The epoxiconazole and tebuconazole fungicides impair granulosa cells functions partly through the aryl hydrocarbon receptor (AHR) signalling with contrasted effects in obese, normo-weight and polycystic ovarian syndrome (PCOS) patients.

Polycystic ovarian syndrome (PCOS), frequently associated to obesity, is the main reproductive disorder in women in age to procreate. Some evidence suggests that pesticides can result in alterations of the female reproductive system, including polycystic ovary syndrome (PCOS). Here, we detected two fungicides, Tebuconazole (Tb) and Epoxiconazole (Epox) in the soils and waters of French area. Our hypothesis is that these two triazoles could be associated to the etiology of PCOS. We used the human KGN cell line and primary human granulosa cells (hGCs) from different group of patients: normal weight non PCOS (NW), normal weight PCOS (PCOS NW), obese (obese) and obese PCOS (PCOS obese). We exposed in vitro these cells to Tb and Epox from 0 up to 10 mM for 24 and 48 h and analysed cell viability and steroidogenesis. In hGCs NW, cell viability was reduced from 12.5 µM for Tb and 75 µM for Epox. In hGCs NW, Epox decreased progesterone (Pg) and estradiol (E2) secretions and inhibited STAR, HSD3B and CYP19A1 mRNA expressions from 25 µM and increased AHR mRNA expression from 75 µM. Tb exposure also reduced steroid secretion and STAR and CYP19A1 mRNA expressions and increased AHR mRNA expression but at cytotoxic concentrations. Silencing of AHR in KGN cells reduced inhibitory effects of Tb and Epox on steroid secretion. Tb and Epox exposure decreased more steroid secretion in hGCs from obese, PCOS NW and PCOS obese groups than in NW group. Moreover, we found a higher gene expression of AHR within these three groups. Taken together, both Epox and Tb reduced steroidogenesis in hGCs through partly AHR and Tb was more cytotoxic than Epox. These triazoles alter more strongly PCOS and/or obese hGCs suggesting that human with reproductive disorders are more sensitive to triazoles exposure.

Determination of the elimination half-life of Glyphosate and its main metabolite, AMPA, in chicken plasma.

Glyphosate-based herbicides (GBHs) are the most-used herbicides worldwide. Concerns about their toxicity and ecotoxicity have motivated scientists to assess their potential effects on animals, as well as their toxicokinetic parameters in rats and humans. However, to our knowledge, such data have not been produced for avian models. In this study, toxicokinetic parameters for glyphosate and AMPA were calculated after one unique dietary exposure (40 mg of glyphosate equivalent per kg) and one unique intravenous injection of a GBH, in hens and roosters respectively. Non compartmental analysis was used to show the evolution of glyphosate and AMPA plasma concentrations over time. After one unique intravenous injection of a glyphosate-based herbicide, glyphosate and AMPA were quickly eliminated from plasma and were poorly distributed (Vssglyphosate = 0.30 L/kg). Their terminal half-lives are 4.7 h and 8.10 h, respectively. After dietary exposure, glyphosate and AMPA followed a 6 h absorption phase followed by a 42 h elimination phase. They were poorly distributed (Vssglyphosate = 0.00562 L/kg), and their maximum concentrations (Cmax) were 21285 µg/L and 108 µg/L, respectively. Their terminal elimination half-lives were 8.94 h and 6.93 h, respectively. Taken together, this study provides new data on the elimination rate and approximate biological half-life range of glyphosate in birds.

In vitro exposure to triazoles used as fungicides impairs human granulosa cells steroidogenesis.

Triazoles are the main components of fungicides used in conventional agriculture. Some data suggests that they may be endocrine disruptors. Here, we found five triazoles, prothioconazole, metconazole, difenoconazole, tetraconazole, and cyproconazole, in soil or water from the Centre-Val de Loire region of France. We then studied their effects from 0.001 µM to 1000 µM for 48 h on the steroidogenesis and cytotoxicity of ovarian cells from patients in this region and the human granulosa line KGN. In addition, the expression of the aryl hydrocarbon receptor (AHR) nuclear receptor in KGN cells was studied. Overall, all triazoles reduced the secretion of progesterone, estradiol, or both at doses that were non-cytotoxic but higher than those found in the environment. This was mainly associated, depending on the triazole, with a decrease in the expression of CYP51, STAR, CYP11A1, CYP19A1, or HSD3B proteins, or a combination thereof, in hGCs and KGN cells and an increase in AHR in KGN cells.

