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Biology of Reproduction 61, 741-748 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Reverse Transcription-Polymerase Chain Reaction Analysis of Genes Involved in Gonadal Differentiation in Pigs1

P. Parma3,a, E. Pailhoux2,a, and C. Cotinota

a Laboratoire de Biologie cellulaire et moléculaire, INRA, 78350 Jouy en Josas, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, testis development is initiated in the embryo as a response to the expression of the sex-determining gene, SRY. The time course of SRY expression during gonadal differentiation in the male has been described in detail only in mice and sheep. In this study, we used reverse transcription-polymerase chain reaction analysis to define the SRY transcription profile in pig genital ridges. SRY transcripts were first detectable from 23 days postcoitum (dpc), then declined sharply after 35 dpc. None were detected at 60 dpc. In addition, we analyzed temporal expression of other genes known to be involved in mammalian sex determination: WT-1, SF-1, SOX9, and AMH. A key stage seems to be 28 dpc, in which SOX9 expression switches between the male and female, and AMH expression begins to attest to Sertoli cell differentiation and to correspond to seminiferous cord formation in the male. Expression of gonadotropin receptors and aromatase was also investigated in porcine gonads, and we showed that their transcripts were detected very early on, especially in the male: 25 dpc for the LH receptor (rLH) and aromatase, and 28 dpc for the FSH receptor (rFSH). In the female, aromatase transcripts were not detected until 70 dpc, and rFSH expression occurred later: at 45 dpc at the onset of meiosis. Moreover, no difference was observed between the sexes for the onset of rLH transcription at 25 dpc. Such a thorough study has never been performed on pigs; developmental analysis will be useful for investigating sex-reversed gonads and determining ontogeny in intersexuality, a common pathology in pigs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals the sex determination process is a model of genetic switching between alternative programs during development. The master gene that initiates the morphogenetic events leading to testicular differentiation has been identified as SRY [1–3]. At the morphological level, seminiferous cord formation and Sertoli and Leydig cell differentiation are the most obvious effects of SRY expression in normal XY gonads. In the mouse, expression of Sry occurs only briefly, from 10.5 days postcoitum (dpc) to 12.0 dpc [4, 5]. As Sertoli cells are not recognizable until this stage, Sry must control the activity of other genes, subsequently ensuring the differentiation and maintenance of Sertoli cell characteristics. In other species like the sheep, SRY expression continues for a long time after the differentiation of Sertoli cells, suggesting possible differences in the gene function [6]. At a molecular level, it is not clear whether SRY acts as an activator of secondary testis-determining genes or as a repressor of ovary-determining genes, or whether it is involved in both processes [7, 8]. Nevertheless, several genes seem to be related to SRY [9]. Sox9 in mice is expressed at a low level in the genital ridge of both sexes and is up-regulated in the XY gonads after the onset of Sry expression [10–11]. Conversely, Dax-1 is down-regulated as testes develop, whereas it continues throughout ovary development [12, 13]. Another gene apparently plays a critical role in the cascade of sex-determining genes: steroidogenic factor-1 (SF-1), a member of the nuclear hormone receptor superfamily [14, 15]. It is involved in gonad morphogenesis and in the transcriptional activation of several genes required for the testicular function, including steroidogenic genes [16] and the anti-Müllerian hormone (AMH) [17–19].

After sex determination, sex-specific differentiation takes place largely in accordance with circulating sex hormones. Sertoli cells produce AMH, which causes regression of the Müllerian ducts [20, 21], whereas Leydig cells produce testosterone, which influences the development of both internal and external genitalia [22]. The pig is a very useful animal model for studying genetics and physiology. In addition, the intersex condition (XX male) is frequently observed in this species and represents an interesting "reverse-genetic" approach to molecular studies in mammalian sex determination and differentiation. However, very few data are available on the developmental expression of the genes involved in these processes in pigs. Here we determined, first, the SRY expression pattern during porcine male gonad development and then the pattern of other key genes known to be involved in sexual differentiation. These results, obtained by reverse transcription-polymerase chain reaction (RT-PCR) analysis, are discussed with regard to the physiological stages of gonadal differentiation. Knowledge of precise expression timing in normal animals is the first step before beginning studies on sex-reversed gonads, such as in intersex animals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Embryos and fetuses were obtained from cyclic crossbred Large White x Landrace sows (Sus scrofa) that were artificially inseminated on the first day of estrus. The animals were slaughtered after 21, 23, 25, 28, 32, 35, 45, 52, and 60 days. Day 0 corresponded to the day of insemination. The pregnant uteri were opened, and the embryos or fetuses were then dissected. Gonads and mesonephroi were not separated until 28 dpc. The number of fetuses used per RNA extraction and the number of independent RT experiments performed at each stage are summarized in Table 1.


