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BOR - Papers in Press, published online ahead of print July 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.018812
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BIOLOGY OF REPRODUCTION 69, 1743–1749 (2003)
DOI: 10.1095/biolreprod.103.018812
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

Expression and Binding Activity of Luteinizing Hormone/Chorionic Gonadotropin Receptors in the Primary Corpus Luteum During Early Pregnancy in the Mare1

Marie Saint-Dizier2, Maryse Chopineau, Joëlle Dupont, Peter F. Daels3, and Yves Combarnous

Unité de Physiologie de la Reproduction et des Comportements,3 UMR 6073 INRA-CNRS-Université F. Rabelais de Tours, 37380 Nouzilly, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Luteal steroids are necessary to maintain the first 70–90 days of pregnancy in the mare. At 35 days postovulation, the resurgence of the primary corpus luteum (CL) coincides with the secretion of the fetal hormone eCG. In order to study the responsiveness of the primary CL to eCG, we have examined levels of luteal equine LH/CG receptors (eLH/CG-R) mRNAs by Northern blot analysis and measured concentrations of eLH/CG binding sites on luteal membranes using 125I-eLH saturation binding assays at three stages of gestation: before the onset of eCG secretion (Days 14–31), from onset to maximum eCG secretion (Days 38–62), and during decline of eCG secretion (Days 83–101). Multiple transcripts of eLH/CG-R (7, 5.7, 4.9, 3.9, 2.8, 1.8, 0.6 kilobase [kb]) were identified in the primary CL at all stages examined. Three of them (5.7, 2.8, 0.6 kb) coded for truncated eLH/CG-R lacking the transmembrane domain. The relative intensities of the four major transcripts tended to decrease (5.7 and 3.9 kb) or were steadily expressed (7 and 1.8 kb) during pregnancy. The affinity of eLH/CG binding sites did not change during pregnancy whereas the number of eLH/CG binding sites decreased significantly after the onset of eCG secretion. Nevertheless, levels of binding sites were still at 44.6% (Days 38–62) to 24.7% (Days 83–101) of those measured before the onset of eCG secretion. Taken together, the presence of eLH/CG-R mRNAs and of a substantial part of eLH/CG binding sites with high affinity suggest that the primary CL still expresses a high number of eLH/CG-R and remains responsive to eCG during early pregnancy.

corpus luteum function, luteinizing hormone, mechanisms of hormone action, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Luteal steroids are necessary for the maintenance of pregnancy until Days 70–90 after ovulation in the mare [1]. Initially, stimulation of the corpus luteum (CL) of conception or primary CL by pituitary LH results in an increase in the progesterone level between Day 2 and Day 8 of pregnancy, similar to what is observed during the estrous cycle [2]. Then, in parallel with low levels of pituitary LH [3], the luteal size and secretion of progesterone tend to decrease until Day 35 postovulation [4, 5]. At this time, coincident with secretion of the fetal hormone eCG, the primary CL increases in size and secretes increasing levels of progesterone and newly synthesized estrogens [5, 6]. The so-called resurgence of the primary CL is also characterized by significant changes in expression of steroidogenic enzymes [7]. Thereafter, from Day 40 to Days 160–180 of gestation, the primary CL is maintained and produces progesterone and estrogens [8]. Additional secondary CL may also develop between Days 40–60 and 160 and contribute to the luteal secretion of steroids [9]. Then a gradual transition from an ovarian to a placental source of progesterone occurs and the ovaries become small and completely inactive until the end of pregnancy [2]. There is some strong evidence that eCG is responsible for the resurgence and support of the primary CL from Day 35 of pregnancy in the mare [4, 5, 10, 11]. Nevertheless, the responsiveness of the primary CL to eCG throughout the time of eCG secretion, i.e., approximately 100 days, remains questionable in regard to the very high eCG circulating levels.

