KIRREL is differentially expressed in adipose tissue from 'fertil+' and 'fertil-' cows: in vitro role in ovary?

We have previously shown that dairy cows carrying the 'fertil-' haplotype for one quantitative trait locus affecting female fertility located on the bovine chromosome three (QTL-F-Fert-BTA3) have a significantly lower conception rate and body weight after calving than cows carrying the 'fertil+' haplotype. Here, we compared by Tiling Array the expression of genes included in the QTL-F-Fert-BTA3 in 'fertil+' and 'fertil-' adipose tissue one week after calving when plasma non-esterified fatty acid concentrations were greater in 'fertil-' animals. We observed that thirty-one genes were overexpressed whereas twelve were under-expressed in 'fertil+' as compared to 'fertil-' cows (P < 0.05). By quantitative PCR and immunoblot we confirmed that adipose tissue KIRREL mRNA and protein were significantly greater expressed in 'fertil+' than in 'fertil-'. KIRREL mRNA is abundant in bovine kidney, adipose tissue, pituitary, and ovary and detectable in hypothalamus and mammary gland. Its expression (mRNA and protein) is greater in kidney of 'fertil+' than 'fertil-' cows (P < 0.05). KIRREL (mRNA and protein) is also present in the different ovarian cells with a greater expression in granulosa cells of 'fertil+' than 'fertil-' cows. In cultured granulosa cells, recombinant KIRREL halved steroid secretion in basal state (P < 0.05). It also decreased cell proliferation (P < 0.05) and in vitro oocyte maturation (P < 0.05). These results were associated to a rapid increase in MAPK1/3 and MAPK14 phosphorylation in granulosa cells and to a decrease in MAPK1/3 phosphorylation in oocyte. Thus, KIRREL could be a potential metabolic messenger linking body composition and fertility.

Short oestrous cycles in sheep during anoestrus involve defects in progesterone biosynthesis and luteal neovascularisation.

Anoestrous ewes can be induced to ovulate by the socio-sexual, 'ram effect'. However, in some ewes, the induced ovulation is followed by an abnormally short luteal phase causing a so-called 'short cycle'. The defect responsible for this luteal dysfunction has not been identified. In this study, we investigated ovarian and uterine factors implicated in male-induced short cycles in anoestrous ewes using a combined endocrine and molecular strategy. Before ovulation, we were able to detect a moderate loss of thecal expression of steroid acute regulatory protein (STAR) in ewes that had not received progesterone priming (which prevents short cycles). At and following ovulation, we were able to identify a significant loss of expression of genes coding key proteins involved in the biosynthesis of progesterone (STAR, CYP11A1 and HSD3B1 (HSD3B)) as well as genes coding proteins critical for vascular development during early luteal development (VEGFA and KDR (VEGFR2)), suggesting dysfunction in at least two pathways critical for normal luteal function. Furthermore, these changes were associated with a significant reduction of progesterone production and luteal weight. Additionally, we cast doubt on the proposed uterus-mediated effect of prostaglandin F2α (PGF2α) as a cause of short cycles by demonstrating the dysregulation of luteal expression of the PGF receptor, which mediates the luteal effects of PGF2α, and by finding no significant changes in the circulating concentrations of PGFM, the principal metabolite of PGF2α in ewes with short cycles. This study is the first of its kind to examine concurrently the endocrine and molecular events in the follicular and early luteal stages of the short cycle.

Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa.

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

The pattern of LH secretion and the ovarian response to the 'ram effect' in the anoestrous ewe is influenced by body condition but not by short-term nutritional supplementation.

In sheep, the 'ram effect' induces out-of-season fertility and good nutrition increases prolificacy. This experiment determined if fatness or short-term nutritional supplementation modified the response to the 'ram effect'. A group of 48 Île-de-France ewes were fed diets that produced groups with body-condition scores (BCS) of >3.0 and <2.0. Within each BCS group animals were supplemented daily with 500g of lupins from Day -5 to Day 0 (ram introduction) resulting in four groups: low BCS, supplemented (n=7) and non-supplemented (n=8) and high BCS, supplemented (n=12) and non-supplemented (n=11). The blood concentrations of glucose and insulin and the LH response to gonadotrophin-releasing hormone (GnRH) were determined. After the 'ram effect' the pattern of LH pulsatility, the LH surge and ovarian responses were analysed. Low BCS ewes had lower glucose and insulin (P<0.001) and supplementation increased both (P≤0.001). The increase in LH induced by GnRH was reduced in low BCS ewes (P=0.015) but it was not affected by supplementation. Similarly, LH pulsatility was reduced in low BCS ewes (P<0.05). The LH surge and ovarian cyclicity were not affected but the follow-up cycle was delayed (P=0.034) and progesterone was reduced (P=0.029) in low BCS ewes. There was an effect of BCS on ovulation rate (P<0.05). These results show that the BCS can modify the response to the 'ram effect' and that supplementation has little effect on this response.

