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Regular Article |
a Department of Physiology, University of Turku, 20520 Turku, Finland
b Department of Pharmacology and Toxicology, Biocenter Oulu, University of Oulu, 90100 Oulu, Finland
ABSTRACT
To study the regulation of fetal testicular steroidogenesis in the rat, we examined effects of members of the natriuretic peptide family, that is, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), on testosterone production of dispersed Leydig cells of rat fetuses at Embryonic Day (E) 18.5. All three peptides stimulated testosterone production, with significant effect at concentrations
1 x 10-8 mol/L of ANP, 1 x 10-9 mol/L of BNP, and 1 x 10-6 mol/L of CNP. Likewise, receptors for all three peptides (i.e., NPR-A, NPR-B, and NPR-C) were expressed in the fetal testis as early as E15.5. The natriuretic peptides had no effect on cAMP production by fetal Leydig cells. When tested in combination with two other peptides previously shown to stimulate fetal testicular steroidogenesis, vasoactive intestinal peptide and pituitary adenylate cyclase-stimulating polypeptide (PACAP-27), the combined effects did not differ significantly from the maximum effect with any one of the peptides alone. In conclusion, our present findings provide both functional and molecular evidences for NPR-A, NPR-B, and NPR-C in the fetal testis. Because ANP has previously been detected in fetal plasma and we now demonstrate the expression of BNP and CNP in fetal testes, these findings indicate involvement of the natriuretic peptides in endocrine and paracrine regulation during the early phase of fetal testicular steroidogenesis at E15.5–19.5 (i.e., before the onset of pituitary LH secretion).
developmental biology, Leydig cells, natriuretic peptides, sexual differentiation, testes
INTRODUCTION
The natriuretic peptide family consists of three structurally related peptides: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The ANP and BNP are synthesized and secreted mainly by cardiocytes to act as hormones in the regulation of blood pressure and fluid-volume homeostasis through their natriuretic and diuretic action on the kidney and as vasorelaxants in vascular smooth muscle cells [1, 2]. On the other hand, CNP is found at a low level in plasma [3], and it seems to be produced by extracardiac tissues [4] to act as a local paracrine or autocrine regulator.
Three different receptors for the natriuretic peptides have been identified so far. Two of them, designated as NP-A receptor (NPR-A) or guanylate cyclase-A (GC-A) [5] and as NP-B receptor (NPR-B) or guanylate cyclase-B (GC-B) [6], represent transmembrane proteins containing an extracellular ligand-binding domain and an intracellular guanylate cyclase domain. The NPR-A interacts with ANP and BNP at a higher affinity than with CNP [7], whereas NPR-B specifically binds CNP [8]. Ligand binding to these receptors is proposed to result in activation of the guanylate cyclase enzyme, leading to intracellular accumulation of cGMP that, in turn, acts as the second messenger eliciting the cellular responses to peptide stimulation [1]. However, other signal transduction pathways have also been implicated in association with these receptors [9–11]. The third natriuretic peptide receptor, called ANP-clearance receptor (C-receptor), lacks intrinsic guanylate cyclase activity, and this receptor has been proposed to be biologically silent and to serve as specific clearance binding sites for the natriuretic peptides [12]. The rank order of binding affinity for the C-receptor is ANP > CNP > BNP in both the human and the rat [7].
Natriuretic peptides modulate the function of a variety of steroidogenic cells in the human, rat, and bovine adrenal glands [13–15]; the mouse and rat Leydig cells [16–18]; and the human and rat granulosa cells [19, 20]. All three natriuretic peptides, especially ANP and BNP, stimulate testosterone production by adult Leydig cells [16–18]. Moreover, all three peptides may be produced locally within the testis. The mRNAs for ANP [21] and CNP [4] are present in the adult testis. Using immunohistochemical studies, ANP was localized in the seminiferous tubules [22, 23], whereas BNP and CNP were localized in the Leydig cells [23, 24]. To our knowledge, however, the ontogeny of testicular expression of the natriuretic peptides and their receptors has not been studied, nor have their testicular functions during the early stages of life been determined.
During fetal life, ANP and BNP are intensively expressed in the fetal mouse heart and ANP and CNP in the fetal brain [25]. Relatively high fetal plasma levels of ANP (
1 nmol/L) have been reported in fetal rats at Embyronic Day (E) 20.5 [26]. Previous reports suggested a regulatory role for the natriuretic peptides regarding blood pressure and body fluids of developing embryos, in analogy with the adult [27], but to our knowledge, no reports concerning their possible endocrine effects during fetal life exist.
