Expression of the full-length and alternatively spliced equine luteinizing hormone/chorionic gonadotropin receptor mRNAs in the primary corpus luteum and fetal gonads during pregnancy

in Reproduction
View More View Less
  • 1 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@tours.inra.fr

The full-length equine luteinizing hormone/chorionic gonadotropin (LH/CG) receptor (eLH/CG-RA) cDNA and two alternatively spliced isoforms (eLH/CG-RB,C) were isolated from luteal tissue and characterized using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5′-rapid amplification of cDNA ends. The 680-amino acid full sequence of eLH/CG-RA displayed 87–92% homology with other mammalian LH/CG-Rs. The eLH/CG-RB and eLH/CG-RC cDNA isoforms were truncated from the 3′-end of exon X: eLH/CG-RB spliced out of frame into the last exon whereas eLH/CG-RC contained an in-frame stop codon within a divergent sequence. Consequently, both eLH/CG-RB and eLH/CG-RC cDNA isoforms encoded putative proteins without transmembrane and intracellular domains.

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.

Abstract

The full-length equine luteinizing hormone/chorionic gonadotropin (LH/CG) receptor (eLH/CG-RA) cDNA and two alternatively spliced isoforms (eLH/CG-RB,C) were isolated from luteal tissue and characterized using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5′-rapid amplification of cDNA ends. The 680-amino acid full sequence of eLH/CG-RA displayed 87–92% homology with other mammalian LH/CG-Rs. The eLH/CG-RB and eLH/CG-RC cDNA isoforms were truncated from the 3′-end of exon X: eLH/CG-RB spliced out of frame into the last exon whereas eLH/CG-RC contained an in-frame stop codon within a divergent sequence. Consequently, both eLH/CG-RB and eLH/CG-RC cDNA isoforms encoded putative proteins without transmembrane and intracellular domains.

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.

Introduction

In the mare, luteal and feto-placental steroids are successively necessary to maintain pregnancy. Initially, stimulation of the primary corpus luteum (CL) or CL of conception by pituitary luteinizing hormone (LH) results in a transient increase in the progesterone level, similar to that which occurs during the estrous cycle (Ginther 1992a). From day 35 of pregnancy, coincident with the secretion of the fetal hormone equine chorionic gonadotropin (eCG), the primary CL increases in size and secretes increasing levels of progesterone and newly synthesized estrogens (Bergfelt et al. 1989, Daels et al. 1991). Thereafter, from day 40 to day 160–180 of gestation, the primary CL is maintained and continues to secrete progesterone and estrogens (Squires et al. 1974). The fetal gonads, on the other hand, enlarge slowly until approximately day 100 post-ovulation, then very rapidly between days 100 and 200 of gestation, exceeding the weight of maternal ovaries around day 150 of pregnancy (Ginther 1992b). From days 60 to 80, the fetal gonads in both sexes provide precursor steroids for the placental synthesis of estrone and equine-specific estrogens, equine and equilenin, and contribute to the production of large amounts of estrogens by the feto-placental unit until the end of pregnancy (Pashen & Allen 1979, Raeside et al. 1979). The equine placenta also produces progesterone and 5α-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.

Materials and Methods

Animals

Light breed mares and Welsh pony-mares (3–17 years old) with no known reproductive pathology were used in this study. Mares were maintained on pasture and supplemented with hay, minerals, and water available ad libitum. The reproductive tracts of the mares were monitored daily during estrus and every other day from ovulation until CL collection using palpation and rectal ultrasonography. The day of ovulation was designated as day 0 of pregnancy. Mares were fertilized by artificial insemination.

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).

The 3′-end of the eLH/CG-R cDNA was subsequently isolated using sense primer P3 designed from the 646-bp PCR product described above (Table 1) 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 × PCR buffer, Advantage 2 polymerase mix (BD Biosciences) and cDNA template. A primer pair encoding the housekeeping gene β-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 × Denhardt’s solution, 1% SDS, 5 × sodium saline citrate (SSC), and 16 μl/ml denatured salmon sperm DNA (Invitrogen). Blots were then hybridized with the α-32P-eLH/CG-R cDNA probe overnight at 42 °C in a buffer containing 50% formamide, 2.5 × Denhardt’s solution, 1% SDS, 5 × SSC, 10 × dextran sulfate, and 16 μl/ml denatured salmon sperm DNA. Blots were next washed in 1 × SSC plus 0.5% SDS at room temperature for 20 min, followed by three 20-min washes in 0.2 × 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 × 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.

