Loys Bodin

Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes

Ewes from the Booroola strain of Australian Mérino sheep are characterized by high ovulation rate and litter size. This phenotype is due to the action of the FecBB allele of a major gene named FecB, as determined by statistical analysis of phenotypic data. By genetic analysis of 31 informative half-sib families from heterozygous sires, we showed that the FecB locus is situated in the region of ovine chromosome 6 corresponding to the human chromosome 4q22–23 that contains the bone morphogenetic protein receptor IB (BMPR-IB) gene encoding a member of the transforming growth factor-β (TGF-β) receptor family. A nonconservative substitution (Q249R) in the BMPR-IB coding sequence was found to be associated fully with the hyperprolificacy phenotype of Booroola ewes. In vitro, ovarian granulosa cells from FecBB/FecBB ewes were less responsive than granulosa cells from FecB+/FecB+ ewes to the inhibitory effect on steroidogenesis of GDF-5 and BMP-4, natural ligands of BMPR-IB. It is suggested that in FecBB/FecBB ewes, BMPR-IB would be inactivated partially, leading to an advanced differentiation of granulosa cells and an advanced maturation of ovulatory follicles.

Regulation of ovulation rate in mammals: contribution of sheep genetic models

Ovarian folliculogenesis in mammals from the constitution of primordial follicles up to ovulation is a reasonably well understood mechanism. Nevertheless, underlying mechanisms that determine the number of ovulating follicles were enigmatic until the identification of the fecundity genes affecting ovulation rate in sheep, bone morphogenetic protein-15 (BMP-15), growth and differentiation factor-9 (GDF-9) and BMP receptor-1B (BMPR-1B). In this review, we focus on the use of these sheep genetic models for understanding the role of the BMP system as an intra-ovarian regulator of follicular growth and maturation, and finally, ovulation rate.

Genome-Wide Association Studies Identify Two Novel BMP15 Mutations Responsible for an Atypical Hyperprolificacy Phenotype in Sheep

Some sheep breeds are naturally prolific, and they are very informative for the studies of reproductive genetics and physiology. Major genes increasing litter size (LS) and ovulation rate (OR) were suspected in the French Grivette and the Polish Olkuska sheep populations, respectively. To identify genetic variants responsible for the highly prolific phenotype in these two breeds, genome-wide association studies (GWAS) followed by complementary genetic and functional analyses were performed. Highly prolific ewes (cases) and normal prolific ewes (controls) from each breed were genotyped using the Illumina OvineSNP50 Genotyping Beadchip. In both populations, an X chromosome region, close to the BMP15 gene, harbored clusters of markers with suggestive evidence of association at significance levels between 1E−05 and 1E−07. The BMP15 candidate gene was then sequenced, and two novel non-conservative mutations called FecXGr and FecXO were identified in the Grivette and Olkuska breeds, respectively. The two mutations were associated with the highly prolific phenotype (pFecXGr = 5.98E−06 and pFecXO = 2.55E−08). Homozygous ewes for the mutated allele showed a significantly increased prolificacy (FecXGr/FecXGr, LS = 2.50±0.65 versus FecX+/FecXGr, LS = 1.93±0.42, p<1E−03 and FecXO/FecXO, OR = 3.28±0.85 versus FecX+/FecXO, OR = 2.02±0.47, p<1E−03). Both mutations are located in very well conserved motifs of the protein and altered the BMP15 signaling activity in vitro using a BMP-responsive luciferase test in COV434 granulosa cells. Thus, we have identified two novel mutations in the BMP15 gene associated with increased LS and OR. Notably, homozygous FecXGr/FecXGr Grivette and homozygous FecXO/FecXO Olkuska ewes are hyperprolific in striking contrast with the sterility exhibited by all other known homozygous BMP15 mutations. Our results bring new insights into the key role played by the BMP15 protein in ovarian function and could contribute to a better understanding of the pathogenesis of women′s fertility disorders.

Genetic components of litter size variability in sheep

Classical selection for increasing prolificacy in sheep leads to a concomitant increase in its variability, even though the objective of the breeder is to maximise the frequency of an intermediate litter size rather than the frequency of high litter sizes. For instance, in the Lacaune sheep breed raised in semi-intensive conditions, ewes lambing twins represent the economic optimum. Data for this breed, obtained from the national recording scheme, were analysed. Variance components were estimated in an infinitesimal model involving genes controlling the mean level as well as its environmental variability. Large heritability was found for the mean prolificacy, but a high potential for increasing the percentage of twins at lambing while reducing the environmental variability of prolificacy is also suspected. Quantification of the response to such a canalising selection was achieved.