Photoperiodic variations of the polysialylated form of neural cell adhesion molecule within the hypothalamus and related reproductive output in the ewe.

Cellular mechanisms induced by melatonin to synchronise seasonal reproduction in several species, including sheep, remain unclear. We sought to evaluate the scale and physiological significance of neural plasticity in order to explain the delay between the change of duration of melatonin secretion and the change of reproductive status following a transition from long days (LD, 16 h light/24 h) to short days (SD, 8 h light/24 h) and from SD to LD. Using Western blots in ovariectomised oestradiol-replaced ewes, we evaluated the content of the polysialylated form of neural cell adhesion molecule (PSA-NCAM), a plasticity marker, in the hypothalamus. From day 15 following a transition to SD, most hypothalamic areas showed a decrease of PSA-NCAM level that was particularly significant in the preoptic area (POA). Following a transition to LD, PSA-NCAM content increased at day 15 in most regions except in the premammillary hypothalamic area (PMH) in which a significant decrease was noted. The functional importance of PSA-NCAM variations for seasonal reproduction was assessed for the PMH and POA. PSA-NCAM was degraded by stereotaxic injections of endoneuraminidase N and luteinising hormone (LH) secretion was recorded in treated and control ewes. Degradation of PSA-NCAM in the PMH in SD-treated ewes failed to produce a significant effect on LH secretion, whereas a similar treatment in the POA before a transition to SD delayed activation of the gonadotroph axis in two-thirds of the ewes. Our results suggest that the photoperiod controls variations of the hypothalamic content of a plasticity marker and that these might be important for the regulation of seasonal reproduction, particularly in the POA.

Comparison of gizzard activity between chickens from genetic D+ and D- lines selected for divergent digestion efficiency.

Gizzard motility was compared between chickens from D(+) (high digestion efficiency) and D(-) (low digestion efficiency) genetic lines selected for divergent digestion efficiency, using strain gauge transducers. Motility was recorded continuously during 24 h in 6 birds per line. Two stimuli, a meal distribution after a feed-deprivation period and lighting after a dark period, were tested during the recording period. A functional test with intravenous injection of serotonin performed at the end of the recording day often resulted in a sharp reduction in gizzard motility, without a significant difference between lines. Compared with D(+) birds, gizzard activity in D(-) birds remained high during fasting or dark periods (P < 0.0006), which reduced the effects of stimuli in D(-) birds. So, coordination between bird activity and gizzard motility tended to be reduced in D(-) compared with D(+) birds (P = 0.0018). This coordination was observed to be positively correlated (P = 0.011) with the relative weight (g/kg of BW) of the stomach (gizzard + proventriculus). This experiment pointed out differences in gizzard motility between D(+) and D(-) chicken lines in terms of response to environmental stimuli, characterized by a failure in the gizzard relaxation process in D(-) birds during rest periods.

Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes.

We determined whether kisspeptin could be used to manipulate the gonadotropin axis and ovulation in sheep. First, a series of experiments was performed to determine the gonadotropic responses to different modes and doses of kisspeptin administration during the anestrous season using estradiol-treated ovariectomized ewes. We found that: 1) injections (iv) of doses as low as 6 nmol human C-terminal Kiss1 decapeptide elevate plasma LH and FSH levels, 2) murine C-terminal Kiss1 decapeptide was equipotent to human C-terminal Kiss1 decapeptide in terms of the release of LH or FSH, and 3) constant iv infusion of kisspeptin induced a sustained release of LH and FSH over a number of hours. During the breeding season and in progesterone-synchronized cyclical ewes, constant iv infusion of murine C-terminal Kiss1 decapeptide-10 (0.48 mumol/h over 8 h) was administered 30 h after withdrawal of a progesterone priming period, and surge responses in LH occurred within 2 h. Thus, the treatment synchronized preovulatory LH surges, whereas the surges in vehicle-infused controls were later and more widely dispersed. During the anestrous season, we conducted experiments to determine whether kisspeptin treatment could cause ovulation. Infusion (iv) of 12.4 nmol/h kisspeptin for either 30 or 48 h caused ovulation in more than 80% of kisspeptin-treated animals, whereas less than 20% of control animals ovulated. Our results indicate that systemic delivery of kisspeptin provides new strategies for the manipulation of the gonadotropin secretion and can cause ovulation in noncyclical females.

Development of OPS vitrified pig blastocysts: effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred.

Unhatched blastocysts from Large White hyperprolific gilts (n=103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO+16.5, 18, or 20% EG+0.4M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126-144microm versus 100% for blastocysts >199microm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO+EG and transferred surgically in groups of 20 or 30 per recipient (n=40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50-70%) and embryo survival rates (14.7-25%), although survival was higher (P=0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.

