Patterns of expression of messenger RNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development and characterization of ovarian follicular populations in ewes carrying the Woodlands FecX2W mutation

Woodlands sheep have a putative genetic mutation (FecX2(W)) that increases ovulation rate. At present, the identity of FecX2(W) is unknown. The trait does not appear to be due to the previously described mutations in bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), or bone morphogenetic protein receptor type 1B (BMPR1B) that affect ovulation rate in sheep. Potentially, FecX2(W) could be an unidentified genetic mutation in BMP15 or in the closely related GDF9, which interacts with BMP15 to control ovarian function. Alternatively, FecX2(W) may affect ovulation rate by changing the expression patterns in the molecular pathways activated by genes known to regulate ovulation rate. The objectives of these experiments were to sequence the complete coding region of the BMP15 and GDF9 genes, determine the patterns of expression of mRNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development, and characterize the follicular populations in ewes heterozygous for the Woodlands mutation and their wild-type contemporaries. No differences in the coding sequences of BMP15 or GDF9 genes were identified that were associated with enhanced ovulation rate. The expression patterns of GDF9 and BMPR2 mRNAs were not different between genotypes. However, expression of BMP15 mRNA was less in oocytes of FecX2(W) ewes in large preantral and antral follicles. Expression of ALK5 mRNA was significantly higher in the oocytes of FecX2(W) ewes, whereas expression of BMPR1B was decreased in both oocytes and granulosa cells of FecX2(W) ewes. FecX2(W) ewes also had increased numbers of antral follicles <1 mm in diameter. These follicles were smaller in average diameter, with the oocytes also being of a smaller mean diameter. Given that a mutation in BMP15 or BMPR1B results in increased ovulation rates in sheep, the differences in expression levels of BMP15 and BMPR1B may play a role in the increase in ovulation rate observed in Woodlands ewes with the FecX2(W) mutation.     

Effects of the FecL major gene in the Lacaune meat sheep population

Background

The major prolificacy gene FecL was first described in the Lacaune sheep meat breed Ovi-Test in 1998. A few studies estimated the effect of this gene on prolificacy but little data is available. In 2010, the Ovi-Test cooperative started genotyping FecL in all of their replacement ewe lambs. Thanks to the large amount of genotyping data that is available now, gene effects on litter size and other relevant traits can be estimated more accurately.

Methods

Our study included 5775 ewes genotyped since 2010 and 1025 sires genotyped since 2002. Performances and pedigrees were extracted from the French national database for genetic evaluation and research. Analysis of the effect of the gene on different traits was based on linear or threshold genetic animal models using the ASReml software.

Results

The female population was composed of 71% homozygous wild type ewes (++), 27% heterozygous ewes for the FecL mutation (L+) and 2% homozygous mutant (LL) ewes. On average, L + ewes produced 0.5 more lambs per lambing than ++ ewes. The FecL gene not only affected the mean litter size but also its variability, which was lower for ++ than for L + ewes. Fertility after insemination was higher for L + ewes than for ++ ewes. Lambs from ++ dams were heavier (+300 g) than the lambs of L + dams and the mortality of twin lambs born from ++ dams was lower than those from L + dams. In addition, bias in estimated breeding values for prolificacy when ignoring the existence of this major gene was quantified.

Conclusions

The effect of the FecL gene on prolificacy was estimated more accurately and we show that this gene affects both the mean and the variability of litter size and other traits. This paper also shows that ignoring the existence of this major gene in genetic evaluation of prolificacy can lead to a large overestimation of polygenic breeding values.     