Spexin role in human granulosa cells physiology and PCOS: expression and negative impact on steroidogenesis and proliferation†.

Spexin (SPX) is a novel neuropeptide and adipokine negatively correlated with obesity and insulin resistance. A recent study investigated expression and regulatory function of SPX in the hypothalamus and pituitary; however, the effect on ovarian function is still unknown. The aim of this study was to characterize the expression of SPX and its receptors, galanin receptors 2 and 3 (GALR2/3), in the human ovary and to study its in vitro effect on granulosa cells (GC) function. Follicular fluid (FF) and GC were obtained from normal weight and obese healthy and diagnosed with polycystic ovarian syndrome (PCOS) women. Expression of SPX and GALR2/3 in the ovary was studied by qPCR, western blot, and immunohistochemistry. The level of SPX in FF was assessed by enzyme-linked immunosorbent assay. The in vitro effect of recombinant human SPX on GC proliferation, steroidogenesis, and signaling pathways (MAP3/1, STAT3, AKT, PKA) was analyzed. Moreover, GC proliferation and estradiol (E2) secretion were measured with and without an siRNA against GALR2/3 and pharmacological inhibition of the above kinases. The results showed that both the SPX concentration in FF and its gene expression were decreased in GC of obese and PCOS women, while the protein expression of GALR2/3 was increased. We noted that SPX reduced GC proliferation and steroidogenesis; these effects were mediated by GALR2/3 and kinases MAP3/1, AKT, and STAT3 for proliferation or kinases MAP3/1 and PKA for E2 secretion. The obtained data clearly documented that SPX is a novel regulator of human ovarian physiology and possibly plays a role in PCOS pathogenesis.

Adipokines expression in reproductive tract, egg white and embryonic annexes in hen.

In mammals, molecules mainly secreted by white adipose tissue named adipokines are also synthetized locally in the reproductive tract and are able to influence reproductive functions. In avian species, previous studies indicated that the adipokine chemerin is highly abundant in the albumen, compared to the yolk and this was associated to high chemerin expression in the magnum. In addition, the authors observed that chemerin and its receptors are expressed by allantoic and amniotic membranes and chemerin is present in fluids during the embryo development. Here, we studied other adipokines, including adiponectin, visfatin, apelin, and adipolin in egg white and their known receptors in the active (egg-laying hen) and regressed (hen not laying) oviduct and embryonic annexes during embryo development. By using Western blot, RT-qPCR analysis and immunohistochemistry, we demonstrated the expression of different adipokines in the egg albumen (visfatin) and the reproductive tract (adiponectin, visfatin, apelin, adipolin, and their cognate receptors) according the position of egg in the oviduct. We showed that the expression of adipokines and adipokines receptors was strongly reduced in the regressed oviducts (arrested laying hen). Results indicated that visfatin and adiponectin appeared at ED11 to 14 and increased until ED18 in amniotic fluid whereas it was found from ED7 and was unchanged during embryo development in allantoic fluid. Taken together, adipokines and their receptors are expressed in the egg white, the reproductive tract and the embryonic annexes. Data obtained suggest important functions of theses metabolic hormones during the chicken embryo development. Thus, adipokines could be potential biomarkers to improve the embryo development.

Chronic dietary exposure to a glyphosate-based herbicide in broiler hens has long-term impacts on the progeny metabolism.

