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RESEARCH |
Unité de Physiologie de la Reproduction et des Comportements, UMR 6175 INRA-CNRS-Université F. Rabelais de Tours-Haras Nationaux, 37 380 Nouzilly, France
Correspondence should be addressed to Marie Saint-Dizier, Equipe Hypophyse, Station Physiologie de la Reproduction et du Comportement, Institut National de la Recherche Agronomique (INRA), 37 380 Nouzilly, France; Email: dizier{at}tours.inra.fr
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Abstract |
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In order to study the responsiveness of the primary corpus luteum (CL) and fetal gonads to eCG, the expression of eLH/CG-R mRNAs was examined by RT-PCR and Northern blot analysis during early and mid-pregnancy. All three eLH/CG-R cDNA isoforms (eLH/CG-RA,B,C) were expressed from day 14 to day 83 of pregnancy in the primary CL and from day 44 to day 222 in fetal gonads. Interestingly, the primary CL at days 89 and 151 expressed only truncated eLH/CG-R cDNA isoforms. The relative values of Northern hybridized major 7, 5.7, 3.9 and 1.8 kb eLH/CG-R mRNA transcripts tended to decrease in the primary CL whereas the unique major 1.8 kb eLH/CG-R mRNA was steadily expressed in fetal gonads during pregnancy. These results show that the expression of eLH/CG-R mRNAs occurs in the fetal gonads before ceasing in the primary CL and suggest that eCG may be involved in the gradual transition from a luteal to a feto-placental output of steroids during equine pregnancy.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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-reduced metabolites starting around day 70, but using maternal sources of cholesterol (Pashen & Allen 1979). Thus, a gradual transition from a luteal to a feto-placental output of steroids occurs, after which the maternal ovaries become small and completely inactive for the remainder of gestation (Holtan et al. 1979). There is convincing evidence that eCG is responsible for the resurgence and support of the primary CL during early pregnancy in the mare (Urwin & Allen 1982, Bergfelt et al. 1989, Daels et al. 1998). Furthermore, the primary CL has been shown to be responsive to eCG during the time of eCG secretion (Saint-Dizier et al. 2003). However, whether or not eCG plays a role in the initial development and steroid production of fetal gonads remains unknown. In pregnant mares and pony mares, the fetal hormone eCG, secreted by the endometrial cups, is first detectable in plasma on days 35–40 of gestation, rises rapidly to reach a peak between days 55 and 70, then decreases slowly to low or undetectable levels by days 120 to 150 of pregnancy (Ginther 1992a, Squires 1993). Whereas the endometrial cups act mainly as endocrine glands in the early stages of cup development, they also act from day 42 as exocrine glands since considerable amounts of eCG pour out of the uterine glands into the space between the cups and the allantochorion (Ginther 1992a). The large amounts of eCG secreted into the uterine lumen raise the possibility that this hormone enters the feto-placental circulation and stimulates the initial hypertropy and steroidogenesis of fetal gonads between days 60 and 120–150 of pregnancy. Nevertheless, it is still unknown whether the fetal gonads are responsive to eCG or to fetal pituitary LH, if present.
The protein structure of eCG is fully identical to that of equine LH (Bousfield et al. 1996) and both hormones bind to the putative LH/CG receptor in equine tissues (eLH/CG-R) (Stewart & Allen 1979 , 1981, Guillou & Combarnous 1983). We have previously reported the presence of several eLH/CG-R mRNA transcripts in the primary CL by Northern blot (Saint-Dizier et al. 2003). Some of this diversity in transcript size seemed to be due to differences in mRNA sequence since a probe for the trans-membrane domain only hybridized to a subset of these transcripts. However, the differences in eLH/CG-R mRNA splicing involved in this process remain unknown. Thus, the aims of this study were first to clone and sequence the full-length eLH/CG-R cDNA and secondly to examine the expression of eLH/CG-R mRNAs in the primary CL and in fetal gonads during early and mid-pregnancy.