A review of the effects of the Booroola gene (FecB) on sheep production

Booroola Merino (B^oM) ewes have a high ovulation rate and litter size which in 1980 was postulated to be due to the effects of a major gene (FecB). This was confirmed in breeding experiments and FecB was subsequently shown to be due to a mutation (BMPR-1B) on chromosome 6. The B^oM originated from an Australian commercial fine wool Merino flock (Booroola) and has been used in crossing experiments and for introgression of FecB into many breeds around the world to improve fecundity. The mutation has recently been found in native sheep breeds in India, China and Indonesia and it is likely that FecB in the Australian B^oM was derived from importations of Garole sheep from India in 1792 and 1793.The effects on production traits of the FecB mutation in a range of genetic comparisons, environments and production systems are reviewed. Comparisons involving B^oM crosses with various other breeds and contrasts of FecB homozygous (BB), heterozygous (B+) and non-carrier (++) genotypes in comparable background genotypes, including non-B^oM, have been summarised from 45 reports. The weighted mean effect for ewes carrying one copy of FecB (B+) was +1.3 (range +0.8 to +2.0) for ovulation rate and +0.7 (range +0.4 to +1.3) for litter size. The effect of a second copy (BB) was generally additive for ovulation rate, with little or no increase in litter size for BB ewes among B^oM crosses. However there was generally a further increase in litter size for BB ewes of about half the effect of one copy (B+) in the Indian and Chinese breeds. Poor lamb survival and lamb growth reduced the number of lambs weaned and total weight of lamb weaned by B+ ewes. Most studies still showed a small advantage for B+ ewes, although several reported negative effects. While embryo survival declines at higher ovulation rates, the effects of FecB per se are equivocal. There is some evidence of a higher non-pregnancy rate among homozygous BB ewes. Most studies reported lower birth weight and growth rate from B^oM cross lambs and lambs from crossbred ewes introgressed with FecB. However it is difficult to separate the effects of low background genetic merit for growth of the B^oM and the lower birth weight and growth rate of lambs from larger litters from the genetic effect of carrying FecB. There was little or no difference in growth rate between BB, B+ and ++ genotype lambs. For other traits including, seasonal oestrous activity, carcass and meat quality and wool production, there was no evidence of major effects of FecB. The opportunities for management and nutritional modification of FecB expression and implications for industry adoption are briefly discussed.     

Alteration of Ganglioside Biosynthesis Responsible for Complex Hereditary Spastic Paraplegia

Hereditary spastic paraplegias (HSPs) form a heterogeneous group of neurological disorders. A whole-genome linkage mapping effort was made with three HSP-affected families from Spain, Portugal, and Tunisia and it allowed us to reduce the SPG26 locus interval from 34 to 9 Mb. Subsequently, a targeted capture was made to sequence the entire exome of affected individuals from these three families, as well as from two additional autosomal-recessive HSP-affected families of German and Brazilian origins. Five homozygous truncating (n = 3) and missense (n = 2) mutations were identified in B4GALNT1. After this finding, we analyzed the entire coding region of this gene in 65 additional cases, and three mutations were identified in two subjects. All mutated cases presented an early-onset spastic paraplegia, with frequent intellectual disability, cerebellar ataxia, and peripheral neuropathy as well as cortical atrophy and white matter hyperintensities on brain imaging. B4GALNT1 encodes β-1,4-N-acetyl-galactosaminyl transferase 1 (B4GALNT1), involved in ganglioside biosynthesis. These findings confirm the increasing interest of lipid metabolism in HSPs. Interestingly, although the catabolism of gangliosides is implicated in a variety of neurological diseases, SPG26 is only the second human disease involving defects of their biosynthesis.     

Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep

The aim of this study was to test the hypothesis that both growth differential factor 9 (GDF9) and bone morphogenetic protein (BMP15; also known as GDF9B) are essential for normal ovarian follicular development in mammals with a low ovulation rate phenotype. Sheep (9-10 per group) were immunized with keyhole limpet hemocyanin (KLH; control), a GDF9-specific peptide conjugated to KLH (GDF9 peptide), a BMP15-specific peptide conjugated to KLH (BMP15 peptide), or the mature region of oBMP15 conjugated to KLH (oBMP15 mature protein) for a period of 7 mo and the effects of these treatments on various ovarian parameters such as ovarian follicular development, ovulation rate, and plasma progesterone concentrations evaluated. Also in the present study, we examined, by immunohistochemistry, the cellular localizations of GDF9 and BMP15 proteins in the ovaries of lambs. Both GDF9 and BMP15 proteins were localized specifically within ovarian follicles to the oocyte, thereby establishing for the sheep that the oocyte is the only intraovarian source of these growth factors. Immunization with either GDF9 peptide or BMP15 peptide caused anovulation in 7 of 10 and 9 of 10 ewes, respectively, when assessed at ovarian collection. Most ewes (7 of 10) immunized with oBMP15 mature protein had a least one observable estrus during the experimental period, and ovulation rate at this estrus was higher in these ewes compared with those immunized with KLH alone. In both the KLH-GDF9 peptide- and KLH-BMP15 peptide-treated ewes, histological examination of the ovaries at recovery (i.e., approximately 7 mo after the primary immunization) showed that most animals had few, if any, normal follicles beyond the primary (i.e., type 2) stage of development. In addition, abnormalities such as enlarged oocytes surrounded by a single layer of flattened and/or cuboidal granulosa cells or oocyte-free nodules of granulosa cells were often observed, especially in the anovulatory ewes. Passive immunization of ewes, each given 100 ml of a pool of plasma from the GDF9 peptide- or BMP15 peptide-immunized ewes at 4 days before induction of luteal regression also disrupted ovarian function. The ewes given the plasma against the GDF9 peptide formed 1-2 corpora lutea but 3 of 5 animals did not display normal luteal phase patterns of progesterone concentrations. The effect of plasma against the BMP15 peptide was more dramatic, with 4 of 5 animals failing to ovulate and 3 of 5 ewes lacking surface-visible antral follicles at laparoscopy. By contrast, administration of plasma against KLH did not affect ovulation rate or luteal function in any animal. In conclusion, these findings support the hypothesis that, in mammals with a low ovulation rate phenotype, both oocyte-derived GDF9 and BMP15 proteins are essential for normal follicular development, including both the early and later stages of growth.     

