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a Equipe de Neuroendocrinologie sexuelle, INRA-PRMD, 37380 Nouzilly, France
b Station d'Amélioration génétique des Animaux, Auzeville BP27, 31326 Castanet-Tolosan cedex, France
c Unité de Zootechnie méditerranéenne, INRA-ENSA, 34060 Montpellier cedex 1, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In conclusion, the data show an association between genotype -- for site MnlI at position 605 and seasonal anovulatory activity in MA ewes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Mediterranean latitudes, great variability exists between breeds and within breeds in terms of the presence and duration of anestrus. Some ewes completely cease ovulatory activity, whereas others show isolated ovulations during anestrus or continue to cycle throughout the year [9, 10]. In the Mérinos d'Arles breed, raised in southern France, the presence of spontaneous ovulatory activity during spring in about 30% of ewes is a repeatable trait under genetic influence [11], suggesting that alleles of specific genes control the presence or absence of spontaneous spring ovulations.
Using this Merino sheep model, we tested the hypothesis of a difference in genomic structure of the gene for Mel1a receptor between two sets of ewes chosen as extremes in terms of the distribution of genetic value taking into account both their own performance and that of their relatives.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Mérinos d'Arles (MA) breed was chosen because some animals exhibit spontaneous ovulation during the normal anestrous season. Furthermore, the availability of a large flock for experimentation made it possible to choose animals with contrasting status in terms of out-of-season ovulatory activity. The very seasonal Ile-de-France breed was used to verify how widespread the mutations and allelic isoforms of Mel1a receptor found in the MA breed are.
Once the experimental animals had been chosen, the protocol involved preparing genomic DNA, amplifying exon II of the Mel1a receptor, and genotyping samples for the presence or absence of the polymorphic MnlI site as demonstrated by Messer et al. [8]. Series of cDNAs were sequenced to establish the mutations associated with the presence or absence of the polymorphic MnlI site. Finally, the characteristics of 125I-melatonin binding to pars tuberalis membranes were examined in the case of the two most important genotypes identified (referred to below as -- and ++). In this case, it was postulated that the Mel1a receptor studied here in the pars tuberalis was the same as that present in the premammillary area.
Choice of Experimental Animals and Management Conditions
MA breed The experimental animals, for which pedigree information was available over 5 generations involving 3044 animals, had been used in a previous experiment in which the genetic value of spontaneous spring ovulatory activity was determined [11]. These ewes were part of the experimental flock of the Domaine du Merle located in the Southeast of France (43.5°N). The spontaneous ovulatory activity of ewes was determined in April, the expected resting season, over 3 consecutive years (1995–1997). Two jugular blood samples were collected at an interval of 8–10 days during the first 2 wk of April. A plasma progesterone level of over 1 ng/ml in one sample indicated that the ewes were in ovulatory activity [12]. A total of 933 ewes, daughters of 176 rams, were studied for 1–3 yr depending on the replacement of culled animals. Thus blood samples were collected from 293, 326, and 314 ewes over 1, 2, and 3 yr, respectively. The experimental ewes in which polymorphism was assessed were selected from among these 933 ewes for their genetic value, which takes into account their own ovulatory activity adjusted by environmental parameters and that of their relatives [11]. Two groups of 36 ewes, a "low group" (L) and a "high group" (H), were chosen as the most extreme ewes based on their genetic value for spring spontaneous ovulatory activity. Ewes of the L group were born from 33 different dams and 17 different sires, while those of the H group were born from 35 different dams and 26 different sires. Of the sires, 14 had daughters in both groups so that 29 ewes of the L group and 21 of the H group shared the same fathers and were half sisters.
Thirty-eight ewes culled out from the flock, according to a general management procedure unrelated to the present experiment, were also kept for genotyping in order to select animals to study 125I-melatonin binding to pars tuberalis membranes.
Animals from the MA breed are referred to as MA ewes.
