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Endocrinology Vol. 139, No. 9 3803-3812
Copyright © 1998 by The Endocrine Society

ARTICLES

Porcine Steroidogenic Factor-1 Gene (pSF-1) Expression and Analysis of Embryonic Pig Gonads during Sexual Differentiation1

Nicolas Pilon, Ramin Behdjani, Isabelle Daneau, Jacques G. Lussier and David W. Silversides

Centre de Recherche en Reproduction Animale, Department of Veterinary Biomedicine, Faculty of Veterinary Medicine, University of Montréal, St.-Hyacinthe, Québec, Canada J2S 7C6

Address all correspondence and requests for reprints to: Dr. David W. Silversides, CRRA, Faculty of Veterinary Medicine, University of Montréal, St.-Hyacinthe, Québec, Canada J2S 7C6. E-mail: silverdw{at}medvet.umontreal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The porcine steroidogenic factor-1 gene (pSF-1) was cloned using a combination of genomic and RT-PCR based cloning methods. pSF-1 consists of an open reading frame of 1383 nt corresponding to a deduced amino acid sequence of 461 aa, similar to bovine and human SF-1. Sequence homologies between pSF-1 and human, bovine and mouse molecules indicate strong evolutionary conservation at both the nt and aa levels. Northern analysis of pSF-1 expression in adult steroidogenic tissues correlated with porcine steroidogenic acute regulatory protein gene (pStAR) and porcine side chain cleavage (pP450scc) gene expression. Notably, pSF-1 expression was readily detected in neonatal testes, absent at 3 weeks of age, and again readily detected at 3 months and in adult testes. pSF-1 expression was weak but detectable in placental tissues at various times of gestation, and was correlated with pStAR and pP450scc expression, indicating classical steroidogenesis in this organ. In developing gonads from 6–12 weeks of gestation, i.e. during the time of sex differentiation in the pig, Northern analysis demonstrated increasing expression of pSF-1 in fetal testes and no expression in ovaries. This expression pattern was paralleled for pStAR, pP450scc, and porcine Müllerian inhibitory substance (pMIS), consistent with pSF-1 involvement in both steroid and protein hormone secretions of the developing testes during sex differentiation. Porcine SRY HMG-box related gene-9 (pSOX-9) expression also paralleled that of pSF-1 in developing testes. In contrast, DSS-AHC critical region on the X chromosome, gene 1 (pDAX-1) was expressed predominantly in the developing ovaries, indicating a possible reciprocal regulation of pSF-1 and pDAX-1 genes in developing pig testes and ovaries.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN EUTHERIAN mammals, development of sex phenotype involves differential gene and hormonal expression between developing male and female fetuses and can be divided into the processes of sex determination and sex differentiation (1, 2, 3). Sex determination represents the step whereby indifferent gonads will develop either as testes or as ovaries. In the male embryo this involves expression of SRY (sex-determining region of the Y chromosome), located on the mammalian Y chromosome. SRY is responsible for initiating testis formation and thus indirectly for expression of the male phenotype (4, 5, 6, 7). The female gonad, in the absence of SRY expression, develops as an ovary. Factors that control the expression of SRY as well as those controlled by SRY are not known at present. Recently, genes that display dosage dependency have been implicated in sex determination, including DAX-1 (DSS-AHC critical region on the X chromosome, gene 1) (8, 9) and SOX-9 (SRY HMG box related, gene 9) (10, 11). In addition, the genes WT-1 (Wilm’s tumor gene 1) (12) and SF-1 (steroidogenic factor-1) (13) were implicated in the process of gonad formation and shown to be expressed in the primitive gonads even before SRY expression.