Statistical analysis

The concentrations of mRNA levels are shown as means±S.E.M. Three stages of pregnancy were considered according to the known pattern of eCG secretion: before the onset of eCG secretion (days 14–31 for CL), during eCG secretion (days 42–89 for CL and days 44–101 for fetal gonads) and after eCG secretion (day 151 for CL and days 151–222 for fetal gonads). The relative levels of mRNAs were compared between stages of pregnancy, irrespective of other variables, with the nonparametric Krus-kall–Wallis test in the primary CL and with the nonparametric Wilcoxon–Mann–Whitney in the fetal gonads. Tests were all performed using StatXact 5 (CYTEL, Cambridge, MA, USA; http://www.cytel.com/). Differences were considered to be significant when P < 0.05.

Results

Nucleotide sequence analysis of eLH/CG-R cDNA isoforms

Based on the conserved sequences of the human, porcine, bovine and murine LH/CG-R cDNAs, the primer pair P1 and P2 was designed and used in RT-PCR reaction using total RNA from a pool of corpora lutea as template. A 646-bp PCR product was obtained, subcloned, then sequenced and found to be homologous with known mammalian LH/CG-R cDNAs. The sequence information from the 646-bp fragment combined with RT-PCR and 5′-RACE products generated a 2047-bp eLH/CG-R cDNA (eLH/CG-RA: Fig. 1), 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 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).

In fetal gonads, the expression of eLH/CG-RA, eLH/CG-RB and eLH/CG-RC mRNAs was detected as early as day 44 of pregnancy and at the following days examined until day 222 of pregnancy (Fig. 3a and 3b). 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 β-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.

Expression of eLH/CG-R mRNA isoforms detected by Northern blot analysis

Northern blot analysis with a cDNA probe covering the major part of the extracellular domain of the eLH/CG-R (EC-probe) revealed seven eLH/CG-R mRNA transcripts at 7, 5.7, 4.9, 3.9, 2.8, 1.8 kb and 0.6 kb in the primary CL (Fig. 4), 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).

Semiquantitative analysis of eLH/CG-R mRNA isoforms

The relative value of the major 1.8 kb mRNA transcript expressed in fetal gonads was quantified and compared with relative values of major eLH/CG-R transcripts expressed in the primary CL during pregnancy (Fig. 6). 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).

Discussion

Three different eLH/CG-R cDNA isoforms (eLH/CG-RA,B,C) were isolated from a pool of equine corpora lutea and identified by nucleotide sequence analysis of RT-PCR and 5′-RACE products. The full-length eLH/CG-R cDNA (eLH/CG-RA) as well as truncated forms encoding receptors without transmembrane and intracellular domains (eLH/CG-RB,C) were expressed from day 14 to day 83 in the primary CL and from day 44 to day 222 of pregnancy in fetal gonads. In contrast, the primary CL at days 89 and 151 expressed only truncated forms of the eLH/CG-R cDNA. The relative values of major mRNA transcripts tended to decrease in the primary CL whereas the unique major 1.8 kb mRNA in fetal gonads was steadily expressed during pregnancy. These results show that the expression of eLH/CG-R mRNAs occurs in fetal gonads before it ends in the primary CL and thus parallels the gradual change from a luteal to a feto-placental steroidogenesis during equine pregnancy.

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.

Table 1

Nucleic acid sequences of primers used for the isolation of the equine LH/CG-R cDNA and for RT-PCR. Location of nucleotides, when specified, corresponds to the eLH/CG-RA cDNA sequence on Fig. 1. Primer P4 is a degenerate primer where B = T,G,C; Y = C,T; K = T,G; R = A,G.