In pregnant mares and pony mares, it is well known that eCG 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 level by Days 120–150 of pregnancy [2, 12]. The protein structure of eCG is fully identical to that of equine LH as a single gene encodes for their ß subunits [13] and another gene encodes for their common {alpha} subunits [14]. Based on studies performed on membranes from cyclic mare CL or stallion testes, eCG and eLH bind to the putative LH/CG receptor in equine tissues (eLH/CG-R) and are devoid of FSH binding in contrast with what is observed in other species [15–17]. However, eCG is the most heavily glycosylated of mammalian pituitary and placental glycoprotein hormones and contains much more and bulkier sialylated carbohydrates than eLH [18]. Moreover, eCG binds to the eLH/CG-R with only one tenth or less the affinity of eLH [15–17, 19] and this could be due to its bulky carbohydrates [20]. The carbohydrate content of equine gonadotropins also influences the circulating half-life, which is much longer for eCG (6 days) than for eLH (4–5 h) in the mare [21, 22]. Furthermore, although plasma concentrations of eCG show individual variations, mean eCG values during early pregnancy (up to 35 µg/ml serum) are 50–1000 times greater than eLH levels (5–15 ng/ml) at the ovulatory surge [2, 23]. In this context, it is necessary to examine the ability of eCG to modulate the luteal function, which depends in part on the expression of eLH/CG-R in the primary CL. Indeed, a general property of G protein-coupled receptors [24], including the LH/CG-R [25], is that prolonged agonist stimulation causes receptor downregulation due to internalization and lysosomal degradation of receptors [26, 27]. In the pseudopregnant rat, the exposure of CL to high concentrations of hCG abolishes the capacity of membrane homogenates or isolated cells to bind 125I-hCG [28, 29]. Concomitant with the downregulation of membrane LH/CG-R, a decrease in the level of all mRNA transcripts was observed [28, 29]. Although two studies describe the changes in luteal LH/CG-R concentrations during the postovulatory period (Days 1–14) in the cyclic mare [30, 31], the period of baseline serum LH concentrations (Days 14–35) followed by very high serum eCG concentrations (Day 35 to Days 80–100) in the pregnant mare has not been investigated. The objective of the present study was to examine the expression of LH/CG-R mRNA transcripts and specific binding sites for eLH/CG in the equine primary CL during early pregnancy.


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

Light breed mares and Welsh pony mares (3–17 yr old) with no known reproductive pathology were used in this study. 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 designed as Day 0 of pregnancy. Mares were fertilized by artificial insemination. Animals were manipulated in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.

Sample Collection

Luteal tissues were obtained from one cyclic mare as control (Day 8 postovulation) and from pregnant mares before the onset of eCG secretion (Day 14, n = 2; Day 15, n = 1; Day 26, n = 1; Day 27, n = 1; Day 28, n = 1; Day 31, n = 1), from onset to maximum secretion of eCG (Day 38, n = 1; Day 39, n = 1; Day 40, n = 1; Day 42, n = 1; Day 43, n = 2; Day 44, n = 1; Day 45, n = 1; Day 46, n = 1; Day 56, n = 1; Day 60, n = 1; Day 61, n = 1; Day 62, n = 1), and during the decline of eCG secretion (Day 83, n = 1; Day 89, n = 1; Day 101, n = 1). The ovaries were collected by hemiovariectomy or within 10 min following sodium pentobarbital-induced death as previously described [7]. After dissection from the ovary, pieces of primary CL were immediately snap frozen in liquid nitrogen, then stored at -70°C.

ELH/CG-R cDNA Probes

Two cDNA fragments of the eLH/CG-R were generated using reverse transcriptase polymerase chain reaction (RT-PCR) amplification: a first fragment covering the putative extracellular domain (EC probe) and a second fragment covering the putative transmembrane domain (TM probe) of the eLH/CG-R. Primers for the EC and TM probes were based on the partial eLH/CG-R cDNA sequence (GenBank accession number AY271258). The primers used to generate the EC probe were forward primer, 5'-CTTTCAGAGGACTTAATGAGGT-3', and reverse primer, 5'-TCTAAAAGCACAGCAGTGGCT-3', which correspond to nucleotides 238–259 and 864–884, respectively, when aligned to the bovine LH-R cDNA sequence [32]. The primers used to generate the TM probe were forward primer, 5'-TGATTTGGCTGATTAATATCCTAGC-3', and reverse primer, 5'-GTTGGTAGCTATCAGTTGTGGATTT-3', which correspond to nucleotides 1129–1153 and 1697–1721, respectively, when aligned to the bovine LH-R cDNA. First-strand synthesis was performed according to the manufacturer's recommendations using RNase H- reverse transcriptase (PowerScript; Clontech, Palo Alto, CA), the gene-specific antisense primer, and 2 µg of equine total RNA from a pool of CL (at diestrus and Days 14, 31, and 44 of pregnancy) as template. The individual equine cDNAs were amplified for 30 cycles using the gene-specific primer pairs and Advantage 2 polymerase mix (Clontech), then subcloned into the TA cloning vector pCR II-TOPO (Invitrogen, Carlsbad, CA), and finally sequenced (Genome Express, Meylan, France). The EC and TM probes were both labeled with {alpha}-32P-dCTP (Perkin-Elmer Life Sciences, Boston, MA) using the Rediprime II random prime labeling system (Amersham Pharmacia Biotech, Little Chalfont, U.K.).