Glyphosate-based herbicides (GBH) are the most commonly used herbicides in agriculture. Several studies reported possible adverse effects on human and animal models after a GBH exposure. However, the effects of a temporary maternal exposure on the progeny have been poorly documented, especially in avian models. We investigated the effects of a hen chronic dietary exposure to a GBH on the progeny, obtained during the period following the withdrawal of GBH from the diet. Hens were exposed to a GBH via their food for 6 wk, after which the GBH was removed from their food. Eggs from these hens were collected 3 wk after the GBH was withdrawn for 1 wk. We monitored the growth performances, metabolic parameters, and behavior from the progeny of the hens (Ex-GBH chicks, n = 186) and compared them with those of unexposed control-hen progeny (CT chicks, n = 213). Ex-GBH chicks were more likely to explore their new environment than CT chicks during the open-field test. In addition, they had an increased fattening and blood triglycerides level, whereas their food consumption was similar to CT chicks. Quantitative PCR on the chemerin system and FASN in chicks livers indicate a transcriptional activity in favor of fatty acid synthesis, and lipidomic analysis on chicks abdominal adipose tissue reveal a global increase in monounsaturated fatty acid and a global decrease in polyunsaturated fatty acids. Seven genes involved in the synthesis of fatty acids were identified with the open access LIPIDMAP software, and their disturbance in Ex-GBH chicks was confirmed via qPCR. Taken together, these results suggest that the progeny of hens temporarily exposed to a GBH are more likely to fatten, even with a balanced diet. The removal of GBH from their contaminated environment would therefore not be sufficient to completely restore their health, has it could induce transgenerational effects.

Triazole pesticides exposure impaired steroidogenesis associated to an increase in AHR and CAR expression in testis and altered sperm parameters in chicken.

Since several decades, we observe the decline of various bird populations that could be partly linked to the agricultural intensification and the use of large amount of pesticides. Even if triazoles compounds are the most widely used fungicides, their effects on the reproductive parameters in birds are not clearly known. In the present study, we investigated the in vitro effects of 8 triazoles compounds alone (propiconazole (PP, from 0 to 10 µM), prothioconazole (PT), epoxiconazole (Epox), tetraconazole (TT), tebuconazole (TB), difenoconazole (Dif), cyproconazole (Cypro), metconazole (MC) (from 0 to 1 mM)) on the male chicken reproductive functions by using testis explants, primary Sertoli cells and sperm samples. In testis, all triazoles at the higher concentrations for 48 h inhibited lactate and testosterone secretion mostly in association with reduced expression of HSD3B and/or STAR mRNA levels. These data were also associated with increased expression of the nuclear receptors Aryl Hydrocarbon Receptor (AHR) and Constitutive Androstane Receptor (CAR) mRNA levels in testis and for all triazoles except for PP a reduction in Sertoli cell viability. When focusing on the sperm parameters, we demonstrated that most of the triazoles (MC, Epox, Dif, TB, TT and Cypro) at 0.1 or 1 mM for either 2, 12 or 24 min of exposure decreased sperm motility and velocity and increased the percentage of spermatozoa abnormal morphology. At the opposite, PP increased sperm motility in a dose dependent manner after 2 min of exposure whereas no significant effect was observed in response to PT whatever the dose and the time of exposure. Moreover, these effects were associated with an increase in the production of reactive oxygen species in spermatozoa. Taken together, most of the triazoles compounds impair testis steroidogenesis and semen parameters potentially through an increase in AHR and CAR expression and in oxidative stress, respectively. Data Availability Statement: All the data will be available.

The Mycotoxin De-Epoxy-Deoxynivalenol (DOM-1) Increases Endoplasmic Reticulum Stress in Ovarian Theca Cells.

Deoxynivalenol (DON) is a major mycotoxin present in animal feed and negatively affects growth and reproduction in farm species, including pigs and cattle. The mechanism of DON action involves the ribotoxic stress response (RSR), and it acts directly on ovarian granulosa cells to increase cell death. In ruminants, DON is metabolized to de-epoxy-DON (DOM-1), which cannot activate the RSR but has been shown to increase cell death in ovarian theca cells. In the present study, we determined if DOM-1 acts on bovine theca cells through endoplasmic stress using an established serum-free cell culture model and to assess whether also DON activates endoplasmic stress in granulosa cells. The results show that DOM-1 increased the cleavage of ATF6 protein, increased the phosphorylation of EIF2AK3, and increased the abundance of cleaved XBP1 mRNA. Activation of these pathways led to an increased abundance of mRNA of the ER stress target genes GRP78, GRP94, and CHOP. Although CHOP is widely associated with autophagy, inhibition of autophagy did not alter the response of theca cells to DOM-1. The addition of DON to granulosa cells partially increased ER stress pathways but failed to increase the abundance of mRNA of ER stress target genes. We conclude that the mechanism of action of DOM-1, at least in bovine theca cells, is through the activation of ER stress.