Sexual differentiation is the developmental period where the committed gonad (testis or ovary) regulates the development of internal and external phenotypic reproductive structures. This process is mediated by hormonal secretions from the gonads and, for the male, this includes the production of MIS (Müllerian inhibiting substance) (14, 15) as well as testosterone. MIS is a paracrine protein hormone, produced by Sertoli cells, responsible for the regression of the Müllerian duct, which would otherwise generate the internal female reproductive structures, including oviduct, uterus, cervix, and upper vagina. The sex steroid testosterone, produced by Leydig cells, is required for the retention and development of male reproductive structures, including vas deferens, epididymus, and seminal vesicles. SF-1, first described as a trans-acting factor for steroidogenesis in the gonads and adrenal gland, displays male specific gonadal expression in the mouse throughout the period of sexual differentiation (16). Transcription of MIS may be controlled by SF-1 as the recognition sequence for SF-1 is present in the promoter regions of MIS (17) and is necessary for normal promoter function in vivo (18). Furthermore, SF-1 binding sites are found in the promoter regions of DAX-1 (19), the steroidogenic hydroxylase enzyme genes (20, 21) and in the promoter of the StAR protein (steroidogenic acute regulatory protein) gene (22, 23). StAR codes for a protein implicated in the delivery of cholesterol through mitochondrial membranes to the cytochrome P450 side-chain cleavage enzyme (P450SCC) in the initial enzymatic steps of steroidogenesis. It is now apparent that SF-1 is implicated in the control of reproductive functions at multiple levels of the reproductive axis (24) including sex determination (19), sex differentiation, gonadotropin hormone production (25), and steroidogenesis (reviewed in Ref. 26).

Most current studies of mammalian sex determination and differentiation use the human or mouse as model, although recent studies in the sheep are now described (27). The domestic pig was chosen as a model for these studies because of access to fetal tissues, the fact that the pig is a polytocous animal, and the relative sequence homologies observed between pig and human SRY (28). The pig has a gestation length of 113–115 days (~16 weeks); histological testes determination occurs at about embryonic day 25 (e25) (29), with SRY expression occurring at least by e21 days (28). Overt sexual differentiation begins between 28–35 days (30 and personal observations), and continues throughout the remainder of gestation.

We now describe coding sequences of porcine SF-1 as well as SF-1 expression studies performed on porcine developing gonads during the time of sexual differentiation and in postnatal and adult tissues. Expression of SF-1 during sex differentiation is compared with the expression of additional porcine genes implicated in sex determination and/or differentiation including StAR, P450scc., MIS, SOX-9 and DAX-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of coding sequences for porcine SF-1
Partial genomic sequences for the porcine SF-1 gene were initially derived via genomic PCR cloning to provide homologous sequences for subsequent complementary DNA (cDNA) cloning and eventual screening of a pig genomic library (see below). Heterologous sense (pSF-1.A: 5'-TCTACGACGAGGACCTGGACGA-3'; pSF-1.C: 5'-AACAAGTTTGGGCCCATGTACAAG-3'; and pSF-1.E: 5'-GAAGTTTCTGAATAACCACAGCCT-3') and antisense (pSF-1.1: 5'-TCAAGTCTGCTTGGCCTGCAGCAT-3'; pSF-1.3: 5'-CAGCTCCTTGAAGACCATGCAC-3'; and pSF-1.5: 5'-ACCGTCAGGCACTTCTGGAAGC-3') primers were designed based on bovine (31) and mouse (32) sequences to amplify three genomic fragments including pSF-1.A5, spanning exons 1 and 2; pSF-1.C3, in exon 3; and pSF-1.E1, in exon 6. PCR cycling was performed on 0.5 µg of male genomic pig DNA using 2.5 U Taq DNA polymerase (Amplitaq, Perkin-Elmer/Cetus, Mississauga, Ontario). Thermal cycling included 40 cycles of 45 sec denaturation at 95 C, 45 sec annealing at 65 C and 2 min elongation at 72 C. Products of amplifications were size-separated on a 1% agarose gel and fragments corresponding to the sizes expected were excised from the gel, purified, and cloned into the PCRII plasmid vector (Invitrogen). Clones were sequenced via the dideoxy method (T7-sequencing kit, Pharmacia Biotech, Uppsala, Sweden) and confirmed to be porcine SF-1 genomic sequences via homologies to SF-1 sequences in the mouse and cow.