Northern Blot Analysis

Total cellular RNA (20 µg) was extracted from all tissues using Trizol reagent (Life Technologies, Gaithersburg, MD) and 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 ultraviolet cross-linking. Blots were prehybridized for 2 h at 42°C in a buffer containing 50% formamide, 5x Denhardt solution, 1% SDS, 5x sodium saline citrate (SSC), and 16 µl/ml denatured salmon sperm DNA (Invitrogen). Blots were then hybridized with one of the two eLH-R cDNA probes overnight at 42°C in a buffer containing 50% formamide, 2.5x Denhardt solution, 1% SDS, 5x SSC, 10x dextran sulfate, and 16 µl/ml denatured salmon sperm DNA. Blots were next washed in 1x SSC plus 0.5% SDS at room temperature for 20 min, followed by three 20-min washes in 0.2x SSC plus 0.5% SDS at 68°C. Membranes were exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) at room temperature for 16–18 h before quantification. Membranes were also exposed to autoradiographic films at -70°C for 1–5 days. Membranes were then washed three times for 20 min with boiling solution of 2x SSC plus 0.2% SDS to remove the first probe, then prehybridized and rehybridized with the second probe as described above. Finally, each membrane was washed three times and prehybridized as described above, then hybridized with the human RNA 18S probe from Ambion, Inc. (Austin, TX). Membranes hybridized with the 18S probe were exposed 1 h to a PhosphorImager screen. All hybridization signals were quantified using ImageQuant software (Molecular Dynamics). Each RNA sample was analyzed 2–5 times on different blots. The RNA sample of one CL at diestrous stage (Day 8 postovulation) 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.

Preparation of Luteal Membranes

Mare luteal tissue was weighed, then homogenized in 25 mM Tris-HCl and 10 mM MgCl2 buffer (pH 7.5; 10 ml/g CL) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) with an Ultra-Turrax homogenizer (Ika-Ultra-Turrax T25; Janke and Kunkel, Staufen, Germany) for 10 sec at 4°C. The homogenate was then centrifuged at 120 x g for 30 min at 4°C. The resulting supernatant was further centrifuged at 30 000 x g for 30 min at 4°C. The pellet containing the crude luteal membranes was resuspended by mild homogenization in 25 mM Tris-HCl Buffer, pH 7.5 (1 ml/g CL), and stored at -20°C until use. The protein concentration of the luteal membrane homogenates was determined by the method of Bradford [33].

Radioreceptor Assays

Homologous equine radioreceptor assays were performed on luteal membranes according to a method previously described for equine testicular fractions [17]. Equine LH (National Hormone & Peptide Program; lot #AFP5130A) was radioactively labeled with 125I-Na (Amersham Pharmacia Biotech) using Iodo-Gen (Pierce, Rockford, IL). Binding specificity of eCG on luteal membranes during pregnancy was assessed using a competitive radioreceptor assay: luteal membranes were incubated with a constant amount of 125I-eLH (30 pM; specific activity of 2100–2400 Ci/mmol) and increasing amounts of unlabeled eLH or eCG (eCG NZY-01, medium molecular weight [MW] fraction [34]). Concentrations of eLH/CG binding sites on luteal membranes were measured using a saturation radioreceptor assay: luteal membranes were incubated with saturating amount of 125I-eLH (7 points from 10–700 pM). Nonspecific binding was determined in the presence of 100 IU hCG (Chorulon; Intervet, Boxmeer, Netherlands), which is known to bind to the eLH/CG-R with similar affinity to eLH [31, 35]. All reaction samples were incubated in duplicate in a final volume of 300 µl 10 mM Tris HCl, 6 mM CaCl2, and 0.25% BSA, pH 7.5. After overnight incubation at room temperature, reactions were stopped by adding 1 ml of cold Tris HCl buffer. Bound and free hormones were separated by centrifugation at 4000 x g for 60 min at 4°C and the pellets were counted in a gamma counter. The data on competition and saturation plots were analyzed with the GraphPad PRISM2.01 software package (San Diego, CA) using the nonlinear "one-site competition" or "one-site binding" curve-fitting procedures in order to estimate the concentration of hormone required to reduce 125I-eLH binding by 50% (IC50), equilibrium constant (Kd), and the maximal binding capacity (Bmax). The number of binding sites was then related to the homogenate protein concentration.