There is now compelling evidence that fetal testicular steroidogenesis must start independently of pituitary LH action [28, 29], and a network of nongonadotropic endocrine or paracrine factors is likely to initiate and maintain the early phase of this endocrine activity. We suggested previously that vasoactive intestinal peptide (VIP) as an endocrine factor [30] and pituitary adenylate cyclase-stimulating polypeptide (PACAP-27) as a paracrine factor [31] are candidates for such stimuli of the fetal testis. Here, we have studied the effects of the three natriuretic peptides on testosterone production by dispersed Leydig cells of E18.5 fetal rats. These effects were correlated with the testicular expression of genes of the natriuretic peptides and their receptors. Additionally, we compared the stimulating effects of the four peptides (i.e., ANP, BNP, VIP, and PACAP-27) both individually and in different combinations.
MATERIALS AND METHODS
Animals and Experimental Design
Adult (2–3-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 SDS RM-3 (Special Diet Service; E, soy-free; Whitham, Essex, UK) 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 as Day 0.5 of gestation, and the day of birth was designated as Postnatal Day 1. Mothers were killed by decapitation under light CO2 anesthesia between E15.5 and E21.5. The fetuses were excised and pinned on a silicon rubber mat. The abdominal wall was then opened, and the testes were removed, snap-frozen in liquid nitrogen, and stored at -70°C until analyzed for RNA (see below). Alternatively, the testes were excised under sterile conditions and placed into ice-cold medium (Dulbecco modified Eagle Medium:F12 [1:1, v:v], with 0.365 g/L of L-glutamine; Life Technologies, Gibco BRL, Glasgow, Scotland) plus 0.1% BSA (Sigma Chemical Co., St Louis, MO), 4.5 g/L of glucose, 20 mmol/L of Hepes, and 0.1 g/L of gentamicin (Biological Industries, Bet HaEmek, Israel) to be used for all 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 the 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 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. Thereafter, the medium was removed, and 1.0 ml of fresh medium containing 0.2 mmol/L of 1-methyl-3-isobutylxanthine (MIX; Aldrich Chemie, Steinheim, Germany) and 10-12 to 10-6 mol/L of ANP, BNP, or CNP (Peninsula Labs, Inc., Belmont, CA) or 30 µg/L of highly purified hCG (CR-121, 11 500 IU/mg; National Institutes of Health, Bethesda, MD) were added and the cells incubated for 4 h. Thereafter, the medium was collected, heated at 100°C for 5 min, and stored at -20°C until analyzed (see below). In another set of experiments, the cells were stimulated by different combinations of 10-7 mol/L of ANP, BNP, VIP, and PACAP-27, as stated below.
RNA Extraction
Total RNA was isolated from the adult and fetal tissues by the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method [32].
Reverse Transcriptase-Polymerase Chain Reaction
Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to screen the expression of natriuretic peptides (i.e., ANP, BNP, and CNP) as well as their receptors (i.e., NPR-A, NPR-B, and C-receptor mRNAs). For this purpose, oligonucleotide primer pairs were used as detailed in Table 1. The primers used were designed to amplify a cDNA fragment spanning more than one intron in the case of BNP and more than three introns in the case of NPR-A, according to the rat BNP and NPR-A genes [33, 34], to exclude potential genomic DNA contamination of the samples. For the other peptides and their receptors, because no reports on rat gene structures were available to our knowledge, the primers were designed according to previous references [35–39]. The RT and PCR reactions were performed sequentially in the same tube [40]. Fifty microliters of the RT-PCR mixture contained 1 nmol/L of each oligo primer, 200 mmol/L of deoxynucleotide triphosphates, 1.5 mmol/L of MgCl2, 20 U of RNasin (Promega, Madison, WI), 12.5 U of Avian Myeloma Virus-reverse transcriptase, and 2.5 U of 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), with final extension for 10 min at 72°C. For all reactions, liquid controls were run in parallel with the RNA samples. These control samples yielded negative reactions. The RT-PCR assays were repeated with independent samples from two to three times with similar results.
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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 5x SSPE (1x SSPE: 180 mmol/L of NaCl, 10 mmol/L of sodium phosphate, and 1 mmol/L of EDTA [pH 7.7]), 5x Denhardt solution, 0.5% (w/v) SDS, and 20 mg/L of calf thymus DNA. Hybridization was performed at 42°C overnight in the prehybridization solution after addition of the corresponding 32P-end-labeled antisense oligo probe, as shown in Table 2. For ANP and BNP genes, 32P-labeled cDNA probes were generated using a full-length ANP cDNA and a 390-base pair (bp) fragment of BNP cDNA as templates and a multiprime DNA labeling system (Promega). 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, 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.