A second round of amplifications were performed to generate complete coding sequences of porcine SF-1. Sense primers (heterologous pSF-1.A; and pSF-1.F: 5'-TGCCTGCAGGAAC-CAGCCAAAGG-3') and antisense primers (pSF-1.5'2: 5'-AATCTGTGCCTTCTTTTGCTGCT-3'; and heterologous pSF-1.1) were used to perform RT-PCR using adult pig ovarian polyadenylated [poly(A)+] RNA to generate cDNA clones pSF-1.A5'2 and pSF-1.F1. Total RNA was recovered via the acid phenol method (33) and poly(A)+ RNA was isolated using magnetic poly dT beads (Magnetobeads, Dynal Corp., Great Neck, NY), as previously described (34). The RT reaction was perform using 2 µg of poly(A)+ RNA using a poly dT (deoxythymidine) adapter primer, AD/T7T17 (5'-GGATCCAAGCTTGAATTCTATACGACTCACTATAGGGTTTTT-TTTTTTTTTTTT-3') and reverse transcriptase enzyme according to the supplier’s instructions (Superscript RT, Life Technologies, Grand Island, NY). Of the 30 µl total volume from the first strand cDNA synthesis reaction, 2 µl were used to perform PCR reactions of 40 cycles using primers pSF-1.A and pSF-1.5'2 or primers pSF-1.F and pSF-1.1 under the following cycling conditions: 45 sec denaturation at 95 C, 45 sec annealing at 65 C, and 2 min elongation at 72 C. Fragments of anticipated size were recovered from a 1% agarose gel, cloned into PCRII plasmid vector (Invitrogen) and sequenced.

Lambda genomic library screening
Using the fragment pSF-1.A5'2 for targeting the 5' flanking genomic sequences and the fragment pSF-1.E1 for targeting the 3' flanking genomic sequences, radioactive double stranded probes were generated using the random hexamer priming method (Quick Prime, Pharmacia) and dCTP[{alpha}-32P] (DuPont, Wilmington, DE). Hybridization screening was performed on a commercial pig genomic library in lambda phage (Clontech, Palo Alto, CA), using standard methods. After four rounds of screening, one clone for each probe was isolated. A SacI restriction digest of the phage insert was subcloned into the plasmid vector pCR-Script (Stratagene, La Jolla, CA). Upon further characterization and sequencing, these subclones provided additional 5' and 3' flanking genomic sequences to the porcine SF-1 coding sequences generated.

Generation of probes for Northern analysis
All the porcine probes used for Northern analysis were generated in our labs. The probe for pSF-1 was based on the pSF-1.C3 fragment (591 bp) described above or on the fragment pSF-1.A.5'2 (331 bp, using primer pSF-1.A, as mentioned above, and primer pSF-1.5'2, as follows: 5'-CGAATCTGTGCCTTCTTTTGCTGCT-3'). The probes for pStAR (pStAR.ORF; 855 bp) and pP450SCC (pSCC.A1; 518 bp) were as already described (35). For pDAX-1 (GenBank accession no. AF019044), pMIS (GenBank accession no. U80853) and pSOX-9 (GenBank accession no. AF029696), the primers used for RT-PCR were the following: for pDAX-1 (fragment of 584 bp), pDAX-1.E: 5'-GTCAAGTACTTGCCCTGCTTCCAG-3' and pDAX-1.2: 5'-CAGCATCATATCATCCATGCTGAC-3'; for pMIS (fragment of 503 bp), pMIS.B: 5'-CTATG-AGCAGGCCTTCCTGG-3' and pMIS.3: 5'-GTCATCCGTGTGAAGCAGC-3'; and for pSOX-9 (fragment of 823bp) pSOX-9.A: 5'-CGTATGAATCTCCTGGACCCCTT-3' and pSOX-9.4: 5'-ATGTCCACGTCGCGGAAGTCGAT-3'. PCR fragments were subcloned into the plasmid vector pGEM-T (Promega) for use in generating radioactive (dCTP[{alpha}-32P]) double-stranded probes.