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Materials and Methods |
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Sample collection and RNA isolation
Luteal tissues were obtained from one cyclic mare in dioestrus as control (day 8 post-ovulation) and from pregnant mares before the onset of eCG secretion (days 14, 15, 26, 27, 28 and 31; n = 1 for each day), during eCG secretion (days 42, 43, 44, 45, 46, 56, 60, 61, 62, 83 and 89; n = 1 for each day) and after the end of eCG secretion (day 151, n = 1). The ovaries were collected by hemiovariectomy or within 10 min following sodium pentobarbital-induced euthanasia as previously described (Saint-Dizier et al. 2003). Fetal gonads (both males and females) were collected following induced euthanasia or slaughter of the pregnant mares. The fetal gonads were obtained during eCG secretion (days 44, 45, 47, 62, 70, 81 and 101; n = 1 for each day) and after the end of eCG secretion (days 151, 202 and 222; n = 1 for each day). After dissection, pieces of tissue were immediately snap-frozen in liquid nitrogen then stored at –70 °C.
Total RNA was extracted from fragments of frozen tissue using Trizol reagent (Invitrogen Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The resulting RNA was quantified spectrophotometrically, aliquoted into smaller volumes, and stored at –80 °C.
Isolation and characterization of eLH/CG-R cDNA isoforms
Total cellular RNA (2 µg) from a pool of corpora lutea (at dioestrus and days 15, 42 and 61 of pregnancy) was reverse transcribed into first-strand cDNA using oligo(dT) primer and Rnase H-reverse transcriptase (Superscript II; Invitrogen Life Technologies, Carlsbad, CA, USA) in the presence of deoxynucleotides according to the manufacturer’s recommendations. A 646-base pair (bp) cDNA fragment encoding a part of the extracellular domain of the LH/CG-receptor was first generated using primer pair P1 and P2 based on cDNA sequences similar among the bovine (Lussier et al. 1996), porcine (Loosfelt et al. 1989), human (Minegishi et al. 1990) and murine (McFarland et al. 1989) LH/CG-Rs (Table 1
). First-strand cDNA was subjected to 30 cycles of PCR amplification using Advantage 2 polymerase mix (BD Biosciences, Palo Alto, CA, USA) and primer pair P1 and P2. Reaction times were 1 min denaturation at 95 °C for the first cycle and 15 s per cycle thereafter, 40 s annealing at 60 °C and 1 min extension at 72 °C for the first 29 cycles, and 10 min extension on the final cycle at 72 °C. The amplified PCR product was resolved on a 1.5% agarose gel, isolated, purified and subcloned into the TA cloning vector pCR II-TOPO (Invitrogen Life Technologies) for nucleic acid sequence determination (Genome Express, Meylan, France).
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) and a degenerate antisense primer (P4) based on cDNA sequences in bovine, porcine and human LH/CG-R (Table 1) or an oligo(dT) as antisense primer. First-strand cDNA was amplified for 30 cycles using Advantage 2 polymerase mix (BD Biosciences) and primer pair P3 and P4 or primer pair P3 and oligo(dT) as described above.
As degenerate sense 5' primers based on cDNA sequences in bovine, porcine and human LH/CG-Rs did not anneal to first strand equine LH/CG-R cDNA, we eventually used the rapid amplification of cDNA ends (RACE) technique to isolate the eLH/CG-R cDNA in the 5' direction. This was accomplished essentially as described by the manufacturer (Invitrogen Life Technologies). The gene-specific primers P5, P6 and P7 were designed from the 646-bp PCR product described above and were used in this procedure (Table 1). Briefly, first-strand cDNA was synthesized from luteal total RNA using the primer P5 and the Rnase H-reverse transcriptase Superscript II at 50 °C, and then purified. An oligo-dC tail was added in the 3'-end of the cDNA using terminal deoxynucleotidyl transferase and dCTP. The dC-tailed cDNA was then amplified for 30 cycles using primer P6 and the Abridged Anchor Primer under the following conditions: 3 min at 95 °C for one cycle, 30 s at 95 °C, 40 s at 55 °C and 1 min at 72 °C for 30 cycles, 10 min at 72 °C for one cycle. A second amplification was performed with the primer P7 and Abridged Universal Amplification Primer as internal primers using one-tenth of the first reaction volume as template and under amplification conditions described above.