Primer nameLocationSequence
Forward primer P1146–1675′-CTTTCAGAGGACTTAATGAGGT-3′
Reverse primer P2772–7925′-TCTAAAAGCACAGCAGTGGCT-3′
Forward primer P3570–5945′-GCTGGAGAAGATGCACAACGGAGCC-3′
Reverse primer P42019–20475′-CAGTTAACABTCYKTRTAGCRAGTCTTG-3′
Reverse primer P5536–5605′-TCCTTTAGCTCCAGGGAAATCAGTG-3′
Reverse primer P6516–5355′-TCGTCCCGTTGAATGCATGA-3′
Reverse primer P7444–4685′-TTCGTTATTCATCCCTTGAAAAGCA-3′
Forward primer P8689–7105′-TTGCCACATCATCCTATTCTCT-3′
Reverse primer P91984–20055′-TATATTGGCAGTGCAATGTGGT-3′
Reverse primer P105′-CTCCAATTCCCCTTCATGATAA-3′
Forward β actin5′-CGTGACATTAAGGAGAAGCTGTGC-3′
Reverse β actin5′-CTCAGGAGGAGCAATGATCTTGAT-3′
Figure 1
Figure 1

Nucleic acid and deduced amino acid sequences of intact (eLH/CG-RA) and truncated (eLH/CG-RB and eLH/CG-RC) equine LH/CG-R cDNA. The putative cleavage site of the peptide signal after alignment with the porcine LH/CG-R is indicated by an arrow ( ⇓ ). 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 to GenBank.)

Citation: Reproduction 128, 2; 10.1530/rep.1.00164

Figure 2
Figure 2

Representative amplification of the alternatively spliced eLH/CG-RA, eLH/CG-RB (a) and eLH/CG-RC (b) isoforms by RT-PCR in the primary corpus luteum from one cyclic mare at day 8 post-ovulation (Dioestrus) and from mares at days 14–151 of pregnancy. Expression of the β-actin gene was used as positive control (c). –RT corresponds to a PCR carried out without reverse transcriptase.

Citation: Reproduction 128, 2; 10.1530/rep.1.00164

Figure 3
Figure 3

Representative amplification of the alternatively spliced eLH/CG-RA, eLH/CG-RB (a) and eLH/CG-RC (b) isoforms by RT-PCR in fetal gonads at days 44–222 of pregnancy. Expression of the β-actin gene was used as positive control (c). –RT corresponds to a PCR carried out without reverse transcriptase.

Citation: Reproduction 128, 2; 10.1530/rep.1.00164

Figure 4
Figure 4

Representative Northern blot analysis of total RNA from one CL at the dioestrous stage (Dioes) as internal control and CL from pregnant mares before the onset of eCG secretion (days 14–31), during eCG secretion (days 42–89) and after eCG secretion (day 151) hybridized with the cDNA EC-probe (top panel) and the RNA 18S-probe (bottom panel) for normalization.

Citation: Reproduction 128, 2; 10.1530/rep.1.00164

Figure 5
Figure 5

Representative Northern blot analysis of total RNA from equine fetal gonads during eCG secretion (day 44–101) and after eCG secretion (day 151–222), and from one CL at the dioestrous stage (Dioes) as internal control, hybridized with the cDNA EC-probe (top panel) and the RNA 18S-probe (bottom panel) for normalization.

Citation: Reproduction 128, 2; 10.1530/rep.1.00164

Figure 6
Figure 6

Relative intensities of eLH/CG-R major mRNA transcripts in the primary CL and in fetal gonads (FG) before eCG secretion (CL: days 14–31, n = 6), during eCG secretion (CL: days 42–89, n = 11; FG: days 44–101, n = 7) and after eCG secretion (CL: day 151, n = 1; FG: days 151–222, n = 3). Values are means±s.e.m.

Citation: Reproduction 128, 2; 10.1530/rep.1.00164

Received 5 January 2004
 First decision 31 March 2004
 Accepted 10 May 2004

The authors wish to thank Guy Duchamp for his technical assistance and Peter F Daels for supplies of some fetal and luteal specimens. M S-D was supported by a fellowship from the Institut National de la Recherche Agronomique and the Région Centre.

References

  • Bergfelt DR, Pierson RA & Ginther OJ 1989 Resurgence of the primary corpus luteum during pregnancy in the mare. Animal Reproduction Science 21 261–270.

    • Search Google Scholar
    • Export Citation
  • Bousfield GR, Butnev VY, Gotschall RR, Baker VL & Moore WT 1996 Structural features of mammalian gonadotropins. Molecular and Cellular Endocrinology 125 3–19.