Ile-de-France breed This experiment was carried out at the INRA, Research Center of Nouzilly, France (47°N). The whole Ile-de-France flock from which the experimental animals were obtained is a large flock of about 2500 animals. Sires are purchased at regular intervals from various external flocks and are introduced to avoid inbreeding and maintain genetic links with the national French scheme of genetic improvement of the Ile-de-France breed. This experiment was performed to confirm, in another breed, the polymorphism of the melatonin receptor observed in the MA breed. Thus it was carried out without reference to incidental spontaneous ovulation in spring. Twenty-nine ewes were chosen at random. Animals from the Ile-de-France breed are referred to as IF ewes.
Genomic DNA Preparation
Two blood samples of 10 ml were collected by jugular venipuncture with EDTA as anticoagulant and kept frozen until DNA preparation. The technique of DNA preparation from leukocytes was adapted from Miller et al. [13]. In brief, thawed samples were diluted four times with cold SLR buffer (10 mM Tris HCl, pH 7.6, 5 mM MgCl2, and 10 mM NaCl) and centrifuged at 1000 rpm for 7 min at 4°C. This process was repeated two or three times in order to obtain clean leukocytes. These were recovered with 4.5 ml TE buffer (10 mM Tris, pH 7.6, 0.1 mM EDTA) to which 100 µl of a 10 mg/ml solution of proteinase K (Boehringer, Mannheim, Germany), 250 µl EDTA (0.5 M, pH 8), and 250 µl SDS 10% was added. The mixture was transferred to 50°C for 2 h under mild agitation. Thereafter, proteins were precipitated by adding 2.15 ml saturated NaCl, and samples were centrifuged at 3800 rpm for 20 min at 20°C. Absolute ethanol (2.5 volume) was added to the supernatant, which precipitated DNA. After 5-min centrifugation at 13 000 rpm, DNA precipitate was rinsed three times with 70% ethanol, dried briefly, and then dissolved in 1 ml TE buffer by gentle agitation at 4°C for 2 days. Concentration, as assessed by optical density at 260 nm, was usually 50–150 ng/µl. Solutions of genomic DNA were kept at 4°C without apparent deterioration for months. Because of blood coagulation in one of the L group ewes, DNA preparation failed; and in the end, genotyping involved 35 ewes of the L group and 36 ewes of the H group. In addition, genomic DNA preparation was performed in the 38 culled-out MA ewes in order to select homozygous animals to study melatonin binding (see below) as well as in 29 IF ewes.
Genotyping Samples
Genomic DNA (100–150 ng) was used for polymerase chain reaction (PCR) employing the primers of Messer et al. [8] corresponding to positions 285–304 (sense primer) and 1108–1089 (antisense primer) of the sequence from Reppert et al. [4]. Amplification consisted of 35 PCR cycles using 1 U Taq polymerase (Pharmacia Amersham Biotech, Uppsala, Sweden) (denaturation: 94°C, 1 min; annealing: 62°C, 1 min; extension: 72°C, 2 min), followed by a final extension step at 72°C for 10 min. PCR products (32 of 50 µl) were digested overnight with 2 U MnlI (New England Biolabs, Beverly, MA). After 3-fold concentration by heating samples to 65°C, the resulting fragments were separated by electrophoresis on a 3% agarose gel in parallel with a 100-base pair (bp) DNA marker (Invitrogen, Leek, The Netherlands). Agarose consisted of a mixture of 1.5% NuSieve Agarose (FMC Bioproducts, Rockland, ME) and 1.5% Appligene Agarose (Appligene Oncor, Illkirch, France). While 8 cleavage sites for MnlI are present within the exon II sequence, only 1 was shown to be polymorphic [8]. This site, at position 605 in the reference sequence [4], depends on an enzyme recognition site on noncoding strand, corresponding to positions 612–615 on the coding strand. The presence of the cleavage site resulted in a 236-bp and a 67-bp fragment, while the loss of this site led to a single 303-bp fragment. An allelic isoform with the cleavage site present was referred to as + whereas it was referred to as - when such a site was absent. Theoretical combinations were therefore genotypes ++, +-, and -- (Fig. 1, lanes 2, 3, and 4, respectively).