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  		TABLE 1. Number of fetuses used in RT-PCR analysis

Terminology The term "embryo" as used here denotes the intrauterine conceptus from 21 to 35 days (sizes 8.0–35.0 mm), as presented by Altman and Dittmer [23].

Sexing of Embryos and Fetuses

Before 45 days of gestation, all embryos and fetuses were sexed by PCR analysis. Genomic DNA was obtained from the liver and used to test three markers of the pig Y chromosome, i.e., SRY, ZFY, and DYZ1, as previously described [24, 25].

RNA Extraction and cDNA Production

Total RNA was extracted from the various tissues using RNA-Plus solution (Bioprobe Systems, Montreuil-sous-Bois, France). Twenty micrograms of each sample was treated with 50 U of DNase I, RNase-free (Boehringer-Mannheim, Indianapolis, IN), for 2 h at 37°C. DNase-treated RNA (5 µg) was reverse transcribed in 20 µl at 42°C for 50 min using 200 U of Superscript II RNase H-reverse transcriptase (Gibco-BRL, Cergy Pontoise, France), 1 mM of each dNTP, and 7.5 µM random hexamers (Pharmacia Biotech, Orsay, France) in the presence of 20 U of RNase inhibitor (Boehringer-Mannheim) and 0.5 µCi of [{alpha}-32P]dCTP (Amersham, Les Ulis, France). An aliquot of the reaction (2 µl) was electrophoresed on 1% agarose gel, which was then dried and autoradiographed. These autoradiograms made it possible to verify the quality (length of cDNA products) and quantity (intensity of the smear) of the RT reaction. Only samples with a smear of comparable length and intensity were used for PCR amplification.

RT-PCR

Two microliters of RT reaction was amplified in a 100-µl PCR reaction using 1 U of Taq polymerase (TaKaRa; Prolabo, Fontenay sous Bois, France) for 30 cycles. PCR conditions and primer sequences for each studied gene are reported in Table 2 [6, 24, 26–32]. RT-PCR products were electrophoresed in agarose gel; they were then Southern blotted and probed with a [{gamma}-32P]ATP-radiolabeled internal oligonucleotide. The sequences of probes used for each gene are shown in Table 3.


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  		TABLE 2. Primer sequences, PCR conditions, and references.


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  		TABLE 3. Sequences of internel probes.

Amplification of Porcine Sequences

To obtain sequences corresponding to the studied porcine genes, we designed oligonucleotide primers based on a sequence comparison of these genes in several species (Table 2). We used cDNA synthesized from the fetal gonads or pig genomic DNA as a template for PCR. Each PCR reaction consisted of cDNA obtained from 0.5 µg of total RNA (or 0.5 µg of genomic DNA), 0.15 µM of each primer, 200 µM of each dNTP (TaKaRa), single-strength DNA Taq polymerase buffer (TaKaRa), 2.5 mM MgCl2, and 0.5 U Taq polymerase (TaKaRa) in a volume of 100 µl. PCR amplification proceeded as follows: 30 cycles at 94°C for 1 min, 55–58°C (depending on the primers; cf. Table 2) for 1 min, and 72°C for 1 min. The PCR products of the expected size were sequenced and compared with the GenBank database. The intron-exon boundary was determined by comparing amplification obtained from cDNA and genomic DNA. The unknown sequences in the pig were submitted to the GenBank database (accession numbers: SF-1: AF006572; SOX9: AF006571; AMH: AF006570; WT-1: AF006569).