    • Search Google Scholar
    • Export Citation
  • Cole HH, Hart GH, Lyons WR & Catchpole HR 1933 The development and hormonal content of fetal horse gonads. Anatomical Record 56 275–289.

  • Daels PF, DeMoraes JJ, Stabenfeldt GH, Hughes JP & Lasley BL 1991 The corpus luteum: source of oestrogen during early pregnancy in the mare. Journal of Reproduction and Fertility Supplement 44 501–508.

    • Search Google Scholar
    • Export Citation
  • Daels PF, Albrecht BA & Mohammed HO 1998 Equine chorionic gonadotropin regulates luteal steroidogenesis in pregnant mares. Biology of Reproduction 59 1062–1068.

    • Search Google Scholar
    • Export Citation
  • Davis DP, Rozell TG, Liu X & Segaloff DL 1997 The six N-linked carbohydrates of the lutropin/choriogonadotropin receptor are not absolutely required for correct folding, cell surface expression, hormone binding, or signal transduction. Molecular Endocrinology 11 550–562.

    • Search Google Scholar
    • Export Citation
  • Ginther OJ 1992a Endocrinology of pregnancy. In Reproductive Biology of the Mare, Basic and Applied Aspects, 2nd edn, pp 419–456. Cross Plain: Equiservices.

  • Ginther OJ 1992b Embryology and placentation. In Reproductive Biology of the Mare, Basic and Applied Aspects, 2nd edn, pp 345–418. Cross Plains: Equiservices.

  • Guillou F & Combarnous Y 1983 Purification of equine gonadotropins and comparative study of their acid-dissociation and receptor-binding specificity. Biochimica et Biophysica Acta 755 229–236.

    • Search Google Scholar
    • Export Citation
  • Hay MF & Allen WR 1975 An ultrastructural and histochemical study of the interstitial cells in the gonads of the fetal horse. Journal of Reproduction and Fertility Supplement 23 557–561.

    • Search Google Scholar
    • Export Citation
  • Holtan DW, Squires EL, Lapin DR & Ginther OJ 1979 Effect of ovariectomy on pregnancy in mares. Journal of Reproduction and Fertility Supplement 27 457–463.

    • Search Google Scholar
    • Export Citation
  • Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT, Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Garnier J, et al. 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245 525–528.

    • Search Google Scholar
    • Export Citation
  • Lussier JG, Houde A, Ethier J & Silversides DW 1996 Complementary DNA structure of the bovine LH receptor. Direct submission to Genbank no U20504.

  • McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL & Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245 494–499.

    • Search Google Scholar
    • Export Citation
  • Minegishi T, Nakamura K, Takakura Y, Miyamoto K, Hasegawa Y, Ibuki Y & Igarashi M 1990 Cloning and sequencing of human LH/hCG receptor cDNA. Biochemical and Biophysical Research Communications 172 1049–1054.

    • Search Google Scholar
    • Export Citation
  • Pashen RL & Allen WR 1979 The role of the fetal gonads and placenta in steroid production, maintenance of pregnancy and parturition in the mare. Journal of Reproduction and Fertility Supplement 27 499–509.

    • Search Google Scholar
    • Export Citation
  • Raeside JI, Liptrap RM, McDonell WN & Milne FJ 1979 A precursor role for DHA in a feto-placental unit for oestrogen formation in the mare. Journal of Reproduction and Fertility Supplement 27 493–497.

    • Search Google Scholar
    • Export Citation
  • Richard F, Martinat N, Remy JJ, Salesse R & Combarnous Y 1997 Cloning, sequencing and in vitro functional expression of recombinant donkey follicle-stimulating hormone receptor: a new insight into the binding specificity of gonadotrophin receptors. Journal of Molecular Endocrinology 18 193–202.

    • Search Google Scholar
    • Export Citation
  • Robert P, Amsellem S, Christophe S, Benifla JL, Bellet D, Koman A & Bidart JM 1994 Cloning and sequencing of the equine testicular follitropin receptor. Biochemical and Biophysical Research Communications 201 201–207.

    • Search Google Scholar
    • Export Citation
  • Saint-Dizier M, Chopineau M, Dupont J, Daels PF & Combarnous Y 2003 Expression and binding activity of luteinizing hormone/chorionic gonadotropin receptors in the primary corpus luteum during early pregnancy in the mare. Biology of Reproduction 69 1743–1749.