Statistical Analysis

The concentrations of eLH/CG binding sites and mRNA levels are shown as mean ± SEM. Three stages of pregnancy were considered according to the known pattern of eCG secretion: before the onset of eCG secretion (Days 14–31, n = 7), from the onset to the maximum secretion of eCG (Days 38–62, n = 13), and during the decline of eCG secretion (Days 83–101, n = 3). The Kd, concentrations of eLH/CG binding sites, and levels of mRNAs were compared between stages of pregnancy, irrespective of other variables, with the nonparametric Kruskal-Wallis test using StatXact 5 (CYTEL, Cambridge, MA; http://www.cytel.com/). Differences were considered to be significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sequences of eLH/CG-R cDNA Probes

A 646-bp (EC probe; Fig. 1) fragment and a 593-bp (TM probe; Fig. 2) fragment of the eLH/CG-R cDNA were amplified from a pool of CL. The cDNA sequences of the EC and TM probes showed high homology with reported LH/CG-R cDNA sequences in porcine (93% and 94%, respectively) [36], bovine (92.5% and 92.1%) [32], human (89.3% and 86.5%) [37], and murine (87.5% and 86.5%) samples [38]. The EC probe corresponds to exons 2–9 and codes for a major part of the extracellular domain. The 593-bp TM probe is located in exon 11 and codes for a large part of the transmembrane domain, including transmembrane segments 1–5, extracellular loops 1 and 2, intracellular loops 1 and 2, and a part of the third intracellular loop.



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  		FIG. 1. Nucleotide and deduced amino acid sequence of the 646-bp eLH/CG-R cDNA fragment used in Northern blot analysis as the EC probe. Forward and reverse primers are in italics and bold. Conserved cysteines are indicated by black boxes. Putative sites for N-linked glycosylation are underlined (Genbank accession number AY271258)



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  		FIG. 2. Nucleotide and deduced amino acid sequence of the 593-bp eLH/CG-R cDNA fragment used in Northern blot analysis as the TM probe. Forward and reverse primers are in italics and bold. Proposed membrane-spanning hydrophobic sequences are underlined. (Genbank accession number AY271258)

Identification of LH/CG-R mRNA Transcripts in the Equine CL

Northern blot analysis with the EC probe revealed seven eLH/CG-R mRNA transcripts in the primary CL at all stages examined (Fig. 3): four major bands at 7, 5.7, 3.9, and 1.8 kb and three minor signals at 4.9, 2.8, and 0.6 kb. Hybridization of the same blots with the TM probe revealed two major bands at 7 and 3.9 kb and a minor band at 4.9 kb. However, one of the major bands at 5.7 kb and the minor bands at 2.8 and 0.6 kb, all detected with the EC probe, were not revealed with the TM probe (Fig. 3). Furthermore, two minor bands of 1.8 and 2.1 kb were observed on blots hybridized with the TM probe at the location of the major 1.8-kb signal revealed with the EC probe. However, due to the high abundance of this major 1.8-kb signal, we cannot assume that the minor 2.1-kb mRNA transcript was not revealed with the EC probe. The number and apparent size of mRNA transcripts detected with both EC and TM probes did not change between diestrus (used as internal control) and pregnancy or between stages of pregnancy. No hybridization signals were observed with RNA samples from lung, kidney, spleen, or liver (Fig. 4).



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  		FIG. 3. Representative Northern blot analysis of total RNA from one primary CL at Day 61 of pregnancy hybridized with cDNA probes corresponding to the extracellular (EC probe, left panel) or transmembrane (TM probe, right panel) domains of the eLH/CG-R. The sizes of different mRNA transcripts are indicated in kb



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  		FIG. 4. Representative Northern blot analysis of total RNA from lung (lane 1), kidney (lane 2), spleen (lane 3), liver (lane 4), CL from one cyclic mare at diestrous stage as internal control (Day 8 postovulation: lanes 5, 11), CL from pregnant mares before the onset of eCG secretion (Days 14–31: lanes 6, 12–14), during eCG increasing secretion (Days 38–62: lanes 7–9, 15–17), during eCG decreasing secretion (Days 83–101: lanes 10, 18) hybridized with the cDNA EC probe (top panels) and the RNA 18S probe for normalization (bottom panels)