All amplified PCR products were subcloned into the TA cloning vector pCR II-TOPO (Invitrogen Life Technologies) and sequenced on both strands (Genome Express). Sequence analyses were done on the Infobiogen web site (http://www.infobiogen.fr).
RT-PCR
Single-strand cDNA was synthesized from 5 µg total cellular RNA using Rnase H-reverse transcriptase (Superscript II; Invitrogen Life Technologies) and 250 ng oligo(dT) primer as recommended by the manufacturer. PCR amplifications were performed on a geneAmp PCR System 9700 (Perkin-Elmer, Norwalk, CT, USA) and reaction mixtures contained 10 pmol of each primer, 0.4 mmol dNTPs l–1, 1 x PCR buffer, Advantage 2 polymerase mix (BD Biosciences) and cDNA template. A primer pair encoding the housekeeping gene ß-actin was used as positive control (Table 1). PCR amplifications with RNAs without reverse transcription (RT) as templates were performed in parallel as negative controls. The amplification profile consisted of 5 min at 94 °C followed by 25–35 cycles of 1 min at 94 °C, 1 min at 61 °C and 1 min at 72 °C. The final cycle included a further 10 min at 72 °C to complete extension. The amplified products were separated by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized on a UV transilluminator. Bands were individually dissected and DNA was extracted using a DNA purification kit (Wizard PCR Preps, Promega, Madison, WI, USA), subcloned into the TA cloning vector pCR II-TOPO (Invitrogen Life Technologies) and sequenced (Genome Express).
Northern blot analysis
A cDNA probe covering the extracellular domain of the eLH/CG-R (EC-probe) was generated by RT-PCR with primers EC1 and EC2 (see arrows in Fig. 1) as previously described (Saint-Dizier et al. 2003) and used for Northern blot analysis. Total cellular RNA (15–20 µg) was separated by agarose gel electrophoresis in the presence of 17% formaldehyde, transferred overnight by capillary blot to a nylon membrane (Nytran Super Charge, Schleicher and Schuell, Dassel, Germany) then fixed by UV cross-linking. Blots were prehybridized for 2 h at 42 °C in a buffer containing 50% formamide, 5 x Denhardt’s solution, 1% SDS, 5 x sodium saline citrate (SSC), and 16 µl/ml denatured salmon sperm DNA (Invitrogen). Blots were then hybridized with the -32P-eLH/CG-R cDNA probe overnight at 42 °C in a buffer containing 50% formamide, 2.5 x Denhardt’s solution, 1% SDS, 5 x SSC, 10 x dextran sulfate, and 16 µl/ml denatured salmon sperm DNA. Blots were next washed in 1 x SSC plus 0.5% SDS at room temperature for 20 min, followed by three 20-min washes in 0.2 x SSC plus 0.5% SDS at 68 °C. Membranes were exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA, USA) at room temperature for 16–18 h before quantification. Membranes were then washed three times for 20 min with a boiling solution of 2 x SSC plus 0.2% SDS to remove the probe and rehybridized with the human RNA 18S probe from Ambion, Inc. (Austin, TX, USA). Membranes hybridized with the 18S probe were exposed for 1 h to a PhosphorImager screen. All hybridization signals were quantified using Image-Quant software (Molecular Dynamics). Each RNA sample was analyzed 2 to 5 times on different blots. The RNA sample of one CL at the diestrous stage (day 8 post-ovulation) was used as internal control in each blot. The intensities for LH/CG-R signals were adjusted with 18S signal values in each blot and the LH/CG-R:18S ratio values were normalized between blots according to the LH/CG-R:18S ratio value of the internal control.
