Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes

Litter size (LS) in sheep is determined mainly by ovulation rate (OR). Several polymorphisms have been identified in the growth differentiation factor 9 (GDF9) gene that result in an increase in OR and prolificacy of sheep. Screening the databank of the Brazilian Sheep Breeders Association for triplet delivery, we identified flocks of prolific Ile de France ewes. After resequencing of GDF9, a point mutation (c.943C>T) was identified, resulting in a non‐conservative amino acid change (p.Arg315Cys) in the cleavage site of the propeptide. This new allele was called Vacaria (FecG^v). A flock of half‐sib ewes was evaluated for OR in the first three breeding seasons, and Vacaria heterozygotes had higher OR (P < 0.001), averaging 2.1 ± 0.1 when compared to 1.2 ± 0.1 in wild‐type ewes. The OR was also influenced by age, increasing in the second and third breeding seasons (P < 0.001). In flocks segregating this allele, the LS was higher in mutant sheep (P < 0.001), averaging 1.61 ± 0.07 in heterozygotes and 1.29 ± 0.03 in wild‐type ewes. Analysis of homozygote reproductive tract morphology revealed uterine and ovarian hypoplasia. Ovarian follicles continue to develop up to small antral stages, although with abnormal oocyte morphology and altered arrangement of granulosa cells. After the collapse of the oocyte in most follicles, the remaining cells formed clusters that persisted in the ovary. This SNP is useful to improve selection for dam prolificacy and also as a model to investigate GDF9 post‐translation processing and the fate of the follicular cells that remain after the oocyte demise.     

Decreased oocyte DAZL expression in mice results in increased litter size by modulating follicle-stimulating hormone-induced follicular growth

While the germ cell-specific RNA binding protein, DAZL, is essential for oocytes to survive meiotic arrest, DAZL heterozygous (het) mice have an increased ovulation rate that is associated with elevated inhibin B and decreased plasma follicle-stimulating hormone (FSH). The relationship between decreased oocyte DAZL expression and enhanced follicular development in het mice was investigated using in vitro follicle cultures and in vivo modulation of endogenous FSH, by treating mice with inhibin and exogenous FSH. In vitro, follicles from het mice are more sensitive to FSH than those of wild-type (wt) mice and can grow in FSH concentrations that are deleterious to wild-type follicles. In vivo, despite no differences between genotypes in follicle population profiles, analysis of granulosa cell areas in antral follicles identified a significantly greater number of antral follicles with increased granulosa cell area in het ovaries. Modulation of FSH in vivo, using decreasing doses of FSH or ovine follicular fluid as a source of inhibin, confirmed the increased responsiveness of het antral follicles to FSH. Significantly more follicles expressing aromatase protein confirmed the earlier maturation of granulosa cells in het mice. In conclusion, it is suggested that DAZL expression represses specific unknown genes that regulate the response of granulosa cells to FSH. If this repression is reduced, as in DAZL het mice, then follicles can grow to the late follicular stage despite declining levels of circulating FSH, thus leading to more follicles ovulating and increased litter size.     

Hormonal Responses to Synthetic Luteinizing Hormone and Follicle Stimulating Hormone-Releasing Hormone in Man

The effects of the gonadotrophin-releasing hormone, synthetic decapeptide luteinizing hormone/follicle stimulating hormone-releasing hormone (LH/FSH-RH), have been studied in 18 normal men and five women in the follicular phase of their menstrual cycle. Rapid and dose-dependent (25 to 100 μg) increases in serum immunoreactive LH were seen, which reached a peak 20 to 30 minutes after a rapid intravenous injection. Similar but much smaller increases in serum immunoreactive FSH were seen. These conclusions have been validated by using two different immunoassay systems for each hormone. The LH/FSH-RH therefore causes both LH and FSH release in man as in animals but does not affect growth hormone, thyrotrophin, or ACTH. The gonadotrophin responses were the same in the women as in the men but were insufficient in the men to cause statistically significant changes in the serum levels of the gonadal steroid hormones, testosterone or oestradiol, or in their precursors 17 α-hydroxyprogesterone or progesterone. In the women, however, there was a rise in oestradiol after the 100-μg doses. The use of LH/FSH-RH will provide an important test to define the level of the lesion in hypogonadal patients and also should be valuable in the treatment of some types of male and female infertility. A simple and clinically useful LH/FSH-RH test of pituitary function is described (100 μg given intravenously), and the provisional normal responses of LH and FSH at 20 and 60 minutes are given.     