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Genotyping was performed in the 109 DNA samples prepared from MA ewes and the 29 samples from IF ewes.
Sequencing
The sequencing was performed in individuals whose genotype was already known. Thus the sequences studied were as follows: 1) in MA ewes, group L genotype ++, n = 10; genotype --, n = 10; group H genotype ++, n = 10; 2) in IF ewes, n = 6 for each genotype ++ and --.
Two cDNA preparation procedures were used before sequencing. In one procedure, cDNA was obtained by PCR as indicated above except that 1 U Vent polymerase (New England Biolabs) was used. Complementary DNA was then subcloned into pBluescript plasmid (Stratagene, La Jolla, CA). DNA was then sequenced from both directions using an automatic DNA sequencer (Applied Biosystem, Foster City, CA). This procedure was used with the genomic DNA of 7 MA ewes. In the other procedure, cDNA was first obtained from at least 3 PCR per genomic DNA. Pooled products were purified by electrophoresis on a 1% agarose gel, and the single band recovered from the gel was further purified using the Qiaex II kit (Qiagen, Hilden, Germany). Direct sequencing after this procedure was performed in 35 ewes (23 MA ewes and 12 IF ewes), of which 10 MA ewes were sequenced in both directions and the rest only in the sense direction. Nucleotide sequences were first compared to the sequence of the Mel1a receptor taken as a reference [4]. Any identical base change from the standard sequence that was found at least twice at the same position within the 42 sequences obtained was considered a "mutation." Furthermore, multiple sequence alignments [14] were used to define families of sequences with similar sets of mutations.
Melatonin Binding to Pars Tuberalis Membranes
Melatonin binding to pars tuberalis membranes was studied in 12 animals: 6 MA ewes with genotype ++ and 6 MA ewes with genotype --.
Animals were slaughtered by decapitation by a licensed butcher in the laboratory slaughterhouse. The animals were slaughtered in the afternoon so that the melatonin receptors would be at their highest level of the day [15]. Pars tu-beralis were recovered approximately 2 min later and immediately frozen in liquid nitrogen.
Melatonin was labeled with 125I as previously described [16]. Binding to membranes was studied according to a method validated for ovine pars tuberalis [15] with several modifications. First, in order to improve the comparison of the two groups, pars tuberalis were processed in pairs, one from an animal of genotype ++ and one from an animal of genotype --; the ages of the ewes from a given pair were similar. Second, at the time of the assay, 6 doses of 125I-melatonin, approximately 5–100 pM, were first delivered in quadruplicate in 3-ml tubes, with or without a 200-fold excess of cold melatonin. Third, thawed pars tuberalis were homogenized in 4 ml buffer in order to obtain an equivalent of 1/40 pars tuberalis per 100 µl delivered to each assay tube. Computations using the Scatchard method [17] allowed measurement of the number of binding sites (Bmax) and the dissociation constant (Kd). Protein concentration of each pars tuberalis homogenate was measured with a protein assay kit derived from the Lowry method [18] (Sigma Diagnostics, St. Louis, MO), using BSA as standard. Results were expressed in fmol/mg protein.
Statistical Tests
Distribution of genotypes between the H and L groups was studied using the chi-square method. Binding of 125I-melatonin in pars tuberalis membranes of ewes of genotypes ++ and -- was compared using two-way ANOVA (Statview; Abacus Concepts, Berkeley, CA) with the genotype and the batch of iodine as factors.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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From the 36 group H ewes, 30 were sampled over the 3 yr of the experiment; 27 of these were cyclic each year while 3 were cyclic 2 yr out of 3. Five ewes were sampled in Years 2 and 3 only and were cyclic in both years. Lastly, 1 ewe was sampled only in the third year of the experiment and was found to be cyclic.
From the 35 group L ewes, 32 were sampled during the 3 yr of the experiment and were never found to be cyclic. Three ewes were sampled in Years 2 and 3 and were noncyclic (Table 1).