Semiquantitative Analysis of Major LH/CG-R mRNA Transcripts

Major hybridization signals detected with the EC probe were quantified. The relative intensities of the 5.7- and 3.9-kb mRNA transcripts tended to decrease during early pregnancy from Day 14 to Day 101 (P = 0.07 and P = 0.04 for 5.7- and 3.9-kb mRNA transcripts, respectively) whereas mRNA species of 7 and 1.8 kb were equally expressed (P > 0.1) during the same period of time (Figs. 4 and 5).



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  		FIG. 5. Relative intensities (means ± SEM) of major eLH/CG-R transcripts detected with the EC probe in CL from pregnant mares before the onset of eCG secretion (Days 14–31, n = 7), during eCG increasing secretion (Days 38–62, n = 10), and during eCG decreasing secretion (Days 83–101, n = 3)

Binding Specificity of eCG on Luteal Membranes from Pregnant Mares

In order to investigate the binding specificity of eCG on luteal membranes from pregnant mares, competitive binding studies were performed on five corpora lutea collected at Days 39, 40, 42, 60, and 61 of pregnancy. At these days, increasing levels of both eLH and eCG fully competed with 125I-eLH on luteal membranes, indicating that eLH and eCG share identical binding sites on equine CL during early pregnancy. Nevertheless, on a molar basis, eCG (MW = 44 000) bound to the luteal eLH/CG-R with only 2.5%–3.9% the binding affinity of eLH (MW = 34 000) (Fig. 6).



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  		FIG. 6. Competition of eCG with 125I-eLH for binding to luteal membranes from pregnant mares: each point represents the mean ± SEM of results from five pregnant mares at Days 39, 40, 42, 60, and 61 of pregnancy.

Affinity and Concentration of eLH/CG Binding Sites on Luteal Membranes

Saturation of eLH/CG binding sites on luteal membrane homogenates with increasing amounts of 125I-eLH could be achieved for all CL of pregnant mares (Fig. 7). The dissociation constant (Kd) did not change with the stage of pregnancy (P > 0.05) and was on average 1.8 ± 0.2 x 10-10 M (n = 23). The concentration of membrane-bound eLH/CG binding sites, expressed per milligram of membrane protein, changed significantly during early pregnancy (P < 0.01). From the onset to maximum secretion of eCG (Days 38–62) and during the decline of eCG secretion (Days 83–101), luteal membranes had significantly fewer eLH/CG binding sites than before the onset of eCG secretion (Days 14–31) (P < 0.05) (Fig. 8). A similar pattern was observed for the number of receptors per milligram CL (data not shown). However, the mean concentrations of eLH/CG binding sites measured after the onset of eCG secretion were still at 44.6% (Days 38–62; eLH/CG-R concentration = 142.9 ± 33.9 fmol/mg protein, n = 13) to 24.7% (Days 83–101; eLH/CG-R concentration = 79.2 ± 50.8 fmol/mg protein, n = 3) of the mean concentration of eLH/CG binding sites measured before eCG secretion (Days 14–31; eLH/CG-R concentration = 320.5 ± 59.1 fmol/mg protein, n = 7).



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  		FIG. 7. Representative saturation plot of 125I-eLH binding to luteal membranes from one mare at Day 26 of pregnancy. Membrane homogenates were incubated in duplicate with 125I-eLH in concentrations ranging from 10 to 700 pM with or without 100 IU cold hCG



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  		FIG. 8. Concentrations of eLH/CG binding sites on luteal membranes from pregnant mares before the onset of eCG secretion (Days 14–31, n = 7), during eCG increasing secretion (Days 38–62, n = 13), and during eCG decreasing secretion (Days 83–101, n = 3). Values are means ± SEM; *, P < 0.05 compared with the Days 14–31 group


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed the presence of multiple mRNA transcripts for the eLH/CG-R, three of which code for truncated LH/CG-R lacking the transmembrane domain. We observed a slight decrease in eLH/CG-R mRNA levels concomitant with a significant decrease in membrane eLH/CG binding sites in the primary CL after the onset of eCG secretion during early pregnancy. Nevertheless, levels of eLH/CG binding sites remained at minimum 25% of those measured before the onset of eCG secretion and their affinity did not change during early pregnancy. These results suggest that the primary CL remains responsive to eCG despite the long half-life and the very high concentration of eCG over this period of time during early gestation.