Northern analysis for pSF-1, pMIS, pStAR, and pP450SCC during sexual differentiation and in adult tissues
To study the developmental expression of porcine SF-1 as well as porcine MIS, StAR and P450SCC during gonadal sex differentiation, fetal ovaries, and testes were dissected from fetuses taken postmortem from pregnant sows at a local abattoir. Gestational age was estimated by measuring the crown-rump length (30) to generate the following samples (in gestational weeks): 6, 6.5, 8, 9, 10, 11, 12, and 13. Reproductive and/or steroidogenic adult tissues were also taken, including adrenal gland, placenta, corpus luteum, and adult ovaries as well as adolescent (~3 months) and adult testes. Perinatal pig testes (4–5 days) and testes of young pigs (3 weeks old) were supplied by a local pig producer. Total RNA was extracted via pelleting over a cesium chloride gradient (36). Approximately 10 µg of total RNA for each tissue (20 µg for placenta) was size-fractionated on a 1% formaldehyde-1% agarose gel and transferred via capillarity to a nylon membrane (Hybond-N, Amersham Corp., Arlington Heights, IL). The membrane was stained with methylene blue (37) for estimating the relative RNA loaded per well and the gel image was digitized (Foto/Eclipse, Fotodyne). Membranes were hybridized with radioactive probes overnight at 65 C, then washed at high stringency and exposed overnight at -70 C to a photographic film (Biomax, Eastman Kodak, Rochester, NY).

Northern analysis for pSOX-9 and pDAX-1 during sexual differentiation
Since Northern blot analysis using total RNA was not sensitive enough to detect transcripts for pSOX-9 or pDAX-1 within the developing gonads, an enrichment of poly (A)+ RNA was necessary. This was accomplished using magnetic beads, as described above, on 150 µg of total RNA to give an estimated equivalent of 5 µg of poly (A)+ RNA. Electrophoresis, transfer to nylon membranes, and hybridizations with double-stranded cDNA probes were performed as described above. Membranes were exposed to photographic film for 1 week.

Nucleotide and amino acid comparisons
Comparisons of nucleotide and deduced amino acid sequences were performed via a Lipman and Pearson type olgarithm (38) using MacDNASIS software (Hitachi, Hialeah, FL)


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The nucleic acid coding and deduced amino acid sequences for porcine SF-1 cDNA are shown in Fig. 1Go, along with 840 bp of 5' flanking genomic sequences and 198 bp of 3' flanking genomic sequences. The sites of intron insertions are noted, based on our work (not shown) and by homology with other species. The porcine SF-1 cDNA consists of an open reading frame (ORF) of 1383 bp corresponding to a protein of 461 aa. Percent homologies of porcine SF-1 cDNA at the nt and deduced aa levels (in parentheses) with SF-1 cDNA of other species are as follow: human, 91% (93%); bovine, 93% (96%); and mouse, 88% (92%). Comparisons of porcine SF-1 deduced aa structure with human, bovine and mouse SF-1 proteins are shown in Fig. 2Go.



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  		Figure 1. cDNA sequence, deduced amino acid sequence for porcine SF-1 as well as 5' and 3' flanking genomic sequences. The amino acid sequence appears below the nucleic acid sequence. The first A of the ATG start codon represents nucleotide 1, whereas the corresponding Met represents amino acid 1. The stop codon at nucleotide 856 is indicated by asterisks. Within the 5' flanking genomic sequences, which correspond to intron 1, in-frame stop codons are underlined. Sites of intron insertions, based on this work and homologies to the mouse and human genes, are indicated by vertical lines with solid triangles.

 


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  		Figure 2. Deduced amino acid comparisons of porcine SF-1. The porcine sequences are compared with the deduced amino acid sequences of the cow, human, and mouse SF-1. The porcine sequences as well as those of the cow and human are comprised of 461 amino acids whereas the mouse sequences have 462 amino acids. To aid comparisons, functional motifs as described by Wong et al. (39 ) are indicated, including zinc finger 1 (ZF-1), zinc finger 2 (ZF-2), the FTZ-F1 box, the proline stretch, regions II and III, and the AF-2 motif.