The large variability in melatonin blood levels in ewes is under strong genetic influence

The present study was conducted to assess the degree of genetic determination of the variability in the mean nocturnal plasma concentration of melatonin in sheep. Three hundred twelve ewes born from 18 males and with known genealogy were sampled at the summer and the winter solstices. The nocturnal plasma melatonin concentration was defined as the mean of four plasma samples taken at hourly intervals in the middle of the night (2200-0200). Identity of the father ( P < 0.001) and the solstice ( P < 0.05) were significant. Melatonin concentrations varied considerably among individuals [338.4 ± 197.5 (SD) pg/ml; range 26.6-981.3 pg/ml] and between rams regarding the melatonin concentrations of their daughters (range from 202.9 to 456.3 pg/ml). Inheritance was analyzed by a statistical model that allows discrimination of genetic effects from nongenetic effects and that estimates repeatability and heritability coefficients. Both the repeatability coefficient between solstices (0.60) and heritability coefficient [0.45 ± 0.07 (SE)] were high. These results demonstrate that the variability in plasma melatonin concentration in ewes is under strong genetic control.The present study was conducted to assess the degree of genetic determination of the variability in the mean nocturnal plasma concentration of melatonin in sheep. Three hundred twelve ewes born from 18 males and with known genealogy were sampled at the summer and the winter solstices. The nocturnal plasma melatonin concentration was defined as the mean of four plasma samples taken at hourly intervals in the middle of the night (2200-0200). Identity of the father (P < 0.001) and the solstice (P < 0.05) were significant. Melatonin concentrations varied considerably among individuals [338.4 +/- 197.5 (SD) pg/ml; range 26.6-981.3 pg/ml] and between rams regarding the melatonin concentrations of their daughters (range from 202.9 to 456.3 pg/ml). Inheritance was analyzed by a statistical model that allows discrimination of genetic effects from nongenetic effects and that estimates repeatability and heritability coefficients. Both the repeatability coefficient between solstices (0.60) and heritability coefficient [0.45 +/- 0.07 (SE)] were high. These results demonstrate that the variability in plasma melatonin concentration in ewes is under strong genetic control.

Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by Laser Capture Microdissection

BackgroundSuccessful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult.The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA.Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis.ResultsWe developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and three granulosa cell-specific genes (KL, GATA4, AMH).A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte.Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA.ConclusionsThe ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.

High repeatability of the amplitude and duration of the nycthemeral rhythm of the plasma melatonin concentration in the Ile-de-France ewe.

Abstract: In order to determine individual variability in the amplitude and duration of the nycthemeral rhythm of the plasma melatonin concentration, 12 ovariectomized estradiol‐treated Ile‐de‐France ewes were submitted to a sequence of 34 long days (LD1, 16L: 8D), 21 short days (SD, 8L: 16D), and 21 LD (LD2). Intensive blood sampling (hourly from 1 hr before dusk to 1 hr after dawn) was performed for 4 consecutive nights in each of the photoperiodic situations (LD1, SD, LD2), i.e., a total of 12 nights per ewe. Plasma melatonin was assayed by radioimmunoassay. Instantaneous amplitude (difference between each hourly measurement of the plasma melatonin concentration during the nocturnal elevation and the mean diurnal melatonin concentration), mean individual amplitude (same definition but using the mean melatonin concentration during the nocturnal elevation) and duration of elevation of plasma melatonin concentrations were calculated for each ewe and each night and analyzed by variance analysis. Instantaneous amplitude varied significantly with photoperiod, with night intra‐photoperiod, with ewes, and time intra‐night. Mean individual amplitude varied significantly with photoperiod and with ewe but not with night intra‐photoperiod. Dramatic differences between individuals appeared in the amplitude and duration of melatonin rhythms. The lowest mean individual amplitude (all nights of measurement) found was 95 pg/ml and the highest was 544 pg/ml. Overall repeatability coefficient of the mean amplitude of melatonin elevation was 0.71 (P < 0.001). Duration of melatonin elevation varied significantly with photoperiod, with nights intra‐photoperiod and with ewes. During LD periods, mean duration of melatonin elevation was equivalent to duration of the night (from 7 to 8 hr), while during SD it varied with ewes from 9 to 16 hr. Repeatability coefficient of the duration of melatonin elevation in SD was 0.57 (P < 0.05). It is concluded that amplitude and duration (in SD) of the nycthemeral rhythm of the plasma melatonin concentration are highly repeatable individual characteristics in the ewe.

Genetic variability in plasma melatonin in sheep is due to pineal weight, not to variations in enzyme activities.