Seasonal remodeling of the progenitor pool and its distribution in the ewe mediobasal hypothalamus.

Recent studies have reported the presence of adult neurogenesis in the arcuate nucleus periventricular space (pvARH) and in the median eminence (ME), two structures involved in reproductive function. In sheep, a seasonal mammal, decreasing daylight in autumn induces a higher neurogenic activity in these two structures. However, the different types of neural stem and progenitor cells (NSCs/NPCs) that populate the arcuate nucleus and median eminence, as well as their location, have not been evaluated. Here, using semi-automatic image analyzing processes, we identified and quantified the different populations of NSCs/NPCs, showing that, during short days, higher densities of [SOX2 +] cells are found in pvARH and ME. In the pvARH, higher densities of astrocytic and oligodendrocitic progenitors mainly contribute to these variations. The different populations of NSCs/NPCs were mapped according to their position relative to the third ventricle and their proximity to the vasculature. We showed that [SOX2 +] cells extended deeper into the hypothalamic parenchyma during short days. Similarly, [SOX2 +] cells were found further from the vasculature in the pvARH and the ME, at this time of year, indicating the existence of migratory signals. The expression levels of neuregulin transcripts (NRGs), whose proteins are known to stimulate proliferation and adult neurogenesis and to regulate progenitor migration, as well as the expression levels of ERBB mRNAs, cognate receptors for NRGs, were assessed. We showed that mRNA expression changed seasonally in pvARH and ME, suggesting that the ErbB-NRG system is potentially involved in the photoperiodic regulation of neurogenesis in seasonal adult mammals.

The influence of selection in wild pheasant (Phasianus colchicus) breeding on reproduction and the involvement of the chemerin system.

Chemerin is a hormone produced mainly by adipose tissue and liver. We have recently shown that it is locally produced in the reproductive tract in hens, particularly at the magnum level, leading to its accumulation in the egg albumen. We have also determined that chemerin is necessary for egg fertilization, embryo development, and angiogenesis within the chorio-allantoic membrane in chicken species. We, therefore, hypothesize that chemerin, widely present in various gallinacean species, could be a marker of egg fertility in this animal order. To demonstrate this, we used a model close to the hen: the pheasant. By RT-qPCR, we have shown that chemerin and its three receptors CMKLR1, GPR1, and CCRL2 are expressed in the reproductive tract of females. In addition, chemerin is also produced predominantly in the magnum and accumulates in the egg albumen as determined by immunoblot. We then compared two lines of pheasants with different reproductive characteristics: the F11 and F22 breeds. F22 lays more eggs than F11, but have significantly lower fertility and hatchability rates. In addition, F22 exhibit a significantly lower amount of chemerin protein in their magnum (P < 0.01) and in the egg albumen (P < 0.0001) compared to F11. Finally, we observed a positive correlation between the chemerin amount in the albumen of F11 eggs and the hatching rate of the eggs (r = 0.5; P = 0.04) as well as a negative correlation between the chemerin quantity in the albumen of F22 eggs and the rate of unfertilized eggs (r = -0.37; P = 0.04). Finally, chemerin system (ligand and receptors) is also expressed within embryo annexes (chorioallantoic and amniotic membranes) during incubation. These data demonstrate an interspecies conservation of chemerin production in the magnum, its accumulation in the egg albumen and its possible use as a marker for determining the quality of eggs in term of fertility and embryo development.

Chicken white egg chemerin as a tool for genetic selection for egg weight and hen fertility.

