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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
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Abstract |
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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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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. 2).
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). 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. 3B). 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. 3C).
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). 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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Footnotes |
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Received August 26, 1997.
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) 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).
