Anne R. O'Connell

Highly Prolific Booroola Sheep Have a Mutation in the Intracellular Kinase Domain of Bone Morphogenetic Protein IB Receptor (ALK-6) That Is Expressed in Both Oocytes and Granulosa Cells

Abstract The Booroola fecundity gene (FecB) increases ovulation rate and litter size in sheep and is inherited as a single autosomal locus. The effect of FecB is additive for ovulation rate (increasing by about 1.6 corpora lutea per cycle for each copy) and has been mapped to sheep chromosome 6q23–31, which is syntenic to human chromosome 4q21–25. Bone morphogenetic protein IB (BMP-IB) receptor (also known as ALK-6), which binds members of the transforming growth factor-β (TGF-β) superfamily, is located in the region containing the FecB locus. Booroola sheep have a mutation (Q249R) in the highly conserved intracellular kinase signaling domain of the BMP-IB receptor. The mutation segregated with the FecB phenotype in the Booroola backcross and half-sib flocks of sheep with no recombinants. The mutation was not found in individuals from a number of sheep breeds not derived from the Booroola strain. BMPR-IB was expressed in the ovary and in situ hybridization revealed its specific location to the oocyte and the granulosa cell. Expression of mRNA encoding the BMP type II receptor was widespread throughout the ovary. The mutation in BMPR-IB found in Booroola sheep is the second reported defect in a gene from the TGF-β pathway affecting fertility in sheep following the recent discovery of mutations in the growth factor, GDF9b/BMP15.

Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 Are Essential for Ovarian Follicular Development in Sheep

Abstract 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., ∼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.

Physiological effects of major genes affecting ovulation rate in sheep.

Genetic mutations with major effects on ovulation rate in sheep were recently identified in two genes of the transforming growth factor (TGFβ) superfamily and a TGFβ receptor, namely bone morphogenetic protein 15 (BMP15), otherwise known as the growth differentiation factor 9b (GDF9b), GDF9 and activin-like kinase 6 (ALK6) otherwise known as the BMP receptor type IB (BMPRIB). Animals homozygous for the BMP15 or GDF9 mutations are anovulatory whereas animals heterozygous for BMP15 or GDF9 or heterozygous or homozygous for ALK6 have higher than normal ovulation rates. Immunisation of ewes against BMP15 or GDF9 shows that both are essential for normal follicular development and control of ovulation rate. Common features of fertile animals with the BMP15, ALK6 (and possibly GDF9) mutations are changes in oocyte development during early preantral follicular growth, earlier maturation of granulosa cells and ovulation of mature follicles at smaller diameters. In summary, these findings have led to a new paradigm in reproductive biology, namely that the oocyte plays a key role in regulating the ovulation rate.

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

Abstract Woodlands sheep have a putative genetic mutation (FecX2W) that increases ovulation rate. At present, the identity of FecX2W 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, FecX2W could be an unidentified genetic mutation in BMP15 or in the closely related GDF9, which interacts with BMP15 to control ovarian function. Alternatively, FecX2W 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 FecX2W ewes in large preantral and antral follicles. Expression of ALK5 mRNA was significantly higher in the oocytes of FecX2W ewes, whereas expression of BMPR1B was decreased in both oocytes and granulosa cells of FecX2W ewes. FecX2W 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 FecX2W mutation.

Effect of age, weight, and sire on embryo and fetal survival in sheep.