    • Search Google Scholar
    • Export Citation
  • Sokka T, Hamalainen T & Huhtaniemi L 1992 Functional LH receptor appears in the neonatal rat ovary after changes in the alternative splicing pattern of the LH receptor mRNA. Endocrinology 130 1738–1740.

    • Search Google Scholar
    • Export Citation
  • Sokka TA, Hamalainen TM, Kaipia A, Warren DW & Huhtaniemi IT 1996 Development of luteinizing hormone action in the perinatal rat ovary. Biology of Reproduction 55 663–670.

    • Search Google Scholar
    • Export Citation
  • Squires EL 1993 Endocrinology of pregnancy. In Equine Reproduction, pp 495–500. Eds AO McKinnon & JL Voss. Philadelphia: Lea and Febiger.

  • Squires EL, Douglas RH, Steffenhagen WP & Ginther OJ 1974 Ovarian changes during the estrous cycle and pregnancy in mares. Journal of Animal Science 38 330–338.

    • Search Google Scholar
    • Export Citation
  • Stewart F & Allen WR 1979 The binding of FSH, LH and PMSG to equine gonadal tissues. Journal of Reproduction and Fertility Supplement 27 431–440.

    • Search Google Scholar
    • Export Citation
  • Stewart F & Allen WR 1981 Biological functions and receptor binding activities of equine chorionic gonadotrophins. Journal of Reproduction and Fertility 62 527–536.

    • Search Google Scholar
    • Export Citation
  • Urwin VE & Allen WR 1982 Pituitary and chorionic gonadotrophic control of ovarian function during early pregnancy in equids. Journal of Reproduction and Fertility Supplement 32 371–381.

    • Search Google Scholar
    • Export Citation
  • Vu-Hai MT, Huet JC, Echasserieau K, Bidart JM, Floiras C, Pernollet JC & Milgrom E 2000 Posttranslational modifications of the lutropin receptor: mass spectrometric analysis. Biochemistry 39 5509–5517.

    • Search Google Scholar
    • Export Citation
  • Wesson JA & Ginther OJ 1980 Fetal and maternal gonads and gonadotropins in the pony. Biology of Reproduction 22 735–743.

  • You S, Kim H, Hsu CC, El Halawani ME & Foster DN 2000 Three different turkey luteinizing hormone receptor (tLH-R) isoforms I: characterization of alternatively spliced tLH-R isoforms and their regulated expression in diverse tissues. Biology of Reproduction 62 108–116.

    • Search Google Scholar
    • Export Citation
  • Zhang FP, Hamalainen T, Kaipia A, Pakarinen P & Huhtaniemi I 1994 Ontogeny of luteinizing hormone receptor gene expression in the rat testis. Endocrinology 134 2206–2213.

    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

     An official journal of

    Society for Reproduction and Fertility

 

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 637 26 3
PDF Downloads 107 32 4
  • View in gallery

    Nucleic acid and deduced amino acid sequences of intact (eLH/CG-RA) and truncated (eLH/CG-RB and eLH/CG-RC) equine LH/CG-R cDNA. The putative cleavage site of the peptide signal after alignment with the porcine LH/CG-R is indicated by an arrow ( ⇓ ). 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 to GenBank.)

  • View in gallery

    Representative amplification of the alternatively spliced eLH/CG-RA, eLH/CG-RB (a) and eLH/CG-RC (b) isoforms by RT-PCR in the primary corpus luteum from one cyclic mare at day 8 post-ovulation (Dioestrus) and from mares at days 14–151 of pregnancy. Expression of the β-actin gene was used as positive control (c). –RT corresponds to a PCR carried out without reverse transcriptase.

  • View in gallery

    Representative amplification of the alternatively spliced eLH/CG-RA, eLH/CG-RB (a) and eLH/CG-RC (b) isoforms by RT-PCR in fetal gonads at days 44–222 of pregnancy. Expression of the β-actin gene was used as positive control (c). –RT corresponds to a PCR carried out without reverse transcriptase.