Markers of follicle function in Belclare-cross ewes differing widely in ovulation rate.

High prolificacy due to a gene that has a large effect on ovulation rate has been noted in Booroola and Inverdale ewes. High prolificacy in the Belclare breed (a composite developed from stocks selected for very large litter size or high ovulation rate) may be related to the segregation of two genes. The aims of this study were (i) to compare the morphological and functional features of ovulatory follicles from carriers (which could only be heterozygous for the genes of interest) and non-carriers, and (ii) to identify markers of the Belclare genes among secreted or cellular ovarian proteins. Belclare carrier ewes had more ovulatory follicles (4.9 +/- 0.4) than did non-carrier ewes (2.0 +/- 0.2) (P < 0.001). Ovulatory follicles from carriers were also smaller (4.4 +/- 0.1 mm versus 5.7 +/- 0.2 mm, P < 0.001) and contained a significantly reduced number of granulosa cells (P < 0.001). However, the proportion of proliferating granulosa cells in ovulatory follicles was similar in both groups. The in vitro secretion of steroids per follicle was only marginally lower in follicles from Belclare carriers compared with non-carriers. Furthermore, similar concentrations of steroidogenic enzymes were present in both groups, indicating that steroidogenic potential per granulosa cell is similar between carriers and non-carriers. Possible markers of the Belclare genes were identified among cellular proteins of follicular walls by two-dimensional PAGE and image analysis. Two spots at 78 and 49 kDa were always absent in samples from non-carriers. When secreted proteins in follicles from carriers were compared with those from non-carriers, two spots at 53 and 41 kDa were restricted to samples from carriers and three spots at 97, 91 and 45 kDa were unique to samples from non-carriers. Interestingly, the spot at 91 kDa is also affected by the Booroola gene.     

Anti-Müllerian hormone concentration in sheep and its dependence of age and independence of BMP15 genotype: An endocrine predictor to select the best donors for embryo biotechnologies

Embryo biotechnologies contribute significantly to the genetic enhancement of livestock, although their efficiency remains limited in sheep, mainly owing to variable ovarian responses to gonadotropins. At present, anti-Müllerian hormone (AMH), which is produced by the granulosa cells of the small antral follicles, is a reliable endocrine marker of the ovarian follicle reserve in many species. The expression of AMH in granulosa cells was shown to be stimulated by bone morphogenetic proteins (BMPs) in vitro, so a mutation affecting the BMP15 gene might modulate AMH production in vivo. The present study aimed to assess plasma AMH concentrations before puberty in two groups of Rasa Aragonesa ewes that were carrying (R+) or not carrying (++) the prolific FecX^R allele and to relate them with their AMH concentrations at adulthood. Additionally, we sought to establish in both genotypes whether AMH measurements during a laparoscopic ovum pick-up (LOPU) program could be predictive of the number of ovarian follicles (≥3 mm) and recovered cumulus-oocyte complexes (COCs). No differences in AMH were found between the R+ and ++ ewes before puberty or during the adult age. Before puberty, the AMH concentration tended to increase from 3 to 4.5 months and to decline at 6 months to levels similar to those observed later in adults (333.8 ± 73.3, 483.2 ± 135.5, and 184.1 ± 38.2 pg/mL, respectively; P < 0.1), showing a large variability between individuals and between ages. A relationship between the AMH concentrations before puberty and during adulthood was not found, likely reflecting different follicular growth dynamics. In adults, the AMH concentration at the beginning of the FSH treatment was strongly correlated with the number of punctured follicles at LOPU in R+ and ++ ewes (r = 0.75 and 0.78, respectively; P < 0.001), and it was possible to accurate determine AMH cutoff values for both genotypes to identify high-responding ewes. On average, 5.1 extra follicles and 2.7 extra COCs were expected per each 100 pg/mL increase in AMH (P < 0.0001 and P < 0.01, respectively). The repeatability of AMH concentration from session to session was 0.70 (P < 0.0001). Our results demonstrated that, regardless of age, the presence of the FecX^R allele did not affect plasma AMH levels. During adulthood, AMH proved to be a good predictor of the ovarian response to FSH stimulation. Such an indicator could therefore be used to improve the performance of embryo biotechnologies in sheep.     