This study was conducted to determine the origin of the high variability in the mean nocturnal plasma melatonin concentration (MC) in sheep. Two extreme groups of 25 lambs each [low (L) and high (H)] were obtained by calculating their genetic value on the basis of the MC of their parents. The MC of lambs was significantly higher in the H group than in the L group (L: 189.7 ± 24.4 vs. H: 344.1 ± 33.0 pg/ml, P < 0.001). Within each group, 13 lambs were slaughtered during the day (D) and 12 lambs during the night (N). Pineal weight was significantly higher in the H group than in the L group (L: 83.5 ± 6.7 vs. H: 119.1 ± 9.2 mg, P < 0.01) but did not differ between D and N. The amount of melatonin released in vitro per milligram of pineal gland, the arylalkylamine N-acetyltransferase (AANAT) activity, the AANAT protein content, and the level of AANAT mRNA differed significantly between D and N but not with genetic group. Hydroxyindole O-methyltransferase activity did not differ significantly between D and N or between genetic groups. Therefore, the genetic difference in MC between the two groups of lambs was attributed to a difference in pineal size, not in enzymatic activity of the pinealocytes.

Genetic variability in melatonin concentrations in ewes originates in its synthesis, not in its catabolism

We investigated whether the genetic difference in plasma melatonin concentration in ewes was due to differences in the synthesis pathway from the pineal gland or in the catabolism of the hormone. Two groups of ewes [9 low (L) and 10 high (H)] were selected according to the breeding value of their mean nighttime plasma melatonin concentrations estimated at winter and summer solstices. In response to an identical dose of melatonin administered intravenously at 9:00 AM, no differences between groups were observed for any of the kinetic parameters: clearance rate, steady-state volume of distribution, terminal half-lives, and mean residence times. In the second experiment, two series of frequent blood samples were performed, one in the middle of the dark phase with samples taken every 5 min, and the other over 24 h with hourly samples. Highly significant differences between groups in nocturnal melatonin production rate were observed (L: 25.7 ± 2.8 vs. H: 63.1 ± 8.9 μg ⋅ kg-1 ⋅ h-1, P < 0.01). Thus the genetic differences in plasma melatonin concentrations in ewes originate in the synthesis pathway of the melatonin from the pineal gland rather than from differences in the catabolism of the hormone.We investigated whether the genetic difference in plasma melatonin concentration in ewes was due to differences in the synthesis pathway from the pineal gland or in the catabolism of the hormone. Two groups of ewes [9 low (L) and 10 high (H)] were selected according to the breeding value of their mean nighttime plasma melatonin concentrations estimated at winter and summer solstices. In response to an identical dose of melatonin administered intravenously at 9:00 AM, no differences between groups were observed for any of the kinetic parameters: clearance rate, steady-state volume of distribution, terminal half-lives, and mean residence times. In the second experiment, two series of frequent blood samples were performed, one in the middle of the dark phase with samples taken every 5 min, and the other over 24 h with hourly samples. Highly significant differences between groups in nocturnal melatonin production rate were observed (L: 25.7 +/- 2.8 vs. H: 63.1 +/- 8.9 microg . kg-1 . h-1, P < 0.01). Thus the genetic differences in plasma melatonin concentrations in ewes originate in the synthesis pathway of the melatonin from the pineal gland rather than from differences in the catabolism of the hormone.

Endocrine Characterization of the Reproductive Axis in Highly Prolific Lacaune Sheep Homozygous for the FecLL Mutation

A prolific allele named FecLL is known to segregate in the meat breed of the French Lacaune sheep on the basis of ovulation rate record. To gain more knowledge about the physiological effects of FecLL, ewes homozygous for FecLL (L/L) were compared to wild-type ewes (+/+) for ovarian phenotype and reproductive endocrine profiles. At the ovarian level, the increased ovulation rate in L/L ewes was associated with an increased number of antral follicles of greater than 3 mm and with preovulatory follicles being, on average, 1 mm smaller. Intrafollicular estradiol and testosterone concentrations were not significantly different between the two genotypes. In contrast, L/L large follicles (≥6 mm) had lower intrafollicular progesterone concentration. At the molecular level, expressions of ovarian markers, such as CYP19A1, CYP11A1, CYP17A1, LHR, and INHA, were not significantly different between the two genotypes. In contrast, FSHR and STAR mRNA levels increased in granulosa cells from L/L ewes. Plasma concentrations of estradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone measured across a synchronized estrous cycle revealed a significant increase in estradiol levels during the follicular phase, a precocious LH surge, and an increase in progesterone level during the luteal phase of L/L ewes compared to +/+ ewes. Circulating concentrations of FSH were not different between the two genotypes. The precocious LH surge was associated with an increase in frequency of LH pulsatility during the follicular phase. At the pituitary level, mRNA levels for LHB, FSHB, GNRHR, and ESR1 were not significantly different between the two genotypes. In contrast, ESR2 mRNA expression was decreased in L/L ewes compared to +/+ ewes. Based on ovarian phenotype and endocrine profiles, these findings suggest that the mutation in the FecL gene affects ovarian function in a different way compared to other known prolific mutations affecting the bone morphogenetic protein signaling system in the ovine species.