Sequencing

PCR products obtained from both genomic DNA and cDNA were purified from agarose gel using silica (Sigma S-5631, St. Quentin Fallavier, France) and cloned into pBluescript KS II+ (Stratagene, Montigny-le-Bretonneux, France). DNA sequencing was performed on both strands using a T7 sequencing kit (Pharmacia) in the presence of [{alpha}-35S]dATP (Amersham). The reaction mixture was run on a 6% denaturing polyacrylamide gel that had been dried and exposed to an x-ray film overnight.

Quantitative RT-PCR

The 295-base pair (bp) SOX9 PCR product, derived from exons 2 and 3, was cloned into pBluescript KS II+ (Stratagene). A 124-bp DNA fragment (pBR322 digested by HaeIII) was then inserted into an internal AluI site. The new PCR product (competitor), which could be amplified using the same primers, differed in size (419 bp). It was amplified and purified, and several dilutions were carried out. For competition with the SOX9 target, different aliquots of SOX9 competitor were added to the PCR reaction and the products were blotted and probed with a [{gamma}-32P]ATP-radiolabeled internal oligonucleotide (Table 3). Hybridization signals were quantified using a PhosphorImager SI and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Formation of the Gonadal Ridge Primordium (21 dpc)

At this stage (21 dpc), the gonadal anlage was composed of three somatic tissues: the surface epithelium, the primitive cords, and the mesenchyme. Very few primordial germ cells were present under or within the surface epithelium. Around five urogenital ridges were pooled per RNA extraction, and three series of independent assays were performed for each investigated gene. The gonadal primordium was undissociable from the mesonephros, and they were tested together, generating a large dilution of the gonad-specific transcripts; the gonadal compound of the urogenital ridge represented around one tenth of the urogenital ridge. After the formation of the urogenital ridge at 21 days, WT-1 and SOX9 transcripts were detected in both sexes (Figs. 1 and 2). Expression of SF-1 and SOX9 (in males) was still very weak and was not observed in all the samples tested (one third for SF-1 and two thirds for SOX9 in male gonads). Surprisingly, expression of SOX9 in the female primordium was too strong to be only gonad specific. For this reason, we presumed that SOX9 expression was also derived from the mesonephros, and we tested male and female mesonephroi at four different stages: 23, 25, 28, and 35 dpc. From 25 dpc, we cut the urogenital ridge longitudinally; one part contained the primitive gonad and the other only the mesonephros. From 28 dpc, the gonads were easily detached from the mesonephros and were tested separately. Each studied stage showed positive signals in relation to SOX9 expression in the mesonephros. Only the results obtained from the males are shown in Figure 3.



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  		FIG. 1. Developmental expression of SOX9, SF-1, WT-1, and AMH genes during male gonadal differentiation in the pig. Total RNA preparations from fetal gonads were used in an RT, with (+) or without (-) reverse transcriptase. Separate PCR reactions were performed using the same RT products with different specific primer pairs. RT-PCR products were then Southern blotted and hybridized with a {gamma}-32P-labeled internal oligonucleotide corresponding to each investigated gene. Autoradiography was performed during 3–8 h.



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  		FIG. 3. SOX9, SRY, and WT-1 expression in mesonephros of male pig fetuses. Male mesonephroi from 23, 25, 28, and 35 dpc were tested for the presence of different transcripts by RT-PCR; then an aliquot (1/10) of the PCR reaction was deposited in an ethidium bromide-stained agarose gel. A positive signal for the SRY gene was obtained only at 23 dpc when gonads were indissociable from mesonephros. When tested separately (25, 28, 35 dpc), SRY expression was not detected in mesonephroi. All the stages were strongly positive for WT-1 gene, revealing an equal amount of cDNA in each sample. Presence or absence of reverse transcriptase is indicated by (+) and (-), respectively. A PCR negative control without cDNA was included in each experiment (H2O lane). M, Molecular weight (100-bp ladder; Pharmacia).

Development of the "Indifferent" Gonad (23 dpc)

Compared to the gonad at 21 dpc, the primordium at this stage had grown into a longitudinal roundish protrusion, and the number of primordial germ cells had increased. The male and female pig gonads at the age of 23 dpc were structurally similar under light microscopy (data not shown).