Missense mutations in the C-terminal portion of the B4GALNT2-encoded glycosyltransferase underlying the Sd(a−) phenotype

Sd^a is a high-frequency carbohydrate histo-blood group antigen, GalNAcβ1-4(NeuAcα2-3)Galβ, implicated in pathogen invasion, cancer, xenotransplantation and transfusion medicine. Complete lack of this glycan epitope results in the Sd(a?) phenotype observed in 4% of individuals who may produce anti-Sd^a. A candidate gene (B4GALNT2), encoding a Sd^a-synthesizing β-1,4-N-acetylgalactosaminyltransferase (β4GalNAc-T2), was cloned in 2003 but the genetic basis of human Sd^a deficiency was never elucidated. Experimental and bioinformatic approaches were used to identify and characterize B4GALNT2 variants in nine Sd(a?) individuals. Homozygosity for rs7224888:T > C dominated the cohort (n = 6) and causes p.Cys466Arg, which targets a highly conserved residue located in the enzymatically active domain and is judged deleterious to β4GalNAc-T2. Its allele frequency was 0.10–0.12 in different cohorts. A Sd(a?) compound heterozygote combined rs7224888:T > C with a splice-site mutation, rs72835417:G > A, predicted to alter splicing and occurred at a frequency of 0.11–0.12. Another compound heterozygote had two rare nonsynonymous variants, rs148441237:A > G (p.Gln436Arg) and rs61743617:C > T (p.Arg523Trp), in trans. One sample displayed no differences compared to Sd(a+). When investigating linkage disequilibrium between B4GALNT2 variants, we noted a 32-kb block spanning intron 9 to the intergenic region downstream of B4GALNT2. This block includes RP11-708H21.4, a long non-coding RNA recently reported to promote tumorigenesis and poor prognosis in colon cancer. The expression patterns of B4GALNT2 and RP11-708H21.4 correlated extremely well in >1000 cancer cell lines. In summary, we identified a connection between variants of the cancer-associated B4GALNT2 gene and Sd^a, thereby establishing a new blood group system and opening up for the possibility to predict Sd(a+) and Sd(a?) phenotypes by genotyping.     

Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries)

Belclare and Cambridge are prolific sheep breeds, the origins of which involved selecting ewes with exceptionally high litter size records from commercial flocks. The variation in ovulation rate in both breeds is consistent with segregation of a gene (or genes) with a large effect on this trait. Sterile ewes, due to a failure of normal ovarian follicle development, occur in both breeds. New naturally occurring mutations in genes for the oocyte-derived growth factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are described. These mutations are associated with increased ovulation rate in heterozygous carriers and sterility in homozygous carriers in both breeds. This is the first time that a mutation in the gene for GDF9 has been found that causes increased ovulation rate and infertility in a manner similar to inactivating mutations in BMP15, and shows that GDF9 is essential for normal folliculogenesis in sheep. Furthermore, it is shown, for the first time in any species, that individuals with mutations in both GDF9 and BMP15 have a greater ovulation rate than sheep with either of the mutations separately.     