The goal was to estimate the heritabilities and genetic variances for embryo and fetal survival (ES) in sheep along with the effect of premating ewe weight, age, and bilateral or unilateral ovulation on ES. The data consisted of 11,369 records on ovulation rate and litter size. Statistical models for ES included year and ovulation rate as fixed effects, premating ewe weight, and age as covariates, and sire of embryo, maternal grandsire (MGS), and permanent maternal environmental effects of the ewe as random effects. The variance components were estimated using REML. In ewes that survived to yr 6, the mean litter size was 1.87, 2.05, 2.01, 2.07, and 1.91 ± 0.04 in ewes of age 2, 3, 4, 5, and 6 yr, respectively. Litter size was less in ewes of age 2 and 6 yr compared to ewes of age 3, 4, and 5 yr (P < 0.01). Ovulation rate was lower at age 2 yr and increased from age 2 to 6 yr (P < 0.05). Two-year-old ewes had lower ES than 3-yr-old ewes (P < 0.01) and the probability of ES decreased after age 3 yr (P < 0.01). Thus, ES contributes significantly to lower fertility in 2-yr-old ewes. In ewes with high ovulation rates (i.e., 5 corpora lutea, CL), more balanced ovulations (i.e., 2 or 3 CL on each ovary) tended (P = 0.06) to be associated with increased ES. A quadratic relationship was observed between ewe weight and litter size (P < 0.01) and a positive linear relationship between premating ewe weight and ovulation rate (P < 0.01). A quadratic effect of ewe weight on ES was observed, with decreased ES for low and high ewe weights (P < 0.01). The optimal ewe weight for ES increased with ovulation rate, which is consistent with the requirement of greater body reserves for maintaining a larger number of fetuses during gestation. A quadratic relationship between ewe weight and the probability that a ewe is able to maintain a pregnancy was also observed (P < 0.05). Pregnancy loss is due to failure of the embryo or fetus or failure of the dam to maintain the pregnancy. The sire of the embryo only influences the embryo, whereas the MGS influences both the ewe and the embryo. The heritability for the direct additive effect on ES in ewes that lambed was 0.0081 ± 0.0139, and the heritability for the maternal additive effect was 0.0447 ± 0.0242. The permanent maternal environmental variance component was significant and explained 8.5% of the phenotypic variance. Thus, genetically, the dams ability to maintain a pregnancy has 5.5 times the effect on pregnancy loss than the embryos ability to survive, and this, in turn, was only half the size of the permanent environmental effect. Therefore, selection among dams based on the mean embryonic survival of their embryos will provide an effective way to improve embryonic survival.

Identification of a Line of Sheep Carrying a Putative Autosomal Gene Increasing Ovulation Rate in Sheep That Does Not Appear to Interact with Mutations in the Transforming Growth Factor Beta Superfamily

Sheep lines with mutations in single genes that have major effects on ovulation rate have been very useful in gaining a better understanding of pathways important in controlling follicular development and ovulation rate. To date however, all known mutations are in the transforming growth factor beta (TGFB) superfamily. Ovulation rates were measured in 720 progeny of 20 rams that were descendants of a single prolific ewe. Evaluation of ovulation rates of daughters of closely related sires suggests the presence of a segregating major gene Fecundity Davisdale (FECD) that increases ovulation rate between 0.4 and 0.8 in heterozygous daughters. Key features of mutations in genes of the TGFB superfamily pathway, such as synergistic interactions with other family members, infertility in homozygous carriers, and increased responsiveness to exogenous gonadotropins, were not observed in this line; thus, the mutation does not appear to be acting in the TGFB pathway. Hence, there is likely a novel mutation being carried in this line of sheep that alters ovulation rate. Future identification of the causative mutation may provide new insights into regulation of follicular development and ovulation rate.

Mutations in the leptin receptor gene associated with delayed onset of puberty are also associated with decreased ovulation and lambing rates in prolific Davisdale sheep

The aim of this study was to determine if single nucleotide polymorphisms (SNPs) in the leptin receptor (LEPR) gene associated with delayed onset of puberty are associated with changes in other reproductive traits in adult ewes. The ovulation rate of ewes homozygous for the SNPs was ~15% lower (PPLEPR SNPs than their wild-type or heterozygous contemporaries. Partial failure of multiple ovulations was also increased (PLEPR had on average 0.2 fewer lambs at mid-pregnancy and at birth compared with the wild-type or heterozygous ewes (PLEPR were strongly associated with poorer reproductive performance in Davisdale ewes, which is likely to be linked to both a reduced number of ova available for fertilisation and an increased number of ewes failing to become pregnant. Increased partial failure of multiple ovulations in ewes with high ovulation rates (i.e. 3 or greater) may also contribute to the poor reproductive performance.