  • View in gallery

    Representative Northern blot analysis of total RNA from one CL at the dioestrous stage (Dioes) as internal control and CL from pregnant mares before the onset of eCG secretion (days 14–31), during eCG secretion (days 42–89) and after eCG secretion (day 151) hybridized with the cDNA EC-probe (top panel) and the RNA 18S-probe (bottom panel) for normalization.

  • View in gallery

    Representative Northern blot analysis of total RNA from equine fetal gonads during eCG secretion (day 44–101) and after eCG secretion (day 151–222), and from one CL at the dioestrous stage (Dioes) as internal control, hybridized with the cDNA EC-probe (top panel) and the RNA 18S-probe (bottom panel) for normalization.

  • View in gallery

    Relative intensities of eLH/CG-R major mRNA transcripts in the primary CL and in fetal gonads (FG) before eCG secretion (CL: days 14–31, n = 6), during eCG secretion (CL: days 42–89, n = 11; FG: days 44–101, n = 7) and after eCG secretion (CL: day 151, n = 1; FG: days 151–222, n = 3). Values are means±s.e.m.

  • Bergfelt DR, Pierson RA & Ginther OJ 1989 Resurgence of the primary corpus luteum during pregnancy in the mare. Animal Reproduction Science 21 261–270.

    • Search Google Scholar
    • Export Citation
  • Bousfield GR, Butnev VY, Gotschall RR, Baker VL & Moore WT 1996 Structural features of mammalian gonadotropins. Molecular and Cellular Endocrinology 125 3–19.

    • Search Google Scholar
    • Export Citation
  • Cole HH, Hart GH, Lyons WR & Catchpole HR 1933 The development and hormonal content of fetal horse gonads. Anatomical Record 56 275–289.

  • Daels PF, DeMoraes JJ, Stabenfeldt GH, Hughes JP & Lasley BL 1991 The corpus luteum: source of oestrogen during early pregnancy in the mare. Journal of Reproduction and Fertility Supplement 44 501–508.

    • Search Google Scholar
    • Export Citation
  • Daels PF, Albrecht BA & Mohammed HO 1998 Equine chorionic gonadotropin regulates luteal steroidogenesis in pregnant mares. Biology of Reproduction 59 1062–1068.

    • Search Google Scholar
    • Export Citation
  • Davis DP, Rozell TG, Liu X & Segaloff DL 1997 The six N-linked carbohydrates of the lutropin/choriogonadotropin receptor are not absolutely required for correct folding, cell surface expression, hormone binding, or signal transduction. Molecular Endocrinology 11 550–562.

    • Search Google Scholar
    • Export Citation
  • Ginther OJ 1992a Endocrinology of pregnancy. In Reproductive Biology of the Mare, Basic and Applied Aspects, 2nd edn, pp 419–456. Cross Plain: Equiservices.

  • Ginther OJ 1992b Embryology and placentation. In Reproductive Biology of the Mare, Basic and Applied Aspects, 2nd edn, pp 345–418. Cross Plains: Equiservices.

  • Guillou F & Combarnous Y 1983 Purification of equine gonadotropins and comparative study of their acid-dissociation and receptor-binding specificity. Biochimica et Biophysica Acta 755 229–236.

    • Search Google Scholar
    • Export Citation
  • Hay MF & Allen WR 1975 An ultrastructural and histochemical study of the interstitial cells in the gonads of the fetal horse. Journal of Reproduction and Fertility Supplement 23 557–561.

    • Search Google Scholar
    • Export Citation
  • Holtan DW, Squires EL, Lapin DR & Ginther OJ 1979 Effect of ovariectomy on pregnancy in mares. Journal of Reproduction and Fertility Supplement 27 457–463.

    • Search Google Scholar
    • Export Citation
  • Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT, Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Garnier J, et al. 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245 525–528.

    • Search Google Scholar
    • Export Citation
  • Lussier JG, Houde A, Ethier J & Silversides DW 1996 Complementary DNA structure of the bovine LH receptor. Direct submission to Genbank no U20504.

  • McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL & Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245 494–499.

    • Search Google Scholar
    • Export Citation
  • Minegishi T, Nakamura K, Takakura Y, Miyamoto K, Hasegawa Y, Ibuki Y & Igarashi M 1990 Cloning and sequencing of human LH/hCG receptor cDNA. Biochemical and Biophysical Research Communications 172 1049–1054.