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Considering ewes sampled in each of the 3 yr of the experiment, the frequency of ewes cycling each year differed very significantly between the two groups (group H vs. group L: 90% vs. 0%; P < 0.001).
Genotyping and Distribution of Allele Frequencies in Group H and Group L
Digestion by MnlI of the cDNAs obtained after PCR amplification distinguished three types of animals as evidenced on agarose gel after electrophoresis (Fig. 1). Individuals were referred to as having ++, +/-, or -- genotypes.
Groups H and L differed significantly in terms of frequencies of homozygous (++), heterozygous (+-), and homozygous (--) ewes (group H: 52.8%, 47.2%, and 0% vs. group L: 28.5%, 42.9%, and 28.5%, respectively; P < 0.001). Frequency of homozygous (++) ewes was significantly higher (P < 0.05) and frequency of homozygous (--) ewes was significantly lower (P < 0.001) in group H than in group L (Fig. 2).
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Analysis of the frequencies of genotypes in half sibs (21 ewes in group H and 29 ewes in group L) yielded results similar to those observed for the totality of groups H and L (group H: 52.4%, 47.6%, and 0% vs. group L: 27.6%, 48.3%, and 24.1%; P < 0.01).
Sequencing
Sequencing verified, in all cases, the presence (or absence) of the polymorphic MnlI cleavage site as assessed by agarose gel electrophoresis. The absence of a cleavage site at position 605 resulted in a single mutation consisting of the substitution of a C by a T on strand - where the recognition site is situated. A complementary substitution of a G by an A on strand + occurred at position 612.
Of the 30 sequences studied in MA ewes, 4 sequences were found to be identical to the sequence of sheep Mel1a receptor [4] in the part of exon II studied here, i.e., between positions 305 and 1088, excluding the positions of the primers. These sequences were found in 4 ewes with genotype ++, 2 from group L and 2 from group H.
In addition to mutation 612 mentioned above, 9 other mutations were observed, although these were not found to be present simultaneously (see below). Each mutation was found to be unique for a given position as indicated in Table 2 and in each case corresponded to a substitution of a C or G by an A or T. Mutations at positions 706 and 893 resulted in the substitution of a valine by an isoleucine and of an alanine by an aspartic acid in the amino acid sequence, respectively. The other mutations were silent. In all, a total of 10 different mutations were registered.
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Table 3 indicates that 8 of the 10 recorded mutations could be found in the MA ewes of groups H and L with genotype ++. Conversely, only 4 mutations were found in ewes of group L with genotype --, including 2 mutations peculiar to this genotype: one in position 612, responsible for the absence of MnlI site, and the other at position 706, associated with an amino acid change.
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Of 29 IF ewes genotyped, 8, 10, and 11 ewes were found to have genotypes ++ (28%), +/- (34%), and -- (38%), respectively. Direct sequencing, after cDNA amplification by PCR of 6 ewes with genotype ++ and 6 ewes with genotype --, indicated the presence of the same mutations as in MA ewes both in genotype ++ (8 mutations) and genotype -- (4 mutations) (Table 3).
Presence of Different Alleles
Mutation at position 612, responsible for the absence of MnlI site, was always found to be associated with the 3 other mutations at positions 453, 706, and 891 found in genotype --. This result was observed when sequencing was carried out either after cloning or directly after PCR, both in MA and in IF ewes (n = 16).
In contrast with the 4 mutations always observed together in genotype --, the 8 mutations found in genotype ++ were not found to be present simultaneously in each sequence but were found to group together within different sets of mutations.
Sequencing after cloning made it possible to distinguish between three types of sequences in ewes with genotype ++: 1) sequences without mutation; 2) sequences with 5 mutations at positions 606, 783, 801, 891, and 893; and 3) sequences with 7 mutations at positions 426, 453, 555, 606, 783, 801, and 891.