Ram-induced oestrus and ovulation in lactating and weaned Corriedale ewes

Two experiments were conducted in consecutive years in which recently (Experiment 1) or temporarily (Experiment 2) weaned ewes and matched post-partum non-lactating flockmates (DRY) were exposed to a stimulus group of rams and oestrous ewes (10 and 20 in Experiment 1, 20 and 20 in Experiment 2) for 28 days in spring. Lactating ewes (n = 130) in Experiment 1 were isolated from their lambs 4 (W-4), 2 (W-2), 1 (W-1) or 0 (W-0) days in advance and exposed along with a group of 32 DRY flockmates. Lactating ewes in Experiment 2 (n = 230) were allocated to an unreplicated factorial of two levels of temporary weaning before stimulation (B0: control; B24: lambs removed 24 h before stimulation) by four levels of ewe-lamb contact imposed at the start of the stimulation (A0: control; A12, A24 and A36: lamb-ewe separation during the initial 12, 24 or 36 h of exposure); DRY ewes (n = 54) acted as an augmented factorial control. Oestrus (rump marks) and ovulation (laparoscopy on day 5 and on day 28 (Experiment 1) or day 32 (Experiment 2)) were recorded. Ovulation and oestrous responses in Experiment 1 were similar for DRY (90.6% and 55.2%, respectively) and recently weaned ewes (83.8% and 53.7%, respectively). Amongst recently weaned ewes, the immediate ovulation response to the rams and the proportion of ewes still cycling by day 28 tended to be lower (P = 0.065 and P = 0.011) in ewes weaned on the day of ram exposure (71.9% and 54.8% v. 87.8% and 80.0%, respectively). Ovulation rate was lower (P < 0.003) in W-2 ewes (1.3 ± 0.10) than in the other recently weaned groups. In Experiment 2, ovulation (83.3%) and oestrous (68.9%) responses in DRY ewes were higher (P = 0.022 and P = 0.053, respectively) than in lactating ewes (66.2% and 51.0%, respectively). More ewes ovulated (P = 0.036) in B24 (70.5%) than in B0 (61.8%). Ewes having their lambs returned 12 h after the onset of stimulation (A12) had poorer ovulation responses (54.9%) than control ewes (A0, 72.9%, P < 0.05); this was probably associated to lamb restitution after the sunset. Main conclusions were that (i) the presence of the lambs is a depressing factor of both ovulation and oestrous responses to the ram effect in lactating ewes, (ii) the ovulation response of lactating ewes will probably benefit from removing lambs for a period of 24 h before the onset of stimulation, (iii) until additional information becomes available, temporary weaning protocols should be designed avoiding lamb restitution during the night.     

Direct Evidence on the Contribution of a Missense Mutation in GDF9 to Variation in Ovulation Rate of Finnsheep

The Finnish Landrace (Finnsheep) is a well known high-prolificacy sheep breed and has been used in many countries as a source of genetic material to increase fecundity of local breeds. Analyses to date have indicated that mutations with a large effect on ovulation rate are not responsible for the exceptional prolificacy of Finnsheep. The objectives of this study were to ascertain if: 1) any of 12 known mutations with large effects on ovulation rate in sheep, or 2) any other DNA sequence variants within the candidate genes GDF9 and BMP15 are implicated in the high prolificacy of the Finnish Landrace breed; using material from lines developed by divergent selection on ovulation rate. Genotyping results showed that none of 12 known mutations (FecB^B, FecX^B, FecX^G, FecX^GR, FecX^H, FecX^I, FecX^L, FecX^O, FecX^R, FecG^E, FecG^H, or FecG^T) were present in a sample of 108 Finnsheep and, thus, do not contribute to the exceptional prolificacy of the breed. However, DNA sequence analysis of GDF9 identified a previously known mutation, V371M, whose frequency differed significantly (P<0.001) between High and Low ovulation rate lines. While analysis of ovulation rate data for Finnsheep failed to establish a significant association between this trait and V371M, analysis of data on Belclare sheep revealed a significant association between V371M and ovulation rate (P<0.01). Ewes that were heterozygous for V371M exhibited increased ovulation rate (+0.17, s.e. 0.080; P<0.05) compared to wild type and the effect was non-additive (ovulation rate of heterozygotes was significantly lower (P<0.01) than the mean of the homozygotes). This finding brings to 13 the number of mutations that have large effects on ovulation rate in sheep and to 5, including FecB^B, FecG^E, FecX^O and FecX^GR, the number of mutations within the TGFβ superfamily with a positive effect on prolificacy in the homozygous state.     