Expression of the SRY gene was first detected in the male developing urogenital ridge at 23 dpc (Fig. 4). At this stage, four urogenital ridges were pooled per RNA extraction, and three independent extractions and RT experiments were performed. No difference was observed among the samples, and all presented a strong SRY-positive signal. In contrast to the other genes studied in the male, such as SF-1 and SOX9, the SRY signal was intense from the first stage of detection, although here also gonads and mesonephros were not separated; consequently, the level of SRY expression at this stage is likely to be elevated. No SRY transcripts were detected in other male embryonic tissue at this stage (Fig. 3 presents mesonephros as an example; and data not shown).



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  		FIG. 4. Time course of Sry expression in developing pig male gonads. Autoradiogram of RT-PCR products, obtained from fetal and adult male gonads. Complementary DNA at various stages was submitted to 30 cycles of amplification, then blotted and hybridized with an internal {gamma}-32P-labeled oligonucleotide. Presence and absence of reverse transcriptase is indicated by (+) and (-), respectively.

In both sexes, all the other genes were expressed clearly except for AMH (Figs. 1 and 2).

Formation of Testicular Cords (26–28 dpc)

The pig gonad became morphologically recognizable as a testis at 26 dpc. The cords then grew in length, and the Leydig cells appeared in the interstitium at 28 dpc. At the same time, the earliest signs of differentiation became apparent in the male tunica albuginea.

From 28 dpc, gonads were tested separately and pooled, 6–10 per assay, because of their small size. At this stage, all genes analyzed in males were expressed strongly—SRY, SOX9, SF-1, and WT-1—except for AMH, whose expression had just begun (Fig. 1). The signal obtained for SRY with the gonad alone was no stronger than signals from earlier stages, when the gonadal transcripts were diluted into the mesonephros (Fig. 4). The opposite phenomenon was observed in the expression of SOX9 and SF-1, whose signals were stronger when the gonads were tested separately (Fig. 1).

A strong expression of gonadotropin receptors was observed at 28 dpc, corresponding to the differentiation of Sertoli and Leydig cells (Fig. 5A). Different PCR products were detected, revealing the existence of a differential splicing process for the FSH receptor (rFSH). This was confirmed by sequencing the amplified bands (data not shown), which corresponded to exons 2–3 (767 bp), exons 2–4 (692 bp), and exons 2–5 (617 bp). The presence of mRNA corresponding to the cytochrome P450 aromatase gene expression was also detected clearly in male gonads at 28 dpc (Fig. 5A).



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  		FIG. 5. Expression pattern of gonadotropin receptors, aromatase, and WT-1 mRNA in pig fetal gonads. Ethidium bromide-stained agarose gels showing RT-PCR products obtained from male (A) and female (B) gonads at 23, 25, 28, 32, and 45 dpc.

Differentiation of the Ovary (35–45 dpc)

The female gonad remained histologically undifferentiated until 28 dpc, and final histological differentiation of the ovary was completed by 44 dpc. Meiosis was first observed at 42 dpc. The level of SOX9 expression decreased markedly in the fetal ovary after 28 dpc and remained weak until 60 dpc (Fig. 2). At 45 dpc, competitive RT-PCR was performed on RNA from male and female gonads. The values obtained were 950 000 and 150 000 cDNA molecules for male and female gonads, respectively, showing that SOX9 expression in the male was six times that in the female (Fig. 6, A and B). During this period, SF-1 and WT-1 transcripts were present in the fetal ovary, while AMH transcripts had not been detected (Fig. 2). The pattern of expression for the LH receptor (rLH) was similar to the pattern in the male (Fig. 5B). In contrast, a weak signal was first detected for the rFSH from 45 dpc, and no transcription was detectable for aromatase until 70 dpc (Fig. 5B and data not shown).



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  		FIG. 2. Developmental expression of SOX9, SF-1, WT-1, and AMH genes during female gonadal differentiation in the pig. The same procedure as described in Figure 1 was used to test female gonads.



