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.

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 daughter's 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.

The role of reproductive loss on flock performance: a comparison of nine industry flocks.

The reproductive performance of a sheep flock is dependent on a multitude of complex interacting factors. Attaining optimal flock performance requires information about how the reproductive steps are linked and relate to readily available measurements of the state of the flock. The goal was to use data from nine commercial flocks (greater than 300,000 records) to investigate and model the key reproductive steps affecting flock reproductive performance. We also developed a maximum-likelihood based methodology to predict flock ovulation rate based on measurements of the number of fetuses at mid-pregnancy (detected by ultrasound-scanning). The model was used to determine how changes in premating liveweight, age, predicted ovulation rate, number of fetuses at mid-pregnancy, lamb survival and lamb growth rate affect the total lamb liveweight at weaning per ewe exposed to the ram in each flock. The data from the commercial flocks were also used to investigate the role of ewe age and premating liveweight on each reproductive step. Sensitivity analyses were conducted to identify the key reproductive steps affecting flock reproductive performance, with a focus on understanding how these steps vary between flocks. The elasticity for embryo survival was 60% of that for lamb survival for these flocks and the elasticities for ovulation rate were highly variable between flocks (0.16 to 0.50 for mature ewes). This indicates that ovulation rate was near-optimal for some flocks, whereas there was potential to significantly improve flock performance in suboptimal flocks. The elasticity for ewe premating liveweight was highly variable between flocks (-0.03 to 0.84 for mature ewes and -0.18 to 1.39 for ewe lambs), indicating that premating liveweight ranged from optimal to suboptimal between flocks. For these suboptimal farms, the opportunity exists to increase flock performance through improved management of ewe premating liveweight. Reproductive loss was significantly greater in ewe lambs than mature ewes, although the difference is dependent on the stage of reproduction and flock. Predicted ovulation rate was 25% lower for ewe lambs and there was a 30% relative decrease in the predicted embryo survival probability from ovulation to scanning for ewe lambs. There was a 10% relative decrease in lamb survival probability from birth to weaning for ewe lambs and lamb growth rate was 25% lower for ewe lambs.

Characterization of local and peripheral immune system in pregnant and nonpregnant ewes.

Modulation of the immune system is known to be important for successful pregnancy but how immune function might differ between the lymph nodes draining the reproductive tract and peripheral lymph nodes is not well understood. Additionally, if immune system changes in response to the presence of an embryo during early pregnancy, and if this response differs in local versus peripheral immune tissue, has not been well characterized. To address these questions, we examined expression of genes important for immune function using NanoString technology in the ampulla and isthmus of the oviduct, endometrium, lymph nodes draining the reproductive tract (lumbo-aortic and medial iliac) as well as a peripheral lymph node (axillary), the spleen, and circulating immune cells from ewes on day 5 of the estrous cycle or pregnancy. Concentrations of estradiol and progesterone in plasma were also determined. Principal component analysis revealed separation of the local from the peripheral lymph nodes (MANOVA P = 3.245e-08, R2 = 0.3) as well as separation of tissues from pregnant and nonpregnant animals [lymph nodes (MANOVA P = 2.337e-09, R2 = 0.5), reproductive tissues (MANOVA P = 2.417e-14, R2 = 0.47)]. Nine genes were differentially (FDR < 0.10) expressed between lymph node types, with clear difference in expression of these genes between the lumbo-aortic and axillary lymph nodes. Expression of these genes in the medial iliac lymph node was not consistently different to either the axillary or the lumbo-aortic lymph node. Expression of IL10RB was increased (FDR < 0.05) by 24% in the reproductive tissue of the pregnant animals compared to nonpregnant animals. Analysis of gene categories revealed that expression of genes of the T-cell receptor pathway in reproductive tract tissues was associated (P < 0.05) with pregnancy status. In conclusion, assessment of gene expression of reproductive and immune tissue provides evidence for a specialization of the local immune system around the reproductive tract potentially important for successful establishment of pregnancy. Additionally, differences in gene expression patterns in reproductive tissue from pregnant and nonpregnant animals could be discerned as early as day 5 of pregnancy. This was found to be associated with expression of genes important for T-cell function and thus highlights the important role of these cells in early pregnancy.

The effect of reproductive loss on the performance of a research flock.