Embryo mortality rate, which can reach up to 40% in avian species, is a major issue for breeding. It is therefore important to identify new embryo development biomarkers for genetic selection to improve reproductive performances. We have recently shown that chemerin is expressed in the oviductal hen magnum, accumulates in egg white, is correlated with embryo survival and could thus be used as a molecular marker of embryo development. Eggs from seven hen breeds (n = 70) were collected during five successive days at the end of the laying period. After weighing eggs, yolk and albumen, an egg white sample from each egg was collected and a blood sample was taken from each hen. Chemerin concentrations in albumen and blood samples were measured by a specific home made ELISA assay. Hen's plasma and egg's albumen chemerin levels were found to be correlated with reproductive parameters such as fecundity, fertility, embryo mortality, hatchability and laying rates. The inter-hen chemerin level variability in albumen was higher than intra-hen except for one breed (R+). We observed significantly different levels of chemerin in egg white between breeds. However, chemerin concentrations in egg white were not significantly associated to variations of hen plasma chemerin levels. Interestingly, we observed negative correlations between albumen chemerin concentrations and egg weight (r = -0.43, p = 0.001), between albumen weight (r = -0.40, p = 0.002), and between yolk weight (r = -0.28, p = 0.03). We also showed negative correlations between egg white chemerin concentrations and fecundity (r = -0.32, p = 0.011) and fertility (r = -0.27, p = 0.04) whereas no significant correlation was observed with the laying rate. Taken together, these results suggest that egg white chemerin concentration might be a good biomarker for genetic selection for egg weight and fertility in hens, provided these data are confirmed on a larger scale.

Chronic dietary exposure to a glyphosate-based herbicide results in total or partial reversibility of plasma oxidative stress, cecal microbiota abundance and short-chain fatty acid composition in broiler hens.

Glyphosate-based herbicides (GBHs) are massively used in agriculture. However, few studies have investigated the effects of glyphosate-based herbicides on avian species although they are largely exposed via their food. Here, we investigated the potential reversibility of the effects of chronic dietary exposure to glyphosate-based herbicides in broiler hens. For 42 days, we exposed 32-week-old hens to glyphosate-based herbicides via their food (47 mg/kg/day glyphosate equivalent, glyphosate-based herbicides, n = 75) corresponding to half glyphosate's no-observed-adverse-effect-level in birds. We compared their performance to that of 75 control animals (CT). Both groups (glyphosate-based herbicides and control animals) were then fed for 28 additional days without glyphosate-based herbicides exposure (Ex-glyphosate-based herbicides and Ex-control animals). Glyphosate-based herbicides temporarily increased the plasma glyphosate and AMPA (aminomethylphosphonic acid) concentrations. Glyphosate and aminomethylphosphonic acid mostly accumulated in the liver and to a lesser extent in the leg muscle and abdominal adipose tissue. Glyphosate-based herbicides also temporarily increased the gizzard weight and plasma oxidative stress monitored by TBARS (thiobarbituric acid reactive substances). Glyphosate-based herbicides temporarily decreased the cecal concentrations of propionate, isobutyrate and propionate but acetate and valerate were durably reduced. The cecal microbiome was also durably affected since glyphosate-based herbicides inhibited Barnesiella and favored Alloprevotella. Body weight, fattening, food intake and feeding behavior as well as plasma lipid and uric acid were unaffected by glyphosate-based herbicides. Taken together, our results show possible disturbances of the cecal microbiota associated with plasma oxidative stress and accumulation of glyphosate in metabolic tissues in response to dietary glyphosate-based herbicides exposure in broiler hens. Luckily, glyphosate-based herbicides at this concentration does not hamper growth and most of the effects on the phenotypes are reversible.

Impact of fibronectin type III domain-containing family in the changes in metabolic and hormonal profiles during peripartum period in dairy cows.