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  		FIG. 6. Quantitative RT-PCR for SOX9 gene. A) Competitive RT-PCR. In two separate experiments, different amounts of competitor were added to a constant quantity of male and female gonadal cDNA (45 dpc). In the male reaction, 5 x 105 and 2, 4, and 16 x 106 molecules were added; in the female reaction 2, 10, and 20 x 104 molecules were used. Presence or absence of reverse transcriptase is indicated by (+) and (-), respectively. After PCR amplification, all samples were electrophoresed, transferred to a membrane, and hybridized with an internal oligonucleotide. B) Logarithmic plot of competitive RT-PCR. Samples shown in A were measured using ImageQuant software after detection using a PhosphorImager, then computed and plotted. The two points of equivalence are indicated by arrows. Open circles: male gonads; closed circles: female gonads.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Time Course of SRY Expression in the Pig

In this study, we found specific RT-PCR products that corresponded to the SRY gene from 23 to 52 dpc; however, after 35 dpc, the signal was weaker. In developing pig embryos, the gonadal ridge first appears at 21 dpc [33]. At the age of 23 dpc, when SRY was first detected, male and female gonads were structurally similar. The histological differentiation of the testis was observed only at the age of 26 dpc through the onset of cord formation [34]; then the Leydig cells appeared in the interstitium at 28 dpc [35]. Until 52 dpc, Sertoli cells changed in form and in number; but from then on they were uniform, and from 60 dpc the organization of the sex cords remained stable until term [36]. In addition, no extragonadal expression of the SRY gene was observed in male porcine fetuses.

Daneau et al. [37] have previously shown that the porcine SRY gene (pSry) is expressed in the genital ridge over several days, including e21 and e23, with a faint expression on e26 and a total absence by e31. These days were numbered from the day of fecundation: 1 or 2 days after the first day of estrus (our Day 0). This difference may in part explain the contradiction between our results and those of this group. It is probable that e21 = 23 dpc and that our results concerning the first wave of pSry transcription do, in fact, agree with results from Daneau et al. [37]. In contrast, we found that pSry transcripts were again present after e31 (33 dpc). The discrepancy with our RT-PCR results is probably due to the different procedures used to obtain cDNA. We performed the RT reaction with random hexamers and consequently produced large quantities of cDNA corresponding to all the different forms of pSry mRNA. In contrast, Daneau and colleagues used specific primers that produced only one type of cDNA at very low yields.

Apart from studies in the pig, relatively little work has been published on the subject of the ontogeny of SRY expression in mammals. To date, the mouse and the sheep have been the only other species in which the Sry genital ridge transcripts and their patterns of expression have been described throughout testicular development [4, 6]. Sheep Sry expression was detected from primitive undifferentiated gonad formation (23 dpc) to 44 dpc; after that, the Sertoli cells had been differentiated and organized into seminiferous tubules secreting AMH (30 dpc). In contrast, mouse Sry expression was detected over a short period (10.5–12 dpc) that corresponded to the start of Sertoli cell differentiation [4]. The expression pattern observed here in the pig is closer to the pattern described in sheep, suggesting either that the role of SRY in these species is not limited to the first steps of Sertoli cell differentiation or that SRY may be also involved in the differentiation of another cellular type in the gonad. It was shown that Sry in mice is expressed in the somatic cells of fetal gonads (Sertoli cells) and in the germ cell line in the adult testis [4, 38, 39]. In contrast, recent work in humans shows that SRY protein is present in embryonic and adult Sertoli cells and germ cells [40]. As in humans, porcine SRY transcripts may originate from both cell types. This may be also explain faint signals observed from 45 dpc until birth.

After birth, pSry was strongly reexpressed at 35 days postpartum prior to puberty, then weakly in mature adult testis. These results were similar to those observed in sheep [6]. In the adult, SRY transcripts and protein have been described in germ cells in mice and humans [39–41]; and we assume that, in pigs, the SRY expression observed in mature testis was also due to the germ cell line—the signal being, in that case, very diluted within the somatic component of the gonad.