Multiple matings modify the estrous length,the moment of ovulation,and the estradiol and LH patterns in ewes

In several species, mating reduces the estrous length and advances ovulation. The aim of this study was to determine if multiple matings reduces the estrous length and modifies the moment of ovulation, as well as the estradiol and LH patterns in ewes. The estrous cycle of Corriedale ewes was synchronized, and the onset of receptivity was monitored every 3 h with rams, avoiding mating. At the estrous onset, ewes were assigned to two experimental groups (n=10 each): 1) estrous was monitored every 3 h with a ram avoiding mating (group CON), and 2) a ram was allowed to mate and ejaculate once every 3 h (group MAT). The ovaries were scanned with transrectal ultrasonography and blood samples were collected for measuring 17β-estradiol and LH concentrations every 3 h until ovulation. Estrus was shorter in MAT than CON ewes (24.7 ± 1.5 h vs. 30.4 ± 1.5 h, respectively; P=0.02); the proportion of animals that ovulated before the end of estrus was greater in CON ewes: (9/10 vs. 3/10, P=0.009). The area under the LH curve (AUC) was greater in MAT than CON ewes (36.1 ± 3.5 ng.h^-1.mL^-1 vs 24.9 ± 3.5 ng.h^-1.mL^-1 P=0.03). However, MAT ewes had a lower 17β-estradiol AUC than CON ewes (41.0 ± 4.9 pg.h^-1.mL^-1 vs 59.4 ± 4.9 pg.h^-1.mL^-1 P=0.01). Mating reduced the estrous length, induced a greater secretion of LH but less total 17β-estradiol secreted and, additionally, ovulation occurred more frequently after the end of estrus in mated ewes.     

Investigation of Prolific Sheep from UK and Ireland for Evidence on Origin of the Mutations in BMP15 (FecXG,FecXB) and GDF9 (FecGH) in Belclare and Cambridge Sheep

This paper concerns the likely origin of three mutations with large effects on ovulation rate identified in the Belclare and Cambridge sheep breeds; two in the BMP15 gene (FecX^G and FecX^B) and the third (FecG^H) in GDF9. All three mutations segregate in Belclare sheep while one, FecX^B, has not been found in the Cambridge. Both Belclare and Cambridge breeds are relatively recently developed composites that have common ancestry through the use of genetic material from the Finnish Landrace and Lleyn breeds. The development of both composites also involved major contributions from exceptionally prolific ewes screened from flocks in Ireland (Belclare) and Britain (Cambridge) during the 1960s. The objective of the current study was to establish the likely origin of the mutations (FecX^G, FecX^B and FecG^H) through analysis of DNA from Finnish Landrace and Lleyn sheep, and Galway and Texel breeds which contributed to the development of the Belclare breed. Ewes with exceptionally high prolificacy (hyper-prolific ewes) in current flocks on Irish farms were identified to simulate the screening of ewes from Irish flocks in the 1960s. DNA was obtained from: prolific ewes in extant flocks of Lleyn sheep (n = 44) on the Lleyn peninsula in Wales; hyper-prolific ewes (n = 41); prolific Galway (n = 41) ewes; Finnish Landrace (n = 124) and Texel (n = 19) ewes. The FecX^G mutation was identified in Lleyn but not in Finnish Landrace, Galway or Texel sheep; FecX^B was only found among the hyper-prolific ewes. The FecG^H mutation was identified in the sample of Lleyn sheep. It was concluded from these findings that the Lleyn breed was the most likely source of the FecX^G and FecG^H mutations in Belclare and Cambridge sheep and that the FecX^B mutation came from the High Fertility line that was developed using prolific ewes selected from commercial flocks in Ireland in the 1960′s and subsequently used in the genesis of the Belclare.     

Analysis of gene expression in granulosa cells of ovine antral growing follicles using suppressive subtractive hybridization

Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5 mm) and small follicles (SF, 3–5 mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.     