This paper reports for the first time the cloning and sequencing of fragments of the eLH/CG-R. Northern blot analysis using a cDNA probe corresponding to the extracellular (EC) region of the eLH/CG-R (EC probe) revealed the presence of seven mRNA transcripts (7, 5.7, 4.9, 3.9, 2.8, 1.8, and 0.6 kb) in the primary CL. This observation is in good agreement with other studies showing several LH/CG-R transcripts variable in size, number, and relative abundance in porcine [36], bovine [39], ovine [40], human [41], and rodent [42] ovaries and testes. The multiple LH/CG-R mRNA transcripts observed in the equine CL may arise from alternate transcriptional start sites, multiple sites of polyadenylation combined with different lengths of polyadenylation, and/or alternate or incorrect splicing of the LH/CG-R gene. In order to address this latter question, we probed blots with a cDNA probe corresponding to the transmembrane (TM) region of the LH/CG-R (TM probe). As the open reading frame of the mammalian LH/CG-R is 2.1 kb, the mRNA species larger than 2.1 kb were expected to be detected with the TM probe. Surprisingly, the major bands of 5.7 kb and 2.8 kb, both detected with the EC probe, were not revealed with the TM probe, whereas the mRNA transcript of 1.8 kb, detected with the EC probe, was also detected with the TM probe. This situation is quite different from that observed in rat ovaries and testes, in which only LH/CG-R mRNAs smaller than 2.1 kb were shown to lack the transmembrane domain [28, 42]. In the equine CL, our results show that the presence of multiple LH/CG-R transcripts arises in part from alternate splicing of the LH/CG-R primary transcript. However, other processes, like alternate transcriptional and/or adenylation sites, seem to be also implicated in large differences in sizes of mRNA transcripts.

Our results indicate that the 5.7- and 2.8-kb mRNA species, if translated, would encode for truncated eLH/CG-R lacking the transmembrane domain. Numerous LH/CG-R cDNAs lacking the transmembrane domain have been cloned and sequenced in other mammalian species (porcine [36], ovine [40], rat [42], bovine [43]). However, little is known on the possible translation and secretion of such splicing isoforms. Studies on mammalian cells transfected with natural or artificially altered LH/CG-R forms lacking the transmembrane domain have shown that the binding affinity for hCG was comparable with cells expressing the full-length receptor form [44–47]. In these studies, the translated products of natural variant forms of the porcine and rat LH/CG-R were in part secreted in the culture medium [44, 45] whereas artificially constructed forms were not [46, 47]. In the sheep ovary, two variants lacking the transmembrane domain have been shown to be translated in vivo and were located in the cytosol [48]. Consequently, the presence of LH/CG-R mRNAs lacking the transmembrane domain during early pregnancy in the equine CL raises the possibility that some truncated eLH/CG-R with hormone binding activity would be secreted in vivo. The roles of such secreted isoforms, if any, might be to capture free eCG in excess and prevent saturation and long-term downregulation of luteal cell surface eLH/CG-R. Whether eLH/CG-R isoforms could modulate luteal eLH/CG-R function remains, however, to be elucidated.

The quantitative levels of major mRNA species tended to decrease (5.7- and 3.9-kb mRNA transcripts) or did not change (7- and 1.8-kb mRNA transcripts) after the onset of eCG secretion, suggesting that eCG has limited or no effects on the LH/CG-R gene expression in luteal cells despite its very high circulating level (>35 µg/ml serum) [2]. This is in contrast with studies performed in the rat ovary, in which a single high dose of hCG induced a marked decrease in rat LH-R transcript levels [28, 29]. In the equine CL, the apparent resistance of the LH/CG-R expression to high levels of eCG may arise from the weak binding affinity of eCG to luteal cells or from a constitutive expression and renewal of the eLH/CG-R during early pregnancy, which would terminate at the time of luteal regression.