Expression of Other Genes Involved in Gonadal Differentiation

SOX9 The gene SOX9 has been reported as a possible key gene in testis differentiation, in not only mammals but also birds [10, 11]. In mammals, SRY could directly up-regulate its expression, whereas in birds, other factors may be involved to ensure male-specific expression. Porcine SOX9 expression during embryonic and fetal development begins at 21 dpc, before SRY and RT-PCR products were obtained repeatedly from 23 dpc in both sexes. As in mice [10], it increased in the testis, especially when the gonad was tested alone from 28 dpc. In the female, expression was intense as early as 21 dpc and mainly resulted from the mesonephros; it then decreased from 28 dpc when the gonad was separately analyzed. From 32 dpc, SOX9 expression was very weak in the fetal ovary. As previously described in mice [11], porcine SOX9 was down-regulated in XX gonads and up-regulated in XY from 28 dpc. This was demonstrated by quantitative RT-PCR data at 45 dpc, when a six-fold difference was observed between expression in males and females. The up-regulation of the SOX9 gene was concomitant with the appearance of differentiated Sertoli cells, since these started to produce cellular-specific factors, such as AMH and FSH receptors, at this time (28 dpc). Our results corroborate the recent study of De Santa Barbara et al. [19], in which an implication of SOX9 in the regulation of AMH gene transcription is demonstrated in vitro. In a partial study beginning once the sex had been determined in the pig, Pilon et al. [42] failed to detect porcine SOX9 transcripts at ew6 (around 45 dpc using our numbering system) by Northern blot analysis. This difference is probably attributable to the lack of sensitivity of the technique used. The investigators found a high SOX9 expression in the testis at ew8 and a detectable but basal expression in the fetal ovaries—findings that agree with our RT-PCR results.

SF-1, AMH, and aromatase SF-1,also known as Ad4BP, was first identified as an orphan receptor that regulates cytochrome P450 steroid hydroxylase gene expression [14–16]. By targeting disruption of the mammalian FTZ-F1 locus, it was established that SF-1 played a major role at all levels of the hypothalamic-pituitary-gonadal axis [16]. It is expressed in all the primary steroidogenic tissues, including the adrenal cortex, testicular Leydig cells, and the theca and granulosa cells and corpus luteum in the ovary [15, 43]. SF-1 mRNA expression in male porcine gonads started at 23 dpc, as did that of SRY, reaching a maximum at 32 dpc and remaining high until 60 dpc. Similar results were observed by Pilon et al. [42] in male gonads at 6–12 wk of gestation. In the mouse, SF-1 transcripts were first detected on embryonic Day 9 (e9) in the urogenital ridge, and the expression was sexually dimorphic by e14.5, with high levels of SF-1 in the testes and trace levels of detectable protein in the ovaries [43]. In the pig, differences in expression levels probably exist between males and females but were observed late, at 45–52 dpc. In male pig fetuses, Leydig cells appeared by Day 28, and testosterone concentrations increased from Day 30, reaching a maximum on Day 35 [44]. After this maximal differentiation stage, Leydig cells underwent involution and decreased in number.

Recently, it has been shown that SF-1 is also involved in regulating AMH and AMH receptor expression and is consequently expressed in Sertoli cells and cells surrounding the Müllerian ducts [18, 19, 45]. The transcripts that we detected by RT-PCR could therefore have originated from both Leydig and Sertoli cells; these two cellular types differentiate around 28 dpc. In situ hybridization or immunohistochemical studies will be necessary to answer the question of origin and reveal a possible sexually dimorphic expression of SF-1 in the pig.

AMH is considered the first functional marker of fetal Sertoli cells. Tran et al. [46] found that, in the pig, the hormone is detectable from 27–28 dpc. Our results agreed with these data, since we detected AMH transcripts from 28 dpc in male gonads; this confirmed that at 28 dpc, Sertoli cell differentiation had taken place. Thereafter, the level of transcription gradually increased in the testes until birth. In contrast, the content of AMH mRNA increased in the ovaries of neonatal animals from birth (unpublished results).