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cAMP and Testosterone Measurements
Extracellular cAMP concentrations in the culture media were assayed using an RIA as described previously [41]. Succinyl-cAMP, radio-iodinated with Na[125I]iodide (IMS 300; Amersham) in our laboratory, served as a tracer [42]. Testosterone was measured in the culture media by RIA as described elsewhere [43]. The assay sensitivity was 2 fmol/tube. The intra-assay coefficient of variation (CV) was less than 6%, and the interassay CV was less than 12%.
Statistical Analysis
All values are presented as the mean ± SEM. A Macintosh version of the superANOVA program (Abacus Concepts, Inc., Berkeley, CA) was used to perform one-factor analysis of variance, followed by factorial tests and Fisher protected least significant difference post-hoc tests. A P value of less than 0.05 was chosen as the limit of statistical significance.
RESULTS
Stimulation of Dispersed Fetal Rat Leydig Cells by Natriuretic Peptides
All three natriuretic peptides stimulated testosterone production by cultured E18.5 Leydig cells (Fig. 1). The lowest concentration with significant (P < 0.01) effect on testosterone production was 1 x 10-8 mol/L for ANP, 1 x 10-9 mol/L for BNP, and 1 x 10-6 mol/L for CNP. The maximally stimulated levels of testosterone production by ANP, BNP, and hCG were comparable, whereas the CNP-stimulated levels of testosterone production remained lower even at the highest concentration of the peptide used (10-6 mol/L). The incubations with submaximally stimulating doses of hCG (30 µg/L) were carried out as a positive control for the stimulability of testosterone production in the experimental conditions used.
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Expression of the Natriuretic Peptide Genes
The RT-PCR and Southern hybridization analyses did not reveal ANP mRNA expression in the fetal or newborn testis at any age examined (Fig. 2, upper panel). However, abundant ANP mRNA was apparent in the adult rat heart and brain that were used as positive controls, reflecting the integrity of the tissue-processing and RNA-detection methods. In contrast, BNP and CNP mRNAs were detected by RT-PCR and Southern hybridization in the fetal and newborn testes as early as E15.5, the earliest age studied. The BNP cDNA fragment with the expected 250-bp size was faintly detectable at E15.5, and a clear message was found at E16.5 and later, together with a smaller transcript (200 bp) (Fig. 2, middle panel). The latter was possibly a result of alternative splicing, but its exact sequence was not studied. In the case of CNP, only a single band of the expected 246-bp size was observed at all ages studied (Fig. 2, lower panel).
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Expression of the Natriuretic Peptide Receptor Genes
Our RT-PCR and Southern hybridization analyses could detect mRNAs for all three natriuretic peptides receptors (i.e., NPR-A, NPR-B and C-receptor) in the fetal rat testis. The primers used amplified only one band of the expected size with each receptor mRNA (Fig. 3). In Figure 3 (lower panel), the apparent slight differences in the size of the fetal testicular transcripts are due to uneven running of the gel.
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Stimulation of Dispersed Fetal Rat Leydig Cells by Different Combinations of Peptides
When we stimulated the E18.5 Leydig cells with any of the peptides (i.e., ANP, BNP, VIP, or PACAP-27) alone or in different combinations, we found that ANP and BNP, as expected, had no effect on cAMP production by fetal Leydig cells (Figs. 4A and 5A). Moreover, the stimulatory effect of any combination of these peptides used (i.e., BNP + VIP and ANP + PACAP-27) or of all four peptides used together did not differ significantly from the effect with any of these peptides when used alone (Figs. 4 and 5).
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DISCUSSION
Recently, we have carried out a series of studies on the regulation of testicular steroidogenesis in the fetal rat [28, 30, 31]. We have suggested that a network of nongonadotropic endocrine or paracrine factors likely initiates and maintains the early phase of this activity before the onset of LH secretion. The gonadotropin-independent onset of Leydig cell steroidogenesis is also emphasized by the normal intrauterine masculinization of LH receptor knockout (LuRKO) mice [29] and of mice with disrupted expression of the transcription coactivator T/ebp (unpublished observation), which is necessary for differentiation of the pituitary gland [44]. Our previous findings showed that VIP as an endocrine factor and PACAP as a paracrine factor are candidates for such stimuli of the fetal testis. The results of the present study strongly support involvement of the natriuretic peptides, especially ANP and BNP, in the early regulation of fetal testicular steroidogenesis as well.
Both ANP and BNP stimulated fetal testicular steroidogenesis in a dose-dependent manner and in a range of similar concentrations (10-9 to 10-8 mol/L), whereas CNP was effective only at an approximately 1000-fold higher concentration. These natriuretic peptides stimulate both the 
4 and 5 pathways of steroidogenesis in adult mouse Leydig cells [16], but to our knowledge, their eventual effects during earlier stages of development have not yet been studied.