Radioreceptor assays revealed common binding sites for eCG and eLH on luteal membranes during early pregnancy, which is in good agreement with data obtained on stallion testicular membranes or luteal membranes from cyclic mares [15–17, 19]. Furthermore, the binding affinity of eCG on equine luteal membranes was not enhanced during pregnancy and remained only 2.5%–3.9% that of eLH, which supports results obtained with membrane homogenates from stallion testis or cyclic mare CL [15–17, 19]. The affinity determined here for the eLH/CG-R (Kd = 1.8 ± 0.2 x 10-10 M) during pregnancy was similar to those described by others in luteal membranes or luteal cells from cyclic mares [30, 31], indicating that the binding site of the luteal eLH/CG-R is not subjected to conformational changes at the time of eCG secretion. This absence of change in the binding affinity of eLH/CG-R is similar to what was observed during bovine [49] and porcine [50] pregnancy. This is, however, in contrast with results obtained in primate CL, in which a decrease in the affinity of LH/CG-R was observed after prolonged exposure to exogenous hCG during simulated early pregnancy [51]. The absence of change in eLH/CG-R affinity in the primary CL suggests that the capacity of eCG to modulate luteal function would depend on the number of eLH/CG binding sites.

The concentration of membrane eLH/CG binding sites determined here for the horse primary CL ranged from 79.2 to 320.5 fmol/mg of protein, which is much higher than what was observed in bovine (5.6–9.6 fmol/mg protein) [49] or porcine (41–95 fmol/mg protein) [50] CL during pregnancy. The high level of membrane-bound LH/CG-R on the equine CL might be a specific mechanism to compensate the weak binding affinity of eCG to its receptor in equine species. Nevertheless, these differences in LH/CG-R concentrations could also arise from differences in techniques used for receptor or/and protein measurements.

In pregnant mares and pony mares, mean plasmatic LH decreases rapidly following the ovulatory surge to baseline levels from Days 6–8 to Day 35 of pregnancy [3]. For this reason and the relatively short plasmatic half-time of circulating pituitary eLH (5 h) [22], the majority if not all luteal LH/CG-R is likely to be unoccupied at this time of pregnancy. Thus, the concentration of luteal eLH/CG-R measured between Days 14 and 31 of pregnancy would correspond to the total number of eLH/CG binding sites on luteal membranes. In contrast, CL collected between Days 38 and 101 of pregnancy were subjected in vivo to high concentrations of circulating eCG before being used as membrane homogenates in 125I-eLH saturation binding assays. It is thus possible that we measured only available and not total eLH/CG binding sites during this period of time. However, according to the fact that eCG binds to the luteal eLH/CG-R with only 2.5%–3.9% the eLH affinity, saturating amounts of 125I-eLH used in the radioreceptor assay must have exchanged with bound eCG on luteal membranes because eLH exhibits more than 30-fold higher affinity than eCG. The total number of eLH/CG binding sites on luteal membrane homogenates nevertheless includes an unknown proportion of eLH/CG-R sequestered within intracellular membrane compartment.

A decrease in luteal eLH/CG binding site concentration was observed between Days 38 and 101 of pregnancy, when the weight of the primary CL remains constant [8]. This suggests that the decrease in eLH/CG-R content was not due to modifications in the luteal cell population. Last, this decrease occurred without any significant change in major transcript levels during the time of eCG secretion, suggesting that this downregulation does not overcome the capacity of equine luteal cells to recycle and/or to resynthesize eLH/CG-R at their membrane surface.

In conclusion, the presence of luteal eLH/CG-R mRNAs and of a substantial part of membrane eLH/CG binding with high affinity after the onset of eCG secretion suggest that the primary CL remains responsive to eCG until the transition from a luteal to a placental source of steroids during early pregnancy in the mare.


   ACKNOWLEDGMENTS
 
We sincerely thank François Lecompte, Guy Duchamp, and Jean-Claude Poirier for technical assistance. We thank A.F. Parlow (National Institutes of Health) and the National Hormone and Peptide Program of the National Institute for Diabetes and Digestive and Kidney Diseases for supplying the eLH.


   FOOTNOTES
 
1 M.S.-D. was supported by a fellowship from the Institut National de la Recherche Agronomique and the Région Centre. Back

2 Correspondence: Marie Saint-Dizier, Equipe Hypophyse, Station Physiologie de la Reproduction et du Comportement, Institut National de la Recherche Agronomique (INRA), 37380 Nouzilly, France. FAX: 33 2 47 42 77 43; dizier{at}tours.inra.fr Back

3 Current Address: Equitechnique, Le Mesnil Vicomte, 61240 Le Merlerault, France Back

Received: 30 April 2003.

First decision: 27 May 2003.

Accepted: 8 July 2003.


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 RESULTS
 DISCUSSION
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