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), spanning over a part of the signal peptide plus the entire extracellular, transmembrane and intracellular domains of the eLH/CG-R. The cDNA sequence (excluding primer sequence) of eLH/CG-RA (accession number AY464091
[GenBank]
to GenBank) showed 92.3, 91.6, 88 and 85.7% homologies with reported cDNA sequences of porcine (Loosfelt et al. 1989), bovine (Lussier et al. 1996), human (Minegishi et al. 1990) and rat (McFarland et al. 1989) LH/CG-R respectively, and showed 64.6% homology with the equine follicle-stimulating hormone (FSH) receptor (FSH-R) cDNA sequence (Robert et al. 1994). The deduced 680-amino acids sequence of eLH/CG-RA cDNA was similarly conserved among mammals (pig 92.5%, cow 91.6%, human 89.1% and rat 87.4%) and showed 58% homology with the equine FSH-R. Alignment with the porcine sequence suggests a putative cleavage site of the signal peptide between Gly8 and Ala9 (Loosfelt et al. 1989). Thus, the eLH/CG-RA cDNA would encode a mature protein of 672 amino acids. The putative 335-amino acid extracellular domain of eLH/CG-R contains 12 cysteine residues conserved in all species and seven potential N-linked glycosylation sites (N-X-S/T), i.e. one more than in the human, porcine, bovine and rat LH/CG-R sequences (Fig. 1). The transmembrane domain is composed of 266 amino acid residues including seven hydrophobic segments potentially spanning the cytoplasmic membrane and 8 conserved cysteine residues. The 71-amino acid COOH-terminal intracellular domain contains three conserved cysteine residues. The putative intracellular loops as well as the intracellular domain display several serine, threonine and tyrosine residues that are potential sites for phosphorylation.
Two other types of clones were isolated by RT-PCR. Using primer pair P3 and P4, seven of the eleven sequenced clones coded for a splice variant of the eLH/CG-RA cDNA and was named eLH/CG-RB. The eLH/CG-RB cDNA spliced out of frame from the 3'-end of exon X (at bp 891) into the coding region of exon XI (at bp 1829), thus encoding a putative protein of 306 amino acids without transmembrane and intracellular domains (Fig. 1). Using primer pair P3 and an oligo(dT), all three sequenced clones encoded a third eLH/CG-R splice variant, named eLH/CG-RC. This variant spliced from the 3'-end of exon X (at bp 891) into a divergent nucleotide sequence, which was not found in the eLH/CG-RA cDNA, and contained an in-frame TGA stop codon (eLH/CG-RC; Fig. 1). This divergent sequence is 590-bp long and contains a poly-A tail at its 3'-end. The eLH/CG-RC cDNA isoform encoded a putative protein of 310 amino acids without transmembrane and intracellular domains.
Expression of eLH/CG-R mRNA isoforms detected by RT-PCR
The expression of intact (eLH/CG-RA) and truncated (eLH/CG-RB and eLH/CG-RC) eLH/CG-R mRNA isoforms was examined in the primary CL and in fetal gonads at different stages of early and mid- pregnancy using qualitative RT-PCR. A primer pair encompassing exon IX through the end of exon XI (primers P8 and P9; see Table 1 and Fig. 1) revealed the expression of both eLH/CG-RA (1318-bp band) and eLH/CG-RB (380-bp band) mRNAs in the primary CL from day 14 to day 83 of pregnancy whereas only the eLH/CG-RB form was detected at day 89 and faintly detected at day 151 (Fig. 2a). A non-specific 500-bp product was also weakly amplified with primer pair P8 and P9 in some samples. The sense primer P8 combined with an antisense primer located in the non coding region of eLH/CG-RC mRNA (primer P10; see Table 1 and Fig. 1) revealed the expression of the eLH/CG-RC mRNA isoform (528-bp band) from day 14 to day 89 of pregnancy. However, no luteal expression of eLH/CG-RC mRNA could be seen at day 151 of pregnancy (Fig. 2b).