 
Northern analysis for pSF-1, as well as pStAR and pP450scc, were performed on 10 µg of total RNA from selected postnatal and adult reproductive and/or steroidogenic tissues and are shown in Fig. 3Go. For pSF-1 as well as for pStAR and pP450scc, transcripts were detected in all the postnatal and adult tissues tested except in testes of 3-week-old piglet, which was very weakly positive for pP450scc. In these studies, two transcripts were seen for pSF-1, including a major transcript of 3.3 kb and an inconsistent minor transcript of 2.9 kb seen in ovarian tissues. Transcripts for pStAR and pP450scc were as previously described (35) with an additional band for pStAR observed at 2.9 kb.



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  		Figure 3. Porcine SF-1, StAR, and P450scc gene expression in postnatal and adult reproductive and/or steroidogenic tissues via Northern blot analysis. Ten micrograms of total RNA was loaded per lane, with sample loading indicated by the 28S RNA band. Tissues studied as follows: corpus luteum at 6, 9, and 12 weeks of gestation, indicated as CL e6w, CL e9w and CL e12w, respectively; adult ovary; newborn testes; testes at 3 weeks (3w) of age; testes at 3 months (3 m) of age; adult testes; and adult adrenal gland. To avoid stripping and reprobing of membranes, separate membranes were generated for each probe. A, The probe is pSF-1.A5'2; hybridizing bands at 3.3 and 2.9 kb are marked. Membrane was exposed to film for 10 days. B, The probe is porcine StAR cDNA; hybridizing bands at 2.9, 1.7, and 1.4 kb are marked. Membrane was exposed to film for 4 h. C, The probe is porcine P450scc cDNA; the hybridizing band at 2.0 kb is marked. Membrane was exposed to film for 4 h.

 
To address the question of steroidogenesis and SF-1 expression in the pig placenta, Northern analysis was performed on RNA isolated from placental tissue taken at various times of gestation. The hybridization pattern from 10 µg total RNA (for pSF-1 probe) and 20 µg of total RNA (for pStAR and pP450scc probes) are depicted in Fig. 4Go.



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  		Figure 4. Porcine SF-1, StAR, and P450scc gene expression in placental tissues at various times during gestation via Northern blot analysis. Placental samples were taken at 6 weeks (e6w), 9 weeks (e9w), and 12 weeks (e6w) of gestation. To avoid stripping and reprobing of membranes, separate membranes were generated for each probe. A, The probe is porcine pSF-1.A5'2; the hybridizing band at 3.3 kb is marked. Sample loading (10 µg total RNA) is indicated by the 28S RNA band. Membrane was exposed to film for 3 weeks. B, The probe is porcine StAR cDNA: hybridizing bands at 2.9, 1.7, and 1.4 are marked. Sample loading Sample loading (20 µg total RNA) is indicated by the 28S RNA band. Membrane was exposed to film overnight. C, The probe is porcine P450scc cDNA; the hybridizing band at 2.0 kb is marked. Sample loading (20 µg total RNA) is indicated by the 28S RNA band. Membrane was exposed to film for 6 h.

 
Northern blot hybridization was also performed on total RNA from developing gonads during the time of sex differentiation. In addition to pSF-1, these studies included pStAR and pP450scc (35) as well as for pMIS, pSOX-9, and pDAX-1. Figure 5Go depicts the expression profiles for pSF-1, pStAR, pP450scc and pMIS in the developing gonads between 6 and 12 weeks, i.e. during the period of sexual differentiation, using 10 µg of total RNA. In the developing testes, increasing expression is seen to about 9 weeks gestation; in contrast, there is a complete absence of expression of these in the developing ovaries at this level of sensitivity. For pSF-1, a major transcript of 2.7 kb and minor transcripts of 1.7 and 1.3 kb were seen. A single transcript of 2.1 kb was seen for pMIS. As expression for pSOX-9 and pDAX-1 was not detected at this level of sensitivity, Northern blot analysis on 5 µg of poly(A)+ RNA from developing gonads was performed for pSOX-9 and pDAX-1 (Fig. 6Go). For pSOX-9, there is a weakly constant and basal expression in the developing ovaries, whereas there is a stronger expression in the developing testes. Two transcripts are detected for pSOX-9, a major transcript of 4.4 kb and a minor transcript of 3.6 kb. For pDAX-1, the hybridization profile displays a constant and basal expression in the developing testes, whereas there is a stronger expression in the developing ovaries. A unique transcript of 1.4 kb is detected for pDAX-1.