The reproductive performance of a sheep flock is dependent on a multitude of complex interacting factors. Achieving optimal flock performance requires knowledge of the reproductive steps and how these are linked and related to available measurements of the state and performance of the flock. The goal was to use previously collected data from a research flock that had undergone selection for fecundity (11,369 lambing records), to model the key reproductive steps affecting flock reproductive performance. The model was used to investigate how changes in liveweight, age, ovulation rate, number of fetuses at midpregnancy, number of lambs born, and birthweight affect the number of lambs weaned and the weaning weight of each lamb in this flock. The data available from the research flock were used to parameterize models of each reproductive step and assess the role of ewe age and premating liveweight on each reproductive step. These models were then linked together as a simulation tool to assess the role of different parameters on flock reproductive performance, which was defined as the total weight of lambs weaned per ewe exposed to the ram. Flock elasticities were calculated that characterize the relative importance of the effect of average premating ewe liveweight (0.81), average ovulation rate (0.33), variance in ovulation rate (-0.095), embryo survival (0.72), lamb survival (1.03), conception failure (0.35), and average ewe age (0.056) on the total kilograms of lamb liveweight at weaning per ewe exposed to the ram. The largest elasticity for lamb survival indicated that a 1% increase in lamb survival is expected to have a 1.03% increase in the total kilograms of lamb liveweight at weaning per ewe exposed to the ram in this flock. Assuming similar costs, interventions to increase lamb survival for this flock will provide the largest increase in the total kilograms of lamb liveweight at weaning per ewe exposed to the ram, which is a key metric of flock performance.

Association of fertility with group mating behavior in ewes.

It was hypothesized that values for fertility variables would vary with number of rams ewes mated with in a group mating management system. In Experiment 1, for adult ewes (n = 872) joined with rams for 35-38 days, 19.0 ± 2.2 %, 19.2 ± 3.5 %, 34.4 ± 1.3 % and 27.4 ± 3.3 % were marked by zero, one, two or three rams during the first 17 days, respectively. In general, as number of rams with which ewes mated increased, number of lambs born (NLB) increased, however, ovulation rate (OR) did not. In mated ewes, embryo survival (ES) increased (P < 0.05) as number of rams with which ewes mated increased. In Experiment 2, ewes that mated with zero or one ram had lesser concentrations of estradiol than ewes that mated with two or three rams when evaluated 30 h after initiation of the follicular phase. Following breeding on the subsequent estrus, number of fetuses at mid-gestation was less in ewes that had mated, in the previous estrus, with zero or one ram compared to two or three rams. In summary, ewes mating with a larger number of rams had greater values for the fertility variables that were evaluated compared with those mating with fewer rams. When ewes mated with fewer rams during the estrous period there were lesser concentrations of estradiol, potentially associated with the decreased ES.

The cooperative effect of growth and differentiation factor-9 and bone morphogenetic protein (BMP)-15 on granulosa cell function is modulated primarily through BMP receptor II.

Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, GDF9B) are oocyte-derived proteins essential for the growth and function of ovarian follicles. Moreover, ovine (o) GDF9 and oBMP15 cooperate to increase both (3)H-thymidine incorporation and alpha-inhibin production and to inhibit progesterone production by rat or ovine granulosa cells. Although the receptors through which these proteins act individually have been determined, the receptor(s) involved in mediating the cooperative effects of GDF9 and BMP15 is (are) unknown. In this study, the effects of the extracellular domains of the types I and II TGFbeta receptors on (3)H-thymidine incorporation by rat granulosa cells stimulated by oGDF9 and oBMP15 were investigated. Stimulation of (3)H-thymidine incorporation was completely blocked by the BMP receptor II (BMPRII) extracellular domain but unaffected by any other type II or any type I receptor. These results suggest that the initial interaction of oGDF9 and oBMP15 is with BMPRII and that a type I receptor is either recruited or already associated with BMPRII to mediate the cooperative effects of these growth factors.

The local regulation of folliculogenesis by members of the transforming growth factor superfamily and its relevance for advanced breeding programmes.

Regulation of the growth and maturation of the ovarian follicle is critical for normal reproductive function. Alterations in this growth can lead to pathological conditions, such as cystic follicles, reduced oocyte quality, or an abnormal endocrine environment leading to poor fertility. Alterations in follicular growth also influence the number of follicles ovulating and thus can change litter size. Both endocrine factors, such as follicle stimulating hormone and luteinizing hormone, as well as local factors, are known to regulate follicular growth and development. This review will focus on the role of local factors in regulation of ovarian follicular growth in ruminants, with a focus on members of the transforming growth factor superfamily. The potential role of these factors in regulating proliferation, apoptosis, steroidogenesis and responsiveness to gonadotrophins will be considered.

Reduced ovulation rate, failure to be mated and fertilization failure/embryo loss are the underlying causes of poor reproductive performance in juvenile ewes.

A ewe that is mated as a juvenile (producing a lamb at 1 year of age) will produce an average of only 0.6 lambs to weaning, compared to an average of 1.2 lambs in adult ewes. Understanding the underlying causes of this low reproductive efficiency and designing methods to improve or mitigate these effects could potentially increase adoption of mating juvenile ewes. In Experiment 1, 2 Cohorts of ewes, born a year apart, were mated in order to lamb at 1 and 2 years of age and the performance of the ewes at each age was compared. Onset of puberty, mating by the fertile ram, ovulation rate, early pregnancy (day 30-35) litter size, number of lambs born and number of lambs weaned were measured. In juvenile ewes, by day 35 of pregnancy, 43% of ova had failed to become a viable embryo and this early loss was the largest contributor to the poor reproductive performance observed. Compared with young adult ewes, ovulation rate was lower (p<0.001), fewer ova were exposed to sperm (p<0.001) and fertilization failure/embryo loss was increased (p<0.001) in juveniles. In Experiment 2, the early pregnancy litter size of juveniles was shown to be greater (p<0.001) in those ewes with a greater ovulation rate (p<0.001). Attaining puberty prior to introduction of the fertile ram was associated with an increased pregnancy rate (p<0.001). In juvenile ewes, failure to mate with the ram, lower ovulation rate and increased fertilisation failure/embryo loss underlie their poor reproductive performance.