Sequencing directly after cDNA amplification made it possible to identify a type of sequence with a single mutation at position 606, either on the two parental chromosomes or on only one of them. Another type of sequence, with 5 mutations that were not the result of the combination of the previous sets of sequences found in ewes with genotype ++, was also observed with mutations at positions 555, 606, 783, 801, and 891; but the contribution of each parental chromosome used as a matrix for PCR primers could not be identified here. From an examination of the sequencing pattern carried out directly after PCR, other allelic isoforms involving the same 8 mutations in genotype ++ were indicated but needed sequencing after cloning to be ascertained.
Melatonin Binding to Pars Tuberalis Membranes from Ewes of Genotype ++ and of Genotype --
The protein content of the pars tuberalis homogenates did not differ between genotypes (1.44 ± 0.12 vs. 1.29 ± 0.07 mg protein for genotype ++ and genotype --, respectively).
The difference between the mean Bmax in the two genotypes (43.2 ± 4.4 vs. 75.4 ± 8.4 fmol/mg protein in genotype ++ and genotype --, respectively) was very highly significant (P < 0.0005). Conversely, Kd values did not differ between groups (general mean: 10.9 ± 0.99 pM).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three main results were obtained. 1) The frequency distribution of genotypes for the melatonin receptor Mel1a gene was found to be very different for the two groups; in particular, a genotype corresponding to the absence of polymorphic MnlI cleavage site on both parental chromosomes, genotype --, was observed only in group L ewes noncycling in spring. 2) This genotype was associated with a single allelic isoform of exon II of the melatonin1a receptor in contrast with genotype ++, which is represented by a series of different isoforms. 3) The Bmax value of the 125I-melatonin binding to the membranes of pars tuberalis was higher in ewes with genotype -- than in those with genotype ++.
Spontaneous ovulation in early spring in the MA ewe has been studied [11], and this trait was found to have a significant heritability (h2 = 0.20) in a model taking into account several physiological parameters such as weight and age of animals. This study and that of other authors [19, 20] suggest the existence of a common overall factor involved in the control of reproductive seasonality. One possible candidate could be melatonin secretion. However, it was demonstrated that although heritability of mean plasma melatonin levels has been found to be high (h2 = 0.45) [21], there was no difference between the mean levels in ewes of groups L and H (unpublished results). Therefore we decided to study the influence of the structure of the gene for Mel1a receptor.
Previous studies have demonstrated that the transduction of the light regime on reproduction occurs through melatonin binding to the premammillary hypothalamus [2]. Although the type of receptor at this level is still unknown, the choice to study Mel1a receptor is supported by the following findings: 1) Mel1a receptor has been cloned in different mammalian species [4] and is widely found in the brain; 2) Mel1b receptor [5], found in the retina and hippocampus, does not seem to be physiologically involved in reproductive seasonality, since the natural knock-out of the gene coding for this receptor does not prevent seasonal reproduction in the golden hamster and the Djungarian hamster [6]; 3) polymorphism of exon II of Mel1a receptor has been observed [7], and the incidence of a mutation leading to the absence of a specific MnlI cleavage site has been found to be variable in breeds with different reproductive seasonalities [8].
Genotyping performed in the MA and IF ewes confirmed the existence of a polymorphic MnlI recognition site at position 612 as in other breeds studied [8]. It led to genotypes ++, +-, and --, the latter corresponding to the absence of a site on both parental chromosomes. Consistent with the results of Messer et al. [8], we found an inverse relationship between the genotype -- frequency of MA and IF breeds (14% vs. 38%) and the ability of ewes to spontaneously ovulate in spring, since in contrast with MA ewes, IF ewes are considered to be a highly seasonal breed [9]. Importantly, the present study extends previous results by demonstrating for the first time an association within breed between the genotype -- and the absence of ovulation in spring. Moreover, the fact that this association was also observed within families (half sibs data) is the very first step toward true evidence of genetic linkage.
These results have led to a more accurate comparison between genotypes -- and ++.