Reduction in BACE1 decreases body weight, protects against diet-induced obesity and enhances insulin sensitivity in mice

Insulin resistance and impaired glucose homoeostasis are important indicators of Type?2 diabetes and are early risk factors of AD (Alzheimer's disease). An essential feature of AD pathology is the presence of BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), which regulates production of toxic amyloid peptides. However, whether BACE1 also plays a role in glucose homoeostasis is presently unknown. We have used transgenic mice to analyse the effects of loss of BACE1 on body weight, and lipid and glucose homoeostasis. BACE1-/- mice are lean, with decreased adiposity, higher energy expenditure, and improved glucose disposal and peripheral insulin sensitivity than wild-type littermates. BACE1-/- mice are also protected from diet-induced obesity. BACE1-deficient skeletal muscle and liver exhibit improved insulin sensitivity. In a skeletal muscle cell line, BACE1 inhibition increased glucose uptake and enhanced insulin sensitivity. The loss of BACE1 is associated with increased levels of UCP1 (uncoupling protein 1) in BAT (brown adipose tissue) and UCP2 and UCP3 mRNA in skeletal muscle, indicative of increased uncoupled respiration and metabolic inefficiency. Thus BACE1 levels may play a critical role in glucose and lipid homoeostasis in conditions of chronic nutrient excess. Therefore strategies that ameliorate BACE1 activity may be important novel approaches for the treatment of diabetes.     

An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157 : H7 with bovine intestinal epithelium

Enterohaemorrhagic Escherichia coli O157 : H7 is a bacterial pathogen that can cause haemorrhagic colitis and haemolytic uremic syndrome. In the primary reservoir host, cattle, the terminal rectum is the principal site of E. coli O157 colonization. In this study, bovine terminal rectal primary epithelial cells were used to examine the role of H7 flagella in epithelial adherence. Binding of a fliC_H7 mutant O157 strain to rectal epithelium was significantly reduced as was binding of the flagellated wild-type strain following incubation with H7-specific antibodies. Complementation of fliC_H7 mutant O157 strain with fliC_H7 restored the adherence to wild-type levels; however, complementation with fliC_H6 did not restore it. High-resolution ultrastructural and imunofluorescence studies demonstrated the presence of abundant flagella forming physical contact points with the rectal epithelium. Binding to terminal rectal epithelium was specific to H7 by comparison with other flagellin types tested. In-cell Western assays confirmed temporal expression of flagella during O157 interaction with epithelium, early expression was suppressed during the later stages of microcolony and attaching and effacing lesion formation. H7 flagella are expressed in vivo by individual bacteria in contact with rectal mucosa. Our data demonstrate that the H7 flagellum acts as an adhesin to bovine intestinal epithelium and its involvement in this crucial initiating step for colonization indicates that H7 flagella could be an important target in intervention strategies.     