Improved vitrification method allowing direct transfer of goat embryos.

The aim of this study was to design a vitrification method suited to field embryo transfer experiments in goat. In a first experiment, a standard vitrification protocol, previously designed for sheep embryos was compared to slow freezing of goat embryos. No significant difference was observed on kidding rate (48% versus 69%, respectively), nor on embryo survival rate (35% versus 45%). Second experiment: all embryos were vitrified. After warming, embryos were either transferred directly (direct transfer), or after in vitro dilution of the cryoprotectants (conventional transfer). The kidding rate was not affected by the transfer method (38% versus 23%, respectively). However, embryo survival rate tended to be higher after direct transfer (26% versus 14%). Third experiment: OPS vitrification was compared to standard vitrification. The kidding rate was not affected (22% versus 39%, respectively), but the embryo survival rate was lower after OPS (14% versus 28%). Fourth experiment: 0.4M sucrose was added with cryoprotectants in vitrification. The kidding rate after direct transfer was significantly enhanced after addition of sucrose (56% versus 27%, respectively), whereas embryo survival rate was not significantly affected (32% versus 18%). Fifth experiment: vitrification with sucrose supplementation was compared to slow freezing. No significant difference was observed after direct transfer on kidding rate (52% versus 31%, respectively), but embryo survival rate tended to be higher after vitrification (34% versus 21%). In conclusion, our results indicate that addition of 0.4M sucrose in association with direct transfer improves significantly the viability of goat vitrified embryos.

[In vivo imaging of ram spermatozoa in the ewe genital tract using fibered confocal microscopy]. / Visualisation in vivo des spermatozoïdes de bélier dans le tractus génital de brebis par microscopie confocale fibrée.

Sperm transit in the female tract is one of the key factors in the success of fertilization after artificial insemination in sheep species. However, its study is limited by the absence of in vivo imaging methods. The imaging of ram sperm in the female genital tract was made possible using the confocal fibered microscopy and fluorescent stains adapted to spermatozoa. Our results show the active role of the uterotubal junction in the selection of sperm during their transit.

Effects of photoperiod on kisspeptin neuronal populations of the ewe diencephalon in connection with reproductive function.

Kisspeptin (Kiss) is a key regulator of reproductive function in both prepubertal and adult mammals. Its expression appears to vary throughout the year in seasonal species. We aimed to determine the impact of a change of photoperiod on the size of Kiss neuronal populations found in the preoptic area (POA) and arcuate nucleus (ARC) of the ewe brain. Using immunocytochemistry, we first examined the proportion of neurones expressing Kiss, using HuC/D as a neuronal marker, at different time-points after transition from long days (LD; 16 : 8 h light/dark cycle) to short days (SD; 8 : 16 h light/dark cycle). Luteinising hormone (LH) secretion was measured in ovariectomised oestradiol replaced ewes from the month preceding the transition to SD until the sacrifice of the animals at days 0, 45 and 112 from this photoperiodic transition. High LH levels were only observed in animals killed at day 112. The number of Kiss neurones/mm(2) doubled in the caudal ARC at day 112. The percentage of neurones showing Kiss immunoreactivity increased significantly in both the POA and ARC in the day 112 group. In a second experiment, ewes kept in LD received an i.c.v. injection of colchicine 20 h before sacrifice. Colchicine treatment increased the number and the percentage of neurones with Kiss in both the POA and caudal ARC. The data obtained suggest that the increase in Kiss neurones detected in the POA and caudal ARC after transition to SD stemmed from an increase in Kiss synthesis. This up-regulation of Kiss content under the shorter day condition appears to be a late event within the cascade activated by a longer secretion of melatonin, which is a critical factor in switching gonadotrophin-releasing hormone secretion to a breeding season profile.

Disruption of lipid rafts induces gonadotropin release in ovine pituitary and LbetaT2 gonadotroph cells.

In order to better understand the cellular mechanisms underlying LH and FSH secretion, we have addressed the contribution of lipid rafts to the secretion of gonadotropins. We used methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering agent, on an LbetaT2 murine gonadotroph cell line and on primary cultures of ovine pituitary cells. We found that in both systems, cholesterol depletion by MbetaCD induced a fast and substantial release of LH in the absence of natural stimulation by GnRH. In ovine pituitary cells, MbetaCD-mediated LH release was shown to be independent of protein synthesis. Twenty-four hours after MbetaCD treatment, there was no loss of cell viability and full recovery of LH secretory capabilities, as determined by GnRH or MbetaCD treatment. In addition, our data suggest the existence of a pool of LH that is not released by GnRH treatment but that is released by MbetaCD treatment. Finally, in ovine pituitary cells, MbetaCD treatment induced FSH secretion. Importantly, these in vitro data are supported by in vivo studies, because MbetaCD injected into the pituitary glands of anaesthetized sheep reproducibly induced a peak of LH release.