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  		Figure 5. Porcine SF-1, StAR, P450scc, and MIS gene expression in fetal gonads during the period of sex differentiation via Northern blot analysis using 10 µg of total RNA. Sample loading (20 µg total RNA) is indicated by the 18S RNA band. For a given gestational time period, ovarian RNA is followed by testicular RNA; e6w refers to embryonic samples 6 weeks of gestational age, whereas e6.5w, e8w, e9w, e10w, e11w, and e12w refer to embryonic samples of 6.5, 8, 9, 10, 11, and 12 weeks gestational age, respectively. To avoid stripping and reprobing of membranes, separate membranes were generated for each probe. A, The probe is porcine pSF-1.C3; hybridizing bands at 2.7, 1.7, and 1.3 kb are marked. Membrane was exposed to film for 1 week. B, The probe is porcine StAR cDNA; hybridizing bands at 2.9, 1.7, and 1.4 kb are marked. Membrane was exposed to film 18 h. C, The probe is porcine P450scc cDNA; the hybridizing band at 2.0 kb is marked. Membrane was exposed to film for 18 h. D, The probe is porcine MIS cDNA; the hybridizing band at 2.1 kb is marked. Membrane was exposed to film for 1 week.

 


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  		Figure 6. Porcine SOX-9 and DAX-1 gene expression in fetal gonads during the time of sex differentiation via Northern blot analysis using 5 µg of poly (A)+ RNA. A, The probe is porcine SOX-9 cDNA; hybridizing bands at 4.4 and 3.6 kb are marked. Sample loading is indicated by the 18S RNA band. Membrane was exposed to film for 1 week. For a given gestational age, ovarian sample is followed by the corresponding testicular sample; e6w refers to samples taken at 6 weeks embryonic gestational age while e8w, e9w, e10w, e11w, and e12w refer to samples taken at 8, 9, 10, 11, and 12 weeks of embryonic gestational age, respectively. B, The probe is porcine DAX-1 cDNA; the hybridizing band at 1.4 kb is marked. Sample loading is indicated by the 18S RNA band. Membrane was exposed to film for 1 week. The loading of RNA is as follows: testes at 6 weeks (e6w), 6.5 weeks (e6.5w), 8 weeks (e8w), 10 weeks (e10w), 11 weeks (e11w), 12 weeks (e12w), and 13 weeks (e13w) of embryonic gestational age; ovaries at 6 weeks (e6w), 8 weeks (e8w), 10 weeks (e10w), and 12 weeks (e12w) embryonic gestational age.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Porcine SF-1 genomic and coding sequences
We have cloned porcine SF-1 coding and flanking genomic sequences and looked at its expression in postnatal and adult reproductive tissues as well as during the time of sex differentiation in the developing gonads. In addition, we have continued expression studies initiated for pStAR and pP450scc (35), and we have looked at the gonadal expression during sex differentiation of other genes involved in sex determination/differentiation including pMIS, pSOX-9, and pDAX-1.