Single-Nucleotide Polymorphisms in the LEPR Gene Are Associated with Divergent Phenotypes for Age at Onset of Puberty in Davisdale Ewes

ABSTRACT Attainment of puberty is a key developmental event influenced by genetic and environmental factors. In examining age at attainment of puberty, we observed closely related rams from the Davisdale line whose daughters differed in age at which they attained puberty. A candidate gene approach was used to identify mutations that may underlie these observed differences. Four rams with divergent phenotypes for their daughters age at onset of puberty were selected for whole-genome sequencing. The coding regions of genes with known roles in regulating reproductive function were searched for single-nucleotide polymorphisms (SNPs) that altered the amino acid sequence of the protein. Of interest were three SNPs in the leptin receptor gene (LEPR). A Sequenom assay was developed to determine the genotype of these SNPs in daughters of 17 sons of a founding sire. A higher percentage of ewe lambs homozygous for the LEPR mutations failed to undergo puberty before 1 yr of age, and those that did undergo puberty during the first breeding season on average were approximately 17 days older than homozygous wild-type ewes. Heterozygous ewes were intermediate for both measurements. Given the predicted change in protein function produced by the mutation in LEPR and the strong associations between the genotype and onset of puberty phenotypes, we propose that this mutation in LEPR underlies the observed difference in age at onset of puberty in the Davisdale line. Furthermore, these animals will likely provide a useful model to better understand the role of leptin in the regulation of puberty.

An earlier rise in systemic progesterone and increased progesterone in the uterine vein during early pregnancy are associated with enhanced embryonic survival in the ewe

Improved livestock production efficiency through greater embryonic survival (ES) is of economic and animal welfare benefit. Physiological characterization of animals that are extreme outliers for ES provides a valuable opportunity to identify a naturally occurring mechanism by which this trait may be enhanced. The objective was to determine the likely cause for the lifetime history of enhanced or reduced ES in a line of ewes selected for high fecundity. To address this question, progesterone concentrations in peripheral plasma as well as ovarian and uterine venous plasma samples were compared between groups of ewes with a lifetime history of either enhanced or reduced ES. The ability of the uterus to synthesize progesterone de novo at Day 5 of gestation was also tested. Ewes with enhanced ES had an earlier rise in progesterone concentration after estrus, irrespective of pregnancy status. In addition, there were increased concentrations of progesterone in the uterine vein in enhanced ES compared with reduced ES ewes on Day 5 of gestation (8.3 ± 0.8 ng/mL and 3.9 ± 1.4 ng/mL, respectively, P < 0.05). However, there were no differences in ovarian venous plasma (enhanced ES, 1725 ± 166 ng/mL; reduced ES, 1665 ± 268 ng/mL) at Day 5 of gestation. Although the endometrial tissue of some ewes (3/8) at Day 5 of gestation expressed three of the key genes necessary for regulation of de novo synthesis of progesterone, expression was not present exclusively in either of the two ES groups and therefore was unlikely to explain differences in the uterine vein progesterone concentrations between the enhanced and reduced ES groups. Collectively, the earlier rise in progesterone concentrations in peripheral plasma during the first week of gestation in the enhanced ES animals was independent of the presence of an embryo. Moreover, increased progesterone concentrations were also observed in the uterine vein at Day 5 of gestation of the enhanced ES ewes. It is proposed that the difference in uterine vein progesterone concentration was likely due to the differences in ovarian venous blood supply rather than de novo synthesis by the uterus.

Activin A and follistatin during the oestrous cycle and early pregnancy in ewes

The activin pathway has been postulated to be involved in regulation of multiple reproductive processes important for survival of the conceptus. These processes include luteinisation of the follicular cells and thus function of the corpus luteum, early embryo development and uterine function including implantation of the conceptus. Therefore, the aim of the current study was to determine whether the concentrations of activin A and follistatin (FST), an activin-binding protein, differed between ewes with a lifetime history of enhanced or reduced embryonic survival (ES). The mRNAs encoding FST and activin A (inhibin beta A subunit; INHBA) were present in the uterus and abundant in the uterine luminal or glandular epithelia by day 18 of gestation. A peak of activin A was observed in the systemic circulation around the time of oestrus, and activin A concentrations were elevated in animals with reduced ES during the oestrous cycle and early gestation. Concentrations of activin A in uterine fluid were approximately twofold greater on day 16 of gestation in ewes with reduced ES compared to those with enhanced ES. No consistent differences in FST were observed between these groups. Treatment of luteinising ovine granulosa cells with activin A in vitro suppressed progesterone secretion providing evidence of a potential pathway whereby increased concentrations of activin A may decrease ES.