    • Search Google Scholar
    • Export Citation
  • Pashen RL & Allen WR 1979 The role of the fetal gonads and placenta in steroid production, maintenance of pregnancy and parturition in the mare. Journal of Reproduction and Fertility Supplement 27 499–509.

    • Search Google Scholar
    • Export Citation
  • Raeside JI, Liptrap RM, McDonell WN & Milne FJ 1979 A precursor role for DHA in a feto-placental unit for oestrogen formation in the mare. Journal of Reproduction and Fertility Supplement 27 493–497.

    • Search Google Scholar
    • Export Citation
  • Richard F, Martinat N, Remy JJ, Salesse R & Combarnous Y 1997 Cloning, sequencing and in vitro functional expression of recombinant donkey follicle-stimulating hormone receptor: a new insight into the binding specificity of gonadotrophin receptors. Journal of Molecular Endocrinology 18 193–202.

    • Search Google Scholar
    • Export Citation
  • Robert P, Amsellem S, Christophe S, Benifla JL, Bellet D, Koman A & Bidart JM 1994 Cloning and sequencing of the equine testicular follitropin receptor. Biochemical and Biophysical Research Communications 201 201–207.

    • Search Google Scholar
    • Export Citation
  • Saint-Dizier M, Chopineau M, Dupont J, Daels PF & Combarnous Y 2003 Expression and binding activity of luteinizing hormone/chorionic gonadotropin receptors in the primary corpus luteum during early pregnancy in the mare. Biology of Reproduction 69 1743–1749.

    • Search Google Scholar
    • Export Citation
  • Sokka T, Hamalainen T & Huhtaniemi L 1992 Functional LH receptor appears in the neonatal rat ovary after changes in the alternative splicing pattern of the LH receptor mRNA. Endocrinology 130 1738–1740.

    • Search Google Scholar
    • Export Citation
  • Sokka TA, Hamalainen TM, Kaipia A, Warren DW & Huhtaniemi IT 1996 Development of luteinizing hormone action in the perinatal rat ovary. Biology of Reproduction 55 663–670.

    • Search Google Scholar
    • Export Citation
  • Squires EL 1993 Endocrinology of pregnancy. In Equine Reproduction, pp 495–500. Eds AO McKinnon & JL Voss. Philadelphia: Lea and Febiger.

  • Squires EL, Douglas RH, Steffenhagen WP & Ginther OJ 1974 Ovarian changes during the estrous cycle and pregnancy in mares. Journal of Animal Science 38 330–338.

    • Search Google Scholar
    • Export Citation
  • Stewart F & Allen WR 1979 The binding of FSH, LH and PMSG to equine gonadal tissues. Journal of Reproduction and Fertility Supplement 27 431–440.

    • Search Google Scholar
    • Export Citation
  • Stewart F & Allen WR 1981 Biological functions and receptor binding activities of equine chorionic gonadotrophins. Journal of Reproduction and Fertility 62 527–536.

    • Search Google Scholar
    • Export Citation
  • Urwin VE & Allen WR 1982 Pituitary and chorionic gonadotrophic control of ovarian function during early pregnancy in equids. Journal of Reproduction and Fertility Supplement 32 371–381.

    • Search Google Scholar
    • Export Citation
  • Vu-Hai MT, Huet JC, Echasserieau K, Bidart JM, Floiras C, Pernollet JC & Milgrom E 2000 Posttranslational modifications of the lutropin receptor: mass spectrometric analysis. Biochemistry 39 5509–5517.

    • Search Google Scholar
    • Export Citation
  • Wesson JA & Ginther OJ 1980 Fetal and maternal gonads and gonadotropins in the pony. Biology of Reproduction 22 735–743.

  • You S, Kim H, Hsu CC, El Halawani ME & Foster DN 2000 Three different turkey luteinizing hormone receptor (tLH-R) isoforms I: characterization of alternatively spliced tLH-R isoforms and their regulated expression in diverse tissues. Biology of Reproduction 62 108–116.

    • Search Google Scholar
    • Export Citation
  • Zhang FP, Hamalainen T, Kaipia A, Pakarinen P & Huhtaniemi I 1994 Ontogeny of luteinizing hormone receptor gene expression in the rat testis. Endocrinology 134 2206–2213.

    • Search Google Scholar
    • Export Citation