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). The signal for eLH/CG-RA mRNA was weak at day 202 of pregnancy although it could not be determined whether this change was an individual variation or a genuine developmental change. The ß-actin signal was detected in all samples (374-bp band; Fig. 2c and 3c) and no signal could be seen with RNAs without RT as templates.
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), as shown previously (Saint-Dizier et al. 2003). The number and apparent size of mRNA transcripts did not change from day 14 to day 89 in the primary CL, whereas no hybridization signal was observed in the day 151 primary CL. Northern blot analysis of total RNA from fetal gonads with the EC-probe revealed two transcripts at 3.9 and 1.8 kb on days 44 and 62, and six transcripts at 7, 5.7, 4.9, 3.9, 2.8 and 1.8 kb from day 70 to day 222 of pregnancy (Fig. 5). Furthermore, whereas four major transcripts at 7, 5.7, 3.9 and 1.8 kb were observed in the primary CL, only the 1.8 kb hybridization signal was predominant in the fetal gonads. However, it is not known if these transcripts are translated. No hybridization signal was observed with RNA samples from adult lung, kidney, spleen or liver (data not shown).
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). The major 1.8 kb mRNA transcript was 1.2 to 3 times less expressed in fetal gonads than in the primary CL or in the dioestrous CL used as internal control in each blot. The relative intensity of the 1.8 kb major transcript tended to increase in fetal gonads at days 70, 81 and 101 of pregnancy. However, there was no significant change in the 1.8 kb mRNA intensity between stages of pregnancy from day 44 to day 222 in fetal gonads. In the primary CL, the relative values of the major 7, 5.7, 3.9 and 1.8 kb mRNA transcripts tended to decrease (P = 0.08, 0.2, 0.06 and 0.14 respectively) between stages of pregnancy from day 14 to day 151 of pregnancy (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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This study reports the first cloning and sequencing of the full-length equine LH/CG-R cDNA. The deduced 680-amino acids sequence showed high homology with reported LH/CG-R sequences in other mammals, especially with the porcine (92.3% homology) and the bovine (91.6%) LH/CG-R proteins. In contrast to all other known mammalian LH/CG-Rs, which contain six conserved consensus sequences for N-linked carbohydrates, the eLH/CG-R displays a seventh putative N-glycosylation site in the N-terminal region of the extracellular domain, created by a Gly31Asn replacement (Gly23Asn replacement in the mature protein) (Loosfelt et al. 1989, McFarland et al. 1989, Minegishi et al. 1990, Lussier et al. 1996). Interestingly, while most mammalian FSH-Rs contain three highly conserved potential sites for N-glycosylation, the equine FSH-R also displays an additional N-glycosylation site in its extracellular domain (Robert et al. 1994). It has been hypothesized that this fourth N-glycosylation site could be involved in preventing eLH/CG binding to the eFSH-R (Richard et al. 1997). Although it is known that five of the six N-glycosylation sites in the porcine LH/CG-R (Vu-Hai et al. 2000) and all six sites in the rat LH/CG-R (Davis et al. 1997) are indeed glycosylated, the potential roles of these glycosidic chains remain unclear. Indeed, the nonglycosylated rat LH/CG-R can be properly folded and expressed at the cell surface, and can bind hormone and transduce signals (Davis et al. 1997). It would be of interest to determine whether all seven N-glycosylation sites of the eLH/CG-R contain carbohydrates. Furthermore, eCG binds to the eLH/CG-R with only one tenth or less the affinity of pituitary eLH in equine tissues (Stewart & Allen 1979 , 1981, Guillou & Combarnous 1983). The possible implication of the equine LH/CG-R extra N-glycosylation site in this differential binding affinity should also be examined.