Because our qualitative RT-PCR and Southern hybridization analyses could not detect ANP mRNA in the fetal testis, it seems that ANP of the fetal atrial and ventricular cardiocytes, producing this peptide as early as E10.5 to E11.5 [45], may act as an endocrine factor to stimulate the fetal testicular steroidogenesis. This is feasible, because circulating ANP levels as high as 2700 ± 180 ng/L (1 nmol/L) have been reported in fetal rat plasma during a previous study [26]. Such a level could be high enough to stimulate fetal testicular steroidogenesis under physiological conditions. Although a direct ANP effect on Leydig cells is likely, indirect action via testicular macrophages is another possibility. The ANP is able to inhibit macrophage tumor necrosis factor-
production [46], which, at least in adult rodent testes, is an inhibitor of steroidogenesis [47].
The BNP was approximately 10-fold more potent than ANP in stimulating testosterone production, and a concentration of 1 x 10-9 mol/L was sufficient to evoke a significant release of testosterone by the fetal Leydig cells. The BNP gene was expressed in the fetal testis as early as E15.5. However, the level of expression in the testis was low during the fetal period, because we were unable to detect it by Northern hybridization analysis (data not shown). In the adult mouse testis, BNP can be localized by immunohistochemistry to Leydig cells [22, 23]. If this also applies to fetal testis, then BNP is produced locally to regulate, in an autocrine fashion, fetal testicular steroidogenesis as early as E15.5.
The CNP is unlikely to play a major role in the stimulation of fetal testicular testosterone production, because it was effective only at a very high concentration. However, the possible testicular role of CNP during fetal life is not necessarily restricted to the regulation of steroidogenesis, and in the fetal testis, this peptide may influence other cells than those of Leydig. In cultured astrocytes, CNP increased the cell volume and intracellular Ca2+ levels [48]. It is also associated with other neuroendocrine structures [49, 50], such as the hypothalamus and pituitary gland.
Our present findings provide both functional and molecular evidence for NPR-A and NPR-B in the fetal testis. The concomitant existence of NPR-A suggests physiological actions of ANP and BNP through this receptor subtype. The rank order of potency for cGMP production via NPR-A is ANP BNP >> CNP [5]. Similarly, the presence of NPR-B, which is specific for CNP, indicates a physiological role for CNP during testicular development.
We have also detected the expression of the third type of natriuretic peptide receptor, the C-receptor, in the fetal testis. Although its exact function is not known, it may be involved in the sequestration and metabolic clearance of natriuretic peptides from the circulation [12]. Whether such a function would be important for the fetal testicular function remains open.
Our results from the stimulation tests using different combinations of the four peptides confirmed that the natriuretic peptides use a second-messenger pathway other than the cAMP and protein kinase (PK) A pathway in stimulating fetal testicular steroidogenesis. Although the natriuretic peptides elicit their effects through the cGMP second-messenger pathway [1], there are some indications that they may alter the intracellular cAMP [9] and phosphoinositide concentrations [10, 11]. In the fetal testis, we found that they had no effect on cAMP production. This indicates that they most probably mediated their effects via the cGMP and PKG pathway, although an effect through the phosphoinositides and PKC pathway cannot be excluded.
Despite using different second-messenger pathways, the final effects of the four peptides tested seem to occur through a common step that could be, for example, phosphorylation of the same protein using different protein kinases. This may explain why the stimulatory effect of any combination of these peptides used (i.e., BNP + VIP and ANP + PACAP-27) or of all four peptides used together did not differ significantly from the effect with any one of the peptides when used alone. It also shows that the stimulability of fetal testicular steroidogenesis is a process that can be saturated by a variety of signaling mechanisms. This may be physiologically important considering the crucial role of sufficient testicular androgen production for male genital differentiation.
In conclusion, we present evidence for a regulatory role of the natriuretic peptides, through endocrine, paracrine, and autocrine mechanisms, in fetal testicular steroidogenesis and/or growth. They represent, in addition to VIP and PACAP, another group of bioactive peptides that may participate in the gonadotropin-independent regulation of rodent fetal testicular steroidogenesis before the onset of pituitary gonadotropin secretion.
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FOOTNOTES
First decision: 19 January 2001.
1 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. 
2 Correspondence: Ilpo Huhtaniemi, Department of Physiology, University
of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. FAX: 358 2 2502610;
ilpo.huhtaniemi{at}utu.fi
Accepted: April 5, 2001.
Received: December 21, 2000.
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