Differential effects of indomethacin on the sheep ovary: Prostaglandin biosynthesis, intracellular calcium, apoptosis, and ovulation

Cells of the apical wall of the dominant follicle and contiguous ovarian surface epithelium become apoptotic with the approach of ovulation in the sheep. It was hypothesized that indomethacin, an established inhibitor of prostaglandin biosynthesis and ovulation, would protect apical ovarian cells from programmed death. The anovulatory potencies of two systemic doses of indomethacin (200 and 800 mg) were tested in gonadotropin-stimulated ewes. A complete blockade of ovulation occurred at the higher dose of indomethacin. Ovulation was not inhibited by 200 mg indomethacin. Both doses of drug suppressed follicular prostaglandin production below pregonadotropin levels. Immunofluorescence detection of digoxigenin end-labeled (fragmented) DNA was used as a marker of apoptosis among ovarian surface epithelial and granulosa cells recovered from the apical hemisphere of preovulatory ovine follicles. Cellular DNA fragmentation was averted in animals given 800 mg indomethacin, whereas apoptosis ensued after 200 mg. A sustained increase in cytosolic calcium is generally a prerequisite to apoptotic DNA fragmentation and cell death. Indeed, intracellular calcium, detected by fluorescence of fura-2, was elevated in ovarian cells of animals destined to ovulate (controls, 200 mg indomethacin) in comparison to (safeguarded) cells of anovulatory ewes (800 mg indomethacin). These observations provide circumstantial evidence that apical ovarian cell degeneration by calcium-mediated apoptosis is a determinant of follicular instability and rupture, but that these events are unrelated to the gonadotropin-induced rise in prostanoid production characteristic of preovulatory follicles.     

A comparison of PMSG and LHRH agonist treatment in the induction of ovulation in the anoestrous ewe

The aim of this experiment was to compare the use of pregnant mares' serum gonadotrophin (PMSG) with that of a luteinizing hormone releasing hormone (LHRH) agonist in the induction of ovulation in anoestrous sheep. Anoestrous ewes were treated with progestagen-impregnated sponges for 12 days. They were given either PMSG at the time of sponge withdrawal or the LHRH agonist D-Ser(But)⁶desGlyNH₂¹⁰LHRH ethylamide 20 h after sponge withdrawal. This protocol was followed over 2 consecutive years. Plasma concentrations of oestradiol and LH were measured, and in the first year a comparison was made of the ovulation rate, conception rate and luteal function of the two groups after artificial insemination. During the first year, all of the PMSG-treated group but none of the agonist-treated group exhibited oestrus. Five of the eight PMSG-treated ewes had embryos in utero at slaughter whilst none was present in the agonist-treated ewes. The secretion of progesterone was greatest in the PMSG-treated ewes (P < 0.001). During the second year, a more frequent blood-sampling regime was employed. Increased plasma concentrations of LH occurred within 3 h of agonist administration. Plasma oestradiol concentrations peaked at 20 h and 45 h after sponge withdrawal in both groups. Both peaks were larger in the agonist-treated group. It is concluded that a single dose of the highly potent LHRH agonist is unable to produce normal luteal function or conception using the present protocol.     

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 FecX^Gr and FecX^O were identified in the Grivette and Olkuska breeds, respectively. The two mutations were associated with the highly prolific phenotype (p_FecX^Gr = 5.98E⁻⁰⁶ and p_FecX^O = 2.55E⁻⁰⁸). Homozygous ewes for the mutated allele showed a significantly increased prolificacy (FecX^Gr/FecX^Gr, LS = 2.50±0.65 versus FecX⁺/FecX^Gr, LS = 1.93±0.42, p<1E⁻⁰³ and FecX^O/FecX^O, OR = 3.28±0.85 versus FecX⁺/FecX^O, 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 FecX^Gr/FecX^Gr Grivette and homozygous FecX^O/FecX^O 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.     

A new polymorphism in the Growth and Differentiation Factor 9 (GDF9) gene is associated with increased ovulation rate and prolificacy in homozygous sheep

Brazilian Santa Inês (SI) sheep are very well‐adapted to the tropical conditions of Brazil and are an important source of animal protein. A high rate of twin births was reported in some SI flocks. Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are the first two genes expressed by the oocyte to be associated with an increased ovulation rate in sheep. All GDF9 and BMP15 variants characterized, until now, present the same phenotype: the heterozygote ewes have an increased ovulation rate and the mutated homozygotes are sterile. In this study, we have found a new allele of GDF9, named FecG^E (Embrapa), which leads to a substitution of a phenylalanine with a cysteine in a conservative position of the mature peptide. Homozygote ewes presenting the FecG^E allele have shown an increase in their ovulation rate (82%) and prolificacy (58%). This new phenotype can be very useful in better understanding the genetic control of follicular development; the mechanisms involved in the control of ovulation rate in mammals; and for the improvement of sheep production.