Northern blot analysis using a cDNA probe encoding the extracellular domain of the eLH/CG-R revealed seven mRNA transcripts at 7, 5.7, 4.9, 3.9, 2.8, 1.8 and 0.6 kb in the primary CL. In a previous study, the presence of multiple eLH/CG-R transcripts seemed to arise in part from alternate splicing of the eLH/CG-R primary transcript since a probe covering the transmembrane domain of the receptor hybridized to only four of these seven transcripts (at 7, 4.9, 3.9 and 1.8 kb) (Saint-Dizier et al. 2003). The present work shows that the alternate splicing of the LH/CG-R primary transcript indeed occurs in the primary CL and in fetal gonads and gives rise to at least two splicing variants (eLH/CG-RB and eLH/CG-RC) in addition to the full-length eLH/CG-R mRNA (eLH/CG-RA). The point of divergence between the full-length eLH/CG-R cDNA and the two truncated cDNA isoforms is the same as the one described for the porcine LH/CG-R cDNA and corresponds to the 3' end of exon X (Loosfelt et al. 1989). One of the three splicing variants described for the porcine LH/CG-R (the D form) has been shown to splice in frame at the transmembrane to intracellular sequence junction and thus contained a putative intracellular domain. In contrast, the eLH/CG-RB form displayed a frameshift at approximately the same point of junction as the porcine D variant and was thus truncated for the putative trans-membrane and intracellular domains. The eLH/CG-RC variant, which completely lacks the exon XI encoding the transmembrane and intracellular domains, was similar to the truncated form found in the turkey ovary (You et al. 2000). Nevertheless, such a variant had not previously been described in mammalian species. The three eLH/CG-R transcripts that have been shown to lack the transmembrane domain by Northern blot analysis were at 5.7, 2.8 and 0.6 kb (Saint-Dizier et al. 2003). The eLH/CG-RB and eLH/CG-RC cDNA isoforms could correspond to the 5.7 kb and/or to the 2.8 kb transcripts observed on Northern blots. Nevertheless, as the open reading frame of the full eLH/CG-R is approximately 2.1 kb, other processes like alternate transcriptional start sites and/or multiple sites and lengths of polyadenylation are probably involved in large differences in LH/CG-R mRNAs sizes.
The detection of eLH/CG-R mRNA isoforms by RT-PCR showed that the expression of the eLH/CG-RA isoform ceased in the primary CL between days 83 and 89 of pregnancy. At days 89 and 151 of pregnancy, only truncated forms of eLH/CG-R mRNAs without transmembrane sequence (eLH/CG-RB and eLH/CG-RC) were detected by RT-PCR. This change in eLH/CG-R mRNA alternative splicing occurs while the feto-placental steroidogenesis is sufficient to support pregnancy (Holtan et al. 1979). However, the primary CL is maintained and continues to secrete progesterone and estrogens until days 160–180 of pregnancy, probably stimulated by eCG (Squires et al. 1974, Ginther 1992a). In a previous study, 125I-eLH saturation binding assays performed on luteal membranes showed that the primary CL at days 83–101 of pregnancy bound 125I-eLH with high affinity and displayed a substantial level of membrane eLH/CG binding sites, which was 24.7% of the level measured at days 14–31 of pregnancy (Saint-Dizier et al. 2003). Furthermore, luteal membranes at day 151 also bound 125I-eLH (data not shown). It is assumed that luteal cells of the primary CL still have membrane eLH/CG-Rs despite only truncated eLH/CG-R mRNAs being synthesized between days 89 and 151 of pregnancy. It is thus supposed that luteal eLH/CG-Rs exhibit long half-lives over this period of time.