The detection of P450 aromatase transcripts in male gonads from 28 dpc was surprising, since during this period androgens are essential to masculinize the internal genital tract and external genitalia. High levels of estrogen secretion, however, are characteristic of steroidogenesis in the pig, in both the newborn and the adult male [47]. Among the species studied, the pig appears to be unique with respect to P450 aromatase expression. Specifically, there are at least two isoforms of porcine P450 aromatase; one is expressed in the placenta and the other in the ovary [32]. The ovarian isoform appears to be less active in estrogen synthesis than the placental isoenzyme. Conley et al. [48] showed that newborn porcine testes express the same isoform as expressed in the ovary. During fetal life, estrogen secretion was first detected in male fetuses at Day 31 of gestation [47]. This was consistent with the presence of P450 aromatase transcript at 28 dpc described in our study. The principal form of estrogen secreted by the porcine fetal testes appeared to be estrone sulfate (E1S), as in early postnatal [47, 49] and adult life [50]. The functional significance of estrogen synthesis during testicular development remains unclear, but it was reported in newborn rats that estradiol induced gonocyte proliferation in vitro [51]. Likewise, it was shown in a recent study by van Pelt et al. [52], using in situ hybridization and immunohistochemistry, that in fetal testes of rats at 16 dpc and in testes of 4-day-old animals, fetal germ cells (gonocytes) contained the estrogen receptor-beta mRNA in their cytoplasm and the estrogen receptor-beta protein in their nucleus. Consequently, the cellular target of estrogens within the testis during fetal life could be the germ cells. Furthermore, targeted disruption of the estrogen receptor-alpha gene in mice has shown that estrogen action is required for fertility in the male [53].

LH and FSH Receptors

In the pig, rLH and rFSH mRNA were clearly detected in males as early as Day 28 of gestation. Previous data indicated two relative maxima of rLH in males, one between Days 35 and 56 postcoitum [54] and the other from 3 to 10 wk of postnatal life [55]. Testicular concentration of rFSH has been also reported as being transiently elevated at 35–56 dpc [54]. In the female, we detected first transcripts of rLH and rFSH at 25 and 45 dpc, respectively. Goxe et al. [54] previously found first positive hybridization signals in fetal pig ovaries at Day 56 for rLH and at Day 90 for rFSH using RNA dot-blot experiments. The use of the sensitive RT-PCR technique in our study enabled us to show that gonadotropin receptor transcription began much earlier. It is noteworthy that gonadal transcripts for LH/FSH receptors are more abundant in the male than in the female throughout gestation [54]. These receptors were synthetized in the gonads in a sexually dimorphic manner, especially for FSH, even in the absence of hormone secretion.

The pig fetal pituitary produces LH from 45 dpc and FSH at 60 dpc [56]. The physiological role of these receptors during the first 50 days of gestation is therefore questionable, since there are undetectable circulating gonadotropin hormones. It seems that shortly after sex determination, Leydig cells differentiate and simultaneously acquire functional gonadotropin receptors and the capacity to secrete testosterone. For Sertoli cells, rFSH are an early marker of cellular differentiation, like AMH.

In conclusion, during porcine XY genital ridge development, a high level of pSry was detected from indifferent gonad formation (22–23 dpc), before the morphological differentiation of Sertoli cells (26 dpc) and the induction of Sertoli cell-specific gene expression (28 dpc), such as that of AMH and rFSH. In contrast to observations in mice, however, pSry gene expression was strongly maintained until 35 dpc, and more weakly until 52 dpc.

A key stage seems to be 28 dpc, since at this stage a male/female switch occurs for SOX9 and Dax-1 genes [57]. This switch was associated with Sertoli cell-specific expression of genes like AMH and rFSH. Simultaneously, Leydig cell differentiation also takes place (presence of rLH and testosterone production). Further investigations will be needed to determine the cellular localization of the transcripts or of the proteins corresponding to SF-1 and aromatase genes. For this reason, immunohistological studies are in progress using specific antibodies to these two proteins. This developmental analysis of normal expression profiles during sex determination in pigs will be also useful for characterizing at a molecular level the sex-reversed gonads of intersex fetuses and determining the first signs of the pathology.


   ACKNOWLEDGMENTS
 
We thank C. Bourgeois and J.M. Gogué for providing pregnant sows and N. Servel for technical assistance. We are also grateful to G.F. Greppi for his encouragement.


   FOOTNOTES
 
1 Grant support: INRA A.I.P: Sex autosomal determination in goats and pigs. P.P. was supported by a Ph.D. fellowship from the Italian Ministry of Research. Back

2 Correspondence. FAX: 33 1 34 65 22 41; pailhoux{at}biotec.jouy.inra.fr Back

3 Current address: Università di Pavia, Dipartimento di Patologia Umana ed Ereditaria, Sezione Biologia Generale e Genetica Medica, Via Forlanini, 14, 27100 Pavia, Italia. Back

Accepted: April 26, 1999.

Received: December 4, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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