The peripartum period in dairy cows is frequently associated with excessive lipolysis due to Negative Energy Balance (NEB). These metabolic disorders are the cause of various pathologies. Some metabolites such as ß-hydroxybutyrate (BHBA) and Non-Esterified Fatty Acids (NEFA) are known to be biomarkers of NEB in dairy cows. The involvement of adipokines, including adiponectin and leptin, during fat mobilization in the peripartum period is well described, but little is known about the impact of myokines at this time. Fibronectin type III domain-containing proteins (FNDC) are myokines and adipokines recently discovered to play a role in metabolic dysfunctions. This study aimed to evaluate some FNDCs (FNDC5, 4, 3A and B) as potential plasma and adipose tissue indicators of NEB in cattle. We measured plasma FNDC concentrations and adipose tissue FNDC gene expression during the peripartum period, 4 weeks before the estimated calving day (4WAP), one (1WPP) and 16 (16WPP) weeks postpartum in two groups of dairy cows with low NEB (LNEB, n = 8) and high NEB (HNEB, n = 13) at 1WPP. Using specific bovine ELISAs, only plasma FNDC5 concentrations varied during the peripartum period in both LNEB and HNEB animals; concentrations were higher at 1WPP as compared to 4WAP and 16 WPP. FNDC5 plasma concentrations was negatively correlated with dry matter intake, live body weight, variation of empty body weight and glucose concentrations, and positively correlated with plasma non-esterified fatty acids and BHBA concentrations. Subcutaneous adipose tissue contained abundant FNDC5 mRNA and protein, as measured by RT-qPCR and immunoblotting, respectively. We also observed that FNDC5 mRNA abundance in subcutaneous adipose tissue was higher at 1 WPP as compared to 4WAP and 16WPP in HNEB cows and higher at 1 WPP as compared to 4 WAP in LNEB cows, and was higher in HNEB than in LNEB animals during early lactation. Finally, we showed that recombinant human irisin (a fragmented product of FNDC5) increased the release of glycerol and abundance of mRNA encoding adipose triglyceride lipase and hormone-sensitive-lipase in bovine and human adipose tissue explants. In conclusion, FNDC5 is expressed in bovine adipose tissue and may be involved in lipid mobilization and regulation of NEB in cattle.

Chemerin is secreted by the chicken oviduct, accumulates in egg albumen and could promote embryo development.

Understanding of the distribution of chemerin and its receptors, Chemokine-like Receptor 1 (CMKLR1), G Protein-coupled Receptor 1 (GPR1) and Chemokine (C-C motif) receptor-like 2 (CCRL2), in the egg and the embryonic annexes is currently lacking, and their role during embryogenesis remains unknown. By immunoblot using monoclonal anti-chicken antibodies and Enzyme Linked Immunosorbent Assays (ELISA), we found that chemerin is expressed 10 times higher in albumen eggs than in blood plasma, and it is also abundant in the perivitelline membrane but undetectable in yolk. Chicken chemerin can inhibit bacterial growth. By Reverse Transcription-quantitative Polymerisation Chain Reaction (RT-qPCR), western-blot, and immunofluorescence, we show that chemerin is locally produced by the oviduct magnum that participates in albumen formation. Using cultures of magnum explants, we demonstrate that progesterone (P4) and oestradiol (E2) treatment increases chemerin secretion into cultured media and expression in magnum. Chemerin and its three receptors are present in amniotic and Chorio Allantoic Membranes (CAM). Only CMKLR1 expression decreased from embryonic day (ED) 7 to ED11 and remained low until ED18. Chemerin concentrations strongly increased in amniotic fluid at D14 when egg albumen crossed the amniotic membrane. In ovo injections of neutralising chemerin and CMKLR1 antibodies (0.01, 0.1 and 1 µg) increased embryo mortality, which occurred mainly at ED12-13, in a dose-dependent manner. Chemerin treatment increased primary CAM viability. Finally, chemerin and CMKLR1 inhibition within the CAM led to a decrease in blood vessel development and associated angiogenic gene expression. Our results show an important function of the chemerin system during embryo development in chickens, suggesting the potential use of this adipokine as a predictive marker for egg fertility or hatchability.

Chronic dietary exposure to a glyphosate-based herbicide results in reversible increase early embryo mortality in chicken.

Glyphosate (Gly) is the active molecule of non-selective herbicides used in conventional agriculture. Some evidence shows that exposure to Glyphosate-Based Herbicides (GBH) can affect both male and female fertility in animal models. However, few data exist on birds that can be easily exposed through their cereal-based diet. To our knowledge, there are no current studies on the effects of chronic dietary exposure to GBH and the potential reversibility on the fertility and embryo development in chickens. In our protocol, hens (32 weeks-old) were exposed to GBH (47 mg kg-1/day-1 glyphosate equivalent corresponding to half of the No-Observed-Adverse-Effect-Level (NOAEL) as defined by European Food Safety Authority in birds, GBH group (GBH), n = 75) or not (Control group (CT), n = 75) for 6 weeks. Then, both CT and GBH groups were fed for 5 more weeks without GBH exposure. During these two periods, we investigated the consequences on the egg performance and quality, fertilization rate, embryo development, and viability