Ten point mutations of the standard sequence of Mel1a exon II were observed within the limits studied here (positions 305–1088 excluding the primers). Five of these ten mutations—at positions 606, 786, 801, 891, and 893—were identical to those already described for the receptor named Mel1aß [7]; and two mutations at positions 606 and 612 have been evidenced by Messer et al. [8]. A mutation at position 612 corresponded to the recognition site for MnlI leading to the absence of cleavage site at position 605. A mutation at position 606 corresponded to an absence of RsaI site also studied [8]. Finally, we demonstrated the existence of four new mutations at positions 426, 453, 555, and 706. More importantly, we put forward the existence of multiple allelic isoforms characterized by a variable number of the ten recorded mutations. Among these alleles, the 16 sequencings of cDNAs genotyped -- strongly indicated the existence of an allele called -. This allele is characterized by the constant presence of the four identical mutations including, by definition, the mutation of the polymorphic MnlI site and also a mutation at position 706, hitherto unknown, in the different + allele combinations. However, the presence of other mutations in parts of the Mel1a sequence not studied here is also plausible.
When the polymorphic MnlI site is present, a series of allelic isoforms, at least 5, have been demonstrated; but it is likely that their number will increase after further sequencing of cloned cDNAs. In the present study, the mutation at position 606 leading to the absence of RsaI site [8] was not found to be of interest for reproductive seasonality. In addition, the genotype ++ was found to be present both in L and H groups, differing by their spontaneous out-of-season ovulatory activity. This suggests that this genotype could be considered neutral toward seasonality, which is also controlled by other environmental parameters such as feeding, incidentally in interaction with photoperiodism.
In expression studies, mutations described in Mel1aß exon II have been found to have no effect on the 125I-melatonin binding to membranes of the pars tuberalis [7]. In our case, the only new mutation changing the amino acid sequence was the mutation at position 706 in the - allele leading to the substitution of a valine at position 220 by an isoleucine in the fifth transmembrane domain. At first sight, this change alone does not seem capable of being a determining factor in the expression of seasonal anestrus in the ewe. Indeed, the presence of an isoleucine at position 220 of the amino acid sequence is observed in various mammalian species, among which some, such as the golden hamster and the Djungarian hamster, have marked reproductive seasonality [4, 22] and others, such as the human and pig, do not [4, 8]. However, isoleucine 220 is close to histine 211, whose mutation modifies the Kd value of 125I-melatonin binding to Mel1aß receptor [23]. The importance of this particular amino acid could be studied by mutagenesis, but it is also possible that as yet unrecorded mutations in the nonstudied part of the Mel1a receptor are involved in the occurrence of seasonal anestrus. However, in a preliminary approach, we therefore examined the parameters of 125I-melatonin binding to membranes of the pars tuberalis in individuals previously genotyped ++ and --.
Experiments in 125I-melatonin binding have shown that Kd values did not differ between genotypes -- and ++ but that the number of binding sites, Bmax, was significantly higher in ewes genotyped -- than in those genotyped ++. This demonstrates, for the first time, a relationship between an allelic isoform of the receptor and one of the 125I-melatonin binding parameters. To interpret the significance of this finding it will be necessary in the future to examine, in both genotypes, other parameters of functional importance such as those involved in the signaling pathway.
In conclusion, we have demonstrated 1) that there is an association between seasonal ovarian inactivity in MA ewes and the homozygous genotype for the absence of a polymorphic MnlI site of the Mel1a exon II; 2) that there is a unique set of mutations, specific to this - allele and a particularly high binding of 125I-melatonin to pars tuberalis membranes in ewes with genotype --; and 3) that due to different combinations of mutations in exon II, the number of allelic isoforms was greater than expected from the description of 
and ß subtypes of the Mel1a receptor [4, 7]. The next step in the present study will be to establish whether the association between genotype and ovarian seasonality is a true genetic linkage. The fact that this association remains within families supports this hypothesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Partly supported by INRA program "Génome et Fonctions." 
2 Correspondence: J. Pelletier, Equipe de Neuroendocrinologie sexuelle, INRA-PRMD, CNRS URA 1291, 37380 Nouzilly, France. FAX: 33 02 47 42 77 43; pelletie{at}tours.inra.fr
Accepted: November 15, 1999.
Received: October 4, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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