Sequence comparisons of the porcine SF-1 cDNA with sequences of other species demonstrates general homology to previously described human, bovine, and mouse sequences (20, 31, 39), with structural motifs being particularly conserved (Fig. 2Go). Therefore, it appears that the SF-1 gene, like SOX-9 (11), MIS (40) and StAR (35) genes, is not undergoing rapid evolutionary changes as seen for SRY (41) and possibly for DAX-1 (42). By comparisons to mouse and human genomic sequences for SF-1 (32, 39, 43), it is most likely that the 5' flanking genomic sequences derived for porcine SF-1 and described in Fig. 1Go represent intron 1 sequences. This is due to the complicated genomic structure at the FTZ-F1 gene locus, which codes for SF-1: exon 1 for SF-1 is not expressed and is found about 3–4 kb upstream from exon 2, which contains the ATG initiating codon for SF-1 (32, 39, 43). Furthermore, the locus is transcriptionally complicated: exon 2 in the mouse is believed to contribute to four distinct transcripts including SF-1, ELP1, ELP2, and ELP3, via differential promoter usage and exon splicing (26). The human gene may involve fewer transcripts due to an in-frame stop codon located just 5' to the SF-1 initiation codon. In this respect, the pig sequences are more similar to the human sequences, as 3 in-frame stop codons proceed the initiation codon for porcine SF-1 (Fig. 1Go). Comparison of the porcine SF-1 promoter with the human and mouse promoters (44) and analysis of intron 1 sequences for potential SF-1 binding sites (45) will be the subjects of future studies.

Postnatal expression of porcine SF-1
The expression profile of pSF-1 in adult tissues parallels that of pStAR and pP450scc. Expression for these three genes can be easily detected in all steroidogenic adult tissues tested including placenta, corpus luteum (CL), testis, ovary, and adrenal gland. Because P450scc is the enzyme that catalyses the first enzymatic step of steroidogenesis, coexpression of pSF-1 and pStAR in these tissues suggests their implication in steroidogenesis. Whether SF-1 expression is absolutely required for steroidogenesis to occur may be tissue dependent (46). We have previously reported that StAR is expressed by the pig placenta (35). Now we demonstrate expression of SF-1, StAR, and P450scc at various times of gestation in the pig placenta, further suggesting that this organ makes use of the classical steroidogenic pathway and underlining the possibility that species differences in mechanisms of placental steroidogenesis can exist. Ovarian expression of SF-1 is well documented in the mouse (32) and human (47) and was found to be dependent on age and stage of reproductive cycle. We detect SF-1 expression in adult porcine ovarian tissues, although a systematic study of the expression of SF-1 in the pig ovary throughout the normal estrous cycle is now needed.

Strong hybridization signals for pSF-1, pStAR, or pP450scc were seen in perinatal, adolescent, and adult testes but were not detected in testes of 3-week-old piglets. This is consistent with findings for SF-1 expression in the neonatal rat testes (48) and correlates with the fact that testosterone is produced by the developing testes until shortly after birth and then not again until puberty.

Multiple and tissue specific transcripts for pSF-1
A survey of postnatal and prenatal tissues revealed differences in SF-1 hybridizing bands. A major band was seen at either 3.3 and 2.7 kb, whereas minor bands were variably seen at 2.9, 1.7, and 1.3 kb, depending on the particular tissue studied and the probe used. These multiple bands could be explained by multiple sites of polyadenylation, by differential splicing and maturation of the SF-1 RNA in a time and tissue dependent fashion, or by the genomic structure of the FTZ-F1 gene locus (26). Further investigations to distinguish between these possibilities are warranted.

Gonadal expression of porcine SF-1, StAR, P450scc, MIS, SOX-9, and DAX-1 during sex differentiation
Expression profiles for genes were performed on developing pig gonads during the time of sex differentiation, including SF-1 as well as genes involved in steroid mediated sex differentiation (StAR and P450scc), genes involved in protein hormone mediated sex differentiation (MIS), and genes implicated in sex determination (DAX-1 and SOX-9). During this period of time, transcripts for pSF-1, pStAR and pP450scc genes are readily detected and with increasing expression intensity in fetal testes. There is no detection of expression (at this level of sensitivity) in fetal ovaries. These results are in keeping with the fact that developing pig testes are steroidogenically active for the production of testosterone by the Leydig cells soon after sex determination and are consistent with results for SF-1 expression in the developing gonads of the mouse (16) and the rat (48).