The proline-rich region of mouse p53 influences transactivation and apoptosis but is largely dispensable for these functions.

The N-terminal proline-rich domain of human p53 has been shown to be important for the induction of apoptosis. However, the corresponding region in mouse and other species is not highly conserved and has been less well studied. In this paper, we have characterized mutants with deletions in this region of mouse p53. Our results demonstrate that deletions in the proline-rich domain have varying effects on function ranging from no effect to severe impairment of cell death activity, depending on precisely which residues are deleted. We also show that the mutants differ in their ability to transactivate different p53 target promoters. Although we have been able to obtain mutants selectively impaired for apoptosis, our data are not generally consistent with this region being a functional domain. The data are more consistent with the interpretation that the region influences function by altering local protein structure which may affect promoter discrimination.

Nuclear localization of Y-box factor YB1 requires wild-type p53.

Nuclear localization and high levels of the Y-box binding protein YB1 appear to be important indicators of drug resistance and tumor prognosis. YB1 also interacts with the p53 tumor suppressor protein. In this paper, we explore a role for p53 in the nuclear localization of YB1. We report that various genotoxic stresses induce nuclear localization of YB1 in a small proportion of treated cells, but only in cells with wild-type p53. We go on to show directly that functional p53 is required for YB1 to translocate to the nucleus. Tumor-associated p53 mutants however are attenuated for YB1 nuclear localization as are mutants mutated in the proline-rich domain of p53. These data link the DNA-damage response of p53 to YB1 nuclear translocation. In addition, we find that YB1 inhibits p53-induced cell death and its ability to trans-activate promoters of genes involved in cell death signaling. Together these data suggest that some forms of p53 cause YB1 to accumulate in the nucleus, which in turn inhibits p53 activity. These results provide a possible explanation for the correlation of nuclear YB1 with drug resistance and poor prognosis in some tumor types, and for the first time implicate p53 in the process of nuclear translocation.

The local regulation of folliculogenesis by members of the transforming growth factor superfamily and its relevance for advanced breeding programmes

Regulation of the growth and maturation of the ovarian follicle is critical for normal reproductive function. Alterations in this growth can lead to pathological conditions, such as cystic follicles, reduced oocyte quality, or an abnormal endocrine environment leading to poor fertility. Alterations in follicular growth also influence the number of follicles ovulating and thus can change litter size. Both endocrine factors, such a sfollicle stimulating hormone and luteinizing hormone, as well as local factors, are known to regulate follicular growth and development. This review will focus on the role of local factors in regulation of ovarian follicular growth in ruminants, with a focus on members of the transforming growth factor superfamily. The potential role of these factors in regulating proliferation, apoptosis, steroidogenesis and responsiveness to gonadotrophins will be considered.

The local regulation of folliculogenesis by members of the transforming growth factor superfamily and its relevance for advanced breeding programmes

Regulation of the growth and maturation of the ovarian follicle is critical for normal reproductive function. Alterations in this growth can lead to pathological conditions, such as cystic follicles, reduced oocyte quality, or an abnormal endocrine environment leading to poor fertility. Alterations in follicular growth also influence the number of follicles ovulating and thus can change litter size. Both endocrine factors, such a sfollicle stimulating hormone and luteinizing hormone, as well as local factors, are known to regulate follicular growth and development. This review will focus on the role of local factors in regulation of ovarian follicular growth in ruminants, with a focus on members of the transforming growth factor superfamily. The potential role of these factors in regulating proliferation, apoptosis, steroidogenesis and responsiveness to gonadotrophins will be considered.(AU)

Evidence that replication of the antitumor adenovirus ONYX-015 is not controlled by the p53 and p14(ARF) tumor suppressor genes.

The adenovirus mutant ONYX-015 is in phase III clinical trials as a novel antitumor therapy. Its apparent efficacy is thought to be due to its ability to replicate selectively in tumor cells defective in the signaling pathway for p53. Recent data have shown that p14(ARF), a positive regulator of p53, inhibits ONYX-015 replication in cells with a wild-type p53, a phenotype that characterizes normal cells. We, however, found that ONYX-015 activates p53 in tumor cells and in normal cells and that this can occur without p14(ARF) induction. We also show that ONYX-015 is not attenuated in cells with functional p53, whether or not p14(ARF) is expressed, and that where attenuation does occur, it is cell type specific.