As shown by RT-PCR, the eLH/CG-RA isoform was expressed in fetal gonads as early as day 44 of pregnancy and in all gonads until day 222 of pregnancy, which was the last time point examined. However, the 1.8 kb transcript was largely predominant in Northern blots performed on the same samples, whereas three additional mRNA transcripts with larger sizes (7, 5.7 and 3.9 kb) were predominantly expressed in the adult primary CL. Since the size of the 1.8 kb transcript is less than the 2.1 kb open reading frame of the full sequence eLH/CG-R, this 1.8 kb transcript must be an incomplete eLH/CG-R mRNA. Furthermore, it is not yet known if this 1.8 kb transcript encodes a functional receptor. In rats, the expression of truncated LH/CG-R mRNA transcripts is first detectable in fetal ovaries and testes as early as embryonic day 13.5 (Sokka et al. 1996) but the full-length LH/CG-R mRNA appears thereafter on embryonic day 15.5 in the testis (Zhang et al. 1994) and on postnatal day 7 in the ovary (Sokka et al. 1992). While the different eLH/CG-R cDNA isoforms were observed at all stages examined in horse fetal gonads, our results do not exclude a possible change in alternative splicing of the eLH/CG-R primary transcript before day 44 and/or after day 222 of pregnancy in fetal ovaries and testes. Furthermore, the major 1.8 kb eLH/CG-R mRNA transcript remained 1.2 to 3 times less expressed in fetal gonads than in the dioestrous or in the primary CL of adult mares during early and mid-pregnancy, which would indicate that a differential regulation of the eLH/CG-R gene transcription occurs between the fetal and the adult life in the mare.
Although gene expression does not necessarily imply that transcripts are translated in proteins or that the receptors are functionally involved in signal transduction, the presence of eLH/CG-R transcripts in horse fetal gonads indicates that the eLH/CG-R, and therefore fetal pituitary eLH and/or eCG themselves, may have a physiological role in early development and steroidogenesis of gonads in this species. Fetal content of pituitary eLH has been found to be low at day 90 of pregnancy and has been shown to increase slowly between days 90 and 150 of pregnancy (Wesson & Ginther 1980). In contrast, the fetal blood concentration of eLH measured by radioimmunoassay was highest between days 100 and 150 of pregnancy, parallel with a rapid growth of fetal gonads (Wesson & Ginther 1980). Since plasma eLH cannot be distinguished from plasma eCG by immunological methods, the high eLH/CG level measured in fetal blood between days 100 and 150 of pregnancy could be due, in part, to the presence of eCG in the fetal circulation. Most of the increasing weight of fetal gonads results from the hyperplasia of the medullar interstitial cells, histologically similar in fetal ovaries and testes (Cole et al. 1933). These interstitial cells are analogous to luteal cells and contain all the organelles normally associated with steroid biosynthesis (Hay & Allen 1975). Since fetal gonads also express the full-length eLH/CG-R cDNA as early as day 44 of gestation, one can hypothesize that eCG, fetal in origin, would stimulate the initial steroidogenesis of fetal gonads.
In conclusion, the presence of the full-length eLH/CG-R cDNA isoform in the primary CL and in fetal gonads during eCG secretion suggests that eCG may be involved in the progressive transition from a luteal to a feto-placental output of steroids during equine pregnancy.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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). Conserved cysteine residues are highlighted by gray boxes. The equine-specific N-glycosylation site is indicated in bold letters double underlined, and other double underlines indicate the six N-linked glycosylation sites conserved among other mammalian LH/CG-Rs. Single underlines indicate the seven predicted transmembrane-spanning domains. The point of divergence between intact and truncated eLH/CG-R cDNA isoforms is indicated by an arrowhead (
) and the point of junction for eLH/CG-RB is indicated by an arrow ( 
). The sites of the primers P8, P9 and P10, used in RT-PCR, are indicated on the cDNA sequence. The primer pair EC1 and EC2, also indicated, was used to generate the eLH/CG-R cDNA EC-probe for Northern blot analysis. (Accession number AY464091