Genetic basis of variation of yield, and yield components in mungbean (Vigna radiata (L.) Wilczek)

Additive, dominance, and epistasis genetic basis of seed yield per plant, number of pods per plant, number of seeds per pod, and 1000 seed weight in mungbean (Vigna radiata (L.) Wilczek) have been examined, using Triple Test Cross (TTC) analysis. The material for TTC test was evaluated in two seasons i.e., kharif (July-October) and spring/summer (March-June), at the research station of the Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistan. Epistasis was present significantly for number of pods per plant and number of seeds per pod when grown in the spring/summer season (March to June). Partition of epistasis showed that additive x additive ('i' type) interaction was an important component of number of pods per plant, and number of seeds per pod was found to be of both types 'i' type, and additive x dominance, and dominance x dominance ('j' and 'l' type) interactions. This indicated that epistasis might be a non-trivial factor in the inheritance of pods per plant, and seeds per pod in mungbean. The expression of epistasis was influenced differentially by particular genotypes, indicating that a limited number of genotypes may not be sufficient to detect non-allelic interactions for a trait in mungbean. Additive and dominance genetic components were significant for all four traits in kharif season (July to October) but only for seed yield and 1000 seed weight in spring/summer season. This suggests that the genes controlling seed yield per plant, and 1000 seed weight are equally sensitive to the environment. The predominance additive gene action in those traits is not significantly influenced by epistasis, suggesting that improvement of the traits can be achieved through standard selection procedures.     

Diversity and divergence in Cistus salvifolius (L.) populations from contrasting habitats

Cistus salvifolius L. is a widespread Mediterranean shrub, occurring over a wide range of environments. Given the degree of habitat differentiation, and geographic isolation of some populations, adaptation to local conditions and hence population divergence might be expected to have occurred. To test this hypothesis morphology and allozyme diversity was measured in 13 populations collected from contrasting habitats around the Mediterranean. Leaf morphology (length, width and petiole length) and internode length varied widely between populations. Leaf width and internode length were negatively correlated with longitude, and leaf length was negatively correlated with mean rainfall. All populations were polymorphic at all allozyme loci studied, and no populations showed significant difference between levels of expected and observed heterozygotes. Allelic diversity (Hs) within populations was high, and populations from the more extreme sites showed no decrease in diversity or predominance of rare genotypes, suggesting there is little selection for characters favouring survival in local conditions. Some populations from highly contrasting habitats, in terms of rainfall, appeared to be genetically similar. However, there were differences between some populations, in areas less than 1 km apart, which have similar geography and climate. Results suggest that the C. salvifolius populations examined may not be as adapted to local environmental conditions as expected. Periodic fires, gene flow, and environmental heterogeneity may all help maintain genetic diversity and hinder adaptation.     

Effects of prolactin on the luteinizing hormone response to gonadotropin- releasing hormone in primary pituitary cell cultures during the ovine annual reproductive cycle

In the sheep pituitary, the localization of prolactin (PRL) receptors in gonadotrophs and the existence of gonadotroph-lactotroph associations have provided morphological evidence for possible direct effects of PRL on gonadotropin secretion. Here, we investigated whether PRL can readily modify the LH response to GnRH throughout the ovine annual reproductive cycle. Cell populations were obtained from sheep pituitaries during the breeding season (BS) and the nonbreeding season (NBS), plated to monolayer cultures for 7 days, and assigned to receive one of the following treatments: 1) nil (control), 2) acute (90- min) bromocriptine (ABr), 3) chronic (7-day) bromocriptine (CBr), 4) ABr and PRL, 5) CBr and PRL, 6) PRL alone, or 7) thyrotropin-releasing hormone. Cells were treated as described above, with the aim of decreasing or increasing the concentrations of PRL in the culture, and simultaneously treated with GnRH for 90 min. The LH concentrations in the medium were then determined by RIA. GnRH stimulated LH in a dose-dependent manner during both stages of the annual reproductive cycle. During the NBS, single treatments did not significantly affect the LH response to GnRH. However, when PRL was combined with bromocriptine, either acutely or chronically, GnRH failed to stimulate LH release at all doses tested (P < 0.01). In contrast, during the BS, the LH response to GnRH was not affected by any of the experimental treatments. These results reveal no apparent effects of PRL alone, but an interaction between PRL and dopamine in the regulation of LH secretion within the pituitary gland, and a seasonal modulation of this mechanism.