For this particular period of the sexual development, we also looked at the expression of pMIS in the fetal gonads. MIS, a member of the TGF-ß family of protein hormones, is secreted by the developing Sertoli cells (14) and results in the regression of the Müllerian duct. pMIS transcripts were detectable by at least 6 weeks gestation in the developing pig testes, with increasing expression seen until about 9 weeks of gestation (Fig. 4DGo). There was no detection of pMIS in the fetal ovaries. In the sheep, MIS expression can be detected in testes as early as e30 days via RT-PCR (27). Interestingly, recognition sequences for SF-1 are seen in the promoter regions of MIS (17) as well as in the promoter region of pStAR and pP450scc (21, 23). As the transcripts of these four genes colocalize to the male porcine gonads during embryogenesis, our results are consistent with the hypothesis that SF-1 is involved in the regulation of both the protein and steroid hormonal components of sex differentiation in the pig.

The Y chromosome located SRY gene is considered the master genetic switch for testes determination (7). More recently, other genes that are not on the Y chromosome and that appear dosage sensitive (or dosage dependent) have been implicated in this process of sex determination; these include SOX-9 and DAX-1. Defects in SOX-9, an autosomal gene located on human chromosome 17, are associated with campomelic dysplasia (CD), a bone dysmorphology syndrome (10). In addition, a high proportion of CD patients who are karyotypically XY exhibit complete or partial male-to-female sex reversal (49). This suggests that the loss of a functional allele for SOX-9 can deregulate male sex determination and differentiation, although it is not yet known precisely how this gene contributes to the molecular pathway of testis development. During the period of sex differentiation in the pig, we observe a strong expression of SOX-9 in the developing pig testes by Northern analysis using poly(A)+ RNA, compared with a detectable but basal expression in the fetal ovaries. This latter observation is in apparent contradiction with in situ hybridization results from mouse fetal gonads (50). The presence and role (if any) of SOX-9 expression in fetal ovaries warrants further investigation.

The gene DAX-1 (8) derives from two genetic loci on the short arm of the X chromosome called AHC (adrenal hypoplasia congenita) and DSS (dosage-sensitive sex reversal). When the DSS locus is duplicated in XY individuals, it causes male-to-female sex reversal (9). Since this description, it has been shown that DAX-1 exhibits a widespread tissue distribution of expression (47); in fact, SF-1 and DAX-1 colocalize in multiple cell types, which has provided circumstantial evidence that their expression is interrelated (51). However, it should be noted that this colocalization of expression is not absolute (52). A putative recognition site for SF-1 is present in the promoter of human DAX-1 (19) and is also present in the porcine DAX-1 promoter (personal observations; GenBank accession no. AF019044). It has been suggested from in vitro data that DAX-1 may interact physically with and by so doing directly inhibit the action of SF-1 (53), although in vivo confirmation of such a direct physical interaction has not been obtained (54). Growing evidence suggests indirect antagonism of SF-1 and DAX-1: SF-1 binding sites are present in the StAR promoter and SF-1 enhances StAR promoter activity (23), whereas DAX-1 binds to the StAR promoter and inhibits StAR gene transcription (55). Our finding of reciprocal expression of SF-1 and DAX-1 genes in the developing porcine testes (which are steroidogenically active) and ovaries (which are not) supports an antagonistic role for these proteins in the control of steroidogenesis within these tissues. Further expression studies of SF-1 under a variety of physiological conditions, in relationship to the expression of DAX-1 as well as other factors including CBP/p300 and steroid receptor coactivator-1 (56, 57), are now needed to arrive at an integrated understanding of SF-1 function.


   Acknowledgments
 
The authors would like to thank Marie-France Allard for her technical assistance; André Hébert, Diane Hébert, Normand Hébert, and Claude Archambault for access to tissue samples; and Micheline Sicotte for help with the manuscript.


   Footnotes
 
1 This work was supported by Natural Sciences and Engineering Council of Canada (NSERC) and Conseil des recherches en peche en agro-alimentaire du Québec (CORPAQ) funding agencies. Sequences for porcine SF-1 were submitted to GenBank and given an accession number of U84399. Back

Received November 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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