-denatured calf thymus DNA, 1% SDS, and 0.1 g/liter yeast transfer RNA. For hybridization, a 32P-labeled complementary RNA (cRNA) probe for the rat VIP2 receptor was synthesized using a Riboprobe system II kit (Promega, Madison, WI) and a fragment of VIP2 receptor complementary DNA (spanning nucleotides 1025–1410), subcloned into T vector pGEM-5Z, 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 2 x SSC-0.1% SDS at room temperature for 15 min and to remove nonspecific hybridization were treated with ribonuclease A (3 mg/liter in 2 x SSC) for 15 min at room temperature, followed by two washes in 0.2 x SSC-0.1% SDS at 64 C for 30 min each time. The filters were exposed to x-ray films (Kodak XAR-5) at -70 C for 7–14 days. Relative mRNA levels at the different ages were obtained by densitometric scanning of the autoradiograms (TINA 2.0 package, Raytest, Straubenhardt, Germany), and the values were normalized by the amount of 18S ribosomal RNA transferred per lane, as estimated by ethidium bromide staining. The molecular size of the mRNA species was determined by comparison with the mobilities of the 18S and 28S ribosomal RNAs.

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

VIP measurements. The testicular homogenates were composed of pools of two pairs of testes on E17.5, E19.5, and E21.5. The frozen tissues were extracted by boiling in 0.9% NaCl for 10 min, followed by homogenization by Ultra-Turrax homogenizer for 1 min. The homogenates were centrifuged at 200 x g for 10 min at 4 C. Each sediment was reextracted by boiling in 0.5 mol/liter acetic acid for 15 min, followed by similar homogenization and centrifugation. The pooled supernatants were freeze-dried and redissolved in 1 ml 0.5 mol/liter sodium phosphate buffer, pH 7.5, for determination of VIP concentration by RIA. The plasma samples were pooled from two to four fetuses. VIP was measured using a RIA kit from Euro-Diagnostica (Malmo, Sweden).

Statistical analysis
All values are the mean ± SEM. A Macintosh version of the superANOVA program (Abacus Concepts, Berkeley, CA) was used for one-factor ANOVA, followed by Duncan’s new multiple range and Fisher’s protected least significant difference post-hoc tests. P < 0.05 was chosen as the limit of statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of dispersed fetal, immature, and adult rat Leydig cells by VIP
VIP stimulated cAMP and testosterone production by cultured E18.5 Leydig cells in a dose-dependent fashion (Fig. 1Go). A dose of 10-9 mol/liter VIP was able to elicit cAMP production (P < 0.05), whereas 10-12 mol/liter VIP was enough to induce significant testosterone release (P < 0.05). When we followed the stimulatory effect over time, it was observed as long as fetal-type Leydig cells are known to be present in the testis (34). The effect was lost between days 16 and 20 postpartum (Fig. 2Go).



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  		Figure 1. Dose-dependent stimulation of cAMP (A) and testosterone (B) production in dispersed fetal (E18.5) Leydig cells by VIP. Fetal rat Leydig cells were dispersed, as described in Materials and Methods, and incubated in the absence and presence of 10-13-10-7 M VIP. cAMP and testosterone in the media were measured by RIA. Each point represents the mean ± SEM of three different experiments (each performed in quadriplicate). The bar indicates testosterone production stimulated by hCG (30 µg/L). The asterisks indicate significant differences from the basal (C) cAMP and testosterone production (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). Please note that the y-axis does not start from zero.

 


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  		Figure 2. The effects of VIP and hCG on cAMP (A) and testosterone (B) production by Leydig cells obtained from testes on postnatal days (D) 13, 16, and 20 and from the adult rat. 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/liter VIP (), and 30 µg/liter hCG (). The concentrations of cAMP in the media were measured by RIA. Each point represents the mean ± SEM (n = 6). The asterisks indicate significant differences from the basal (C) cAMP and testosterone production (***, P < 0.001; ****, P < 0.0001).

 
VIP and VIP receptor gene expression
RT-PCR and Southern hybridization did not reveal VIP mRNA expression in the fetal brain and fetal or newborn testis at any age examined (Fig. 3AGo). However, abundant VIP mRNA was apparent in the adult rat brain used as a positive control, reflecting the integrity of the tissue processing and RNA detection methods. Using the same technique, we could not detect any mRNA for VIP1 receptor in the fetal or newborn testis, whereas it was expressed in the adult brain and liver and abundantly in the adult lung that were used as positive controls (Fig. 3BGo). In contrast, VIP2 receptor mRNA was detected by RT-PCR and Southern hybridization in the fetal and newborn testes as early as E15.5 (Fig. 3CGo).



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  		Figure 3. Southern hybridization analysis of the RT-PCR products of VIP (A), VIP1 receptor (B), and VIP2 receptor (C) transcripts in the fetal rat testes of E15.5–17.5, 1 day postpartum (1 D), fetal brain (FB), adult brain (AB), adult lung, and liquids alone as a negative control. The migration of the expected cDNA products are depicted on the right of the panels. (In B, the hybridization in FB is probably due to leakage from the lung lane.)

 
Northern hybridization analysis
Northern hybridization analysis of total testicular RNA in pre- and postnatal rats of different ages using a cRNA probe for the VIP2 receptor showed a faint signal in fetal (E17.5), neonatal (1-day-old), and infantile (7-day-old) testes (Fig. 4Go). The level of expression increased clearly between days 7–14 postpartum and stayed high thereafter. The VIP2 receptor mRNA transcript detected by Northern hybridization was about 3.5 kb, which is in good agreement with a previous report (27). The apparent slight differences in the sizes of the testicular transcripts are probably due to partially uneven running of the gel, as indicated by the presence of a similar pattern in migration of the 18S ribosomal RNA. However, the possibility that smaller transcripts exist cannot be ruled out. No hybridization was observed in the lane containing the negative control (20 µg total RNA extracted from liver).



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  		Figure 4. A, Northern hybridization analysis of rat testicular VIP2 receptor 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 transfered onto a nylon membrane and hybridized with a cRNA probe for the VIP2 receptor. A single transcript of approximately 3.5 kb was observed in the testes from the different ages and in the brain, but not in liver (negative control). The positions of the 28S and 18S ribosomal RNAs are indicated. The bottom panel shows the amounts of the 18S ribosomal RNAs in the same lanes by ethidium bromide staining. B, The relative steady state VIP2 receptor mRNA levels during fetal and postnatal testicular development. The values were obtained by densitometric scanning of two independent Northern hybridizations expressed in terms of arbitrary densitometric units (AUD), assigning to the most abundant lane (28D) a value of 100%.

 
VIP measurements
The testicular VIP content was unmeasurable at the different fetal ages tested (i.e. <1 fmol/testis), whereas the plasma VIP concentration was 82.9 ± 1.1 pmol/liter on E17.5, decreasing to 44.6 ± 2.6 pmol/liter on E19.5 and to 28.2 ± 2.3 pmol/liter on E21.5 (Table 1Go).


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  		Table 1. Rat plasma VIP concentration at different ages

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We reported previously that in the rat, fetal testicular steroidogenesis starts independently of LH, suggesting that nongonadotropic endocrine or paracrine factors are likely to initiate and maintain the early fetal testicular steroidogenesis before the onset of LH secretion (22). Our previous findings suggest that VIP is a candidate for such an endo/paracrine stimulus of the fetal testis. Further evidence for this view was obtained in the present study, which suggests that extratesticular VIP may act as an endocrine regulator of fetal testicular steroidogenesis via VIP2 receptors. When we followed the effect of VIP through neonatal and early postnatal development, it disappeared between days 16–20 postpartum, i.e. during the time when fetal-type Leydig cells are replaced by adult-type cells (34). Hence, this effect may be described as specific for the fetal-type Leydig cells, in agreement with a recent report (35).

It has been proposed by many investigators that during fetal life, VIP has neurotropic actions only with VIP receptors restricted to the central nervous system (36, 37, 38). Indeed, it has been shown that VIP has unique neurotropic properties, promoting cell mitosis (11) and increasing DNA and protein content in cultured whole mouse embryos (12), but other possible endocrine actions have received less attention.

In the testis, VIP stimulated the steroidogenesis during the fetal and early neonatal periods. This effect seems to be mediated by VIP2 receptors that are expressed at least as early as E15.5 in the fetal testis. By Northern hybridization, the level of expression of the VIP2 receptor mRNA was low during these periods, displaying a marked increase from day 7 postpartum onward. It has been shown that the effect of VIP on neonatal testicular steroidogenesis increases the 3ßHSD and cholesterol side chain-cleavage enzyme activities (39). In adult life, VIP seems to be involved in the regulation of spermatogenesis, but not of steroidogenesis, as VIP2 receptor mRNA was localized by in situ hybridization to spermatocytes but not to the interstitium and Leydig cells (8). This is in agreement with our data from Northern hybridization, where the level of expression increased significantly between days 7–14 postpartum. This is the period when meiotic division starts in germ cells. VIP2 receptor mRNA expression reaches a maximum between days 14–28 postpartum, at which time we encounter the highest relative number of spermatocytes in the testis (40).

VIP stimulated both fetal testicular cAMP production and steroidogenesis in a dose-dependent manner, but with greatly different sensitivities. A dose of 10-9 mol/liter VIP was needed to elicit cAMP production, whereas testosterone release was stimulated by a dose of 10-12 mol/liter VIP. This is much lower than the dose of VIP (10-6–10-8 mol/liter) required to elicit hormone production in adrenocortical, anterior pituitary, and granulosa cells (39). This may reflect either the high sensitivity of fetal Leydig cells to very small increases in cAMP, or the ability of VIP to stimulate fetal testicular steroidogenesis at low doses through other second messenger pathways. Effects of VIP on inositol phosphate generation (16, 17), activation of protein kinase C (18, 19), and mobilization of calcium in astrocytes (20) and hippocampal neurons (21) suggest that this peptide can operate through signal transduction mechanisms other than adenylate cyclase activity. Moreover, when the mouse homolog of the VIP2 receptor was expressed in Xenopus oocytes, VIP was found to activate a calcium-activated chloride current at a concentration of 10-10 mol/liter (41). The VIP2 receptor also recognizes PHI and pituitary PACAP with similar affinity (10). However, PHI could not be detected in the central nervous system before birth (36), whereas PACAP immunoreactivity was detected in rat embryos as early as E14.5 (42). Hence, PACAP and other related peptides may also stimulate these receptors in the embryo.

The circulating levels of VIP in the rat fetus (8 x 10-11 mol/liter on E17.5) are sufficient to stimulate fetal testicular steroidogenesis, and it has been shown that the concentration of VIP in the E11 embryo is 4-fold higher than that on E17 (38). This means that VIP is available in physiologically effective concentrations in the rat fetus at the time when critical developmental events, including embryogenesis and organogenesis, take place.

The origin of VIP in the fetal circulation remains unclear, in particular as we and others (36, 38, 43) were unable to localize either VIP by immunocytochemistry (El Gehani, F., P. Panula, and I. Huhtaniemi, unpublished data) or VIP mRNA by RT-PCR/Southern hybridization in fetal brain and testis. Accordingly, Hill et al. (38) suggested that in rodents, the maternal compartment may provide the neuroendocrine stimulus for embryonic growth through a surge of VIP during early postimplantation development. In contrast, other investigators have shown VIP gene expression in rat fetal brain (44, 45). The earliest expression of VIP in the rat embryo is on E13.5, when the peptide and mRNA are expressed transiently in a high percentage of cells in the rat stellate ganglia (37). Clearly, further studies on the origin of circulating VIP in the fetus are needed.

Our findings on the stimulatory role of VIP on fetal rat testicular steroidogenesis before any gonadotropin action are in line with previous observations suggesting that VIP stimulates cAMP and aromatase activity in fetal rat ovaries before follicle formation and acquisition of responsiveness to FSH (46), pointing to a general role of VIP in regulating steroidogenesis during development. However, a clear sex difference is observed in this respect in adulthood, as VIP does not stimulate adult Leydig cell steroidogenesis (Ref. 35 and present results), but elicits estradiol secretion by adult rat ovaries (47) and granulosa cells (48) in vitro and regulates cholesterol side chain-cleavage cytochrome P450 enzyme gene expression in granulosa cells (49).

In conclusion, our data provide evidence favoring a role for VIP as an endocrine stimulus of fetal testicular steroidogenesis. This action is probably mediated through VIP2 receptors, which are expressed in rat fetal testis as early as E15.5; the maternal compartment is the likely source of circulating VIP at this stage. It is of importance that circulating VIP can be measured at fetal ages (before E20.5) when testicular steroidogenesis displays high activity despite the absence of LH in the fetal circulation (22). Together, these data indicate that fetal testicular testosterone production is initiated without gonadotropins, and that VIP, possibly together with other nongonadotropic factors, may contribute to the early stimulation of fetal Leydig cells.


   Acknowledgments
 
The authors are grateful to Prof. Rolf Håkanson and Dr. P. Pakarinen for helpful discussions and advice during these experiments. The VIP measurements were made in the laboratory of Prof. Håkanson. The technical assistance of Ms. T. Laiho and Ms. B. Carlsson is gratefully acknowledged.


   Footnotes
 
1 This work was supported in part by a grant from the General Secretariat of Education and Scientific Research, Libya (to F.E.-G.); a postdoctoral grant from Direccion General de Investigacion Cientifica y Tecnica, Ministry of Science, Spain (to M.T.-S.); and grants from the Academy of Finland and the Sigrid Jusélius Foundation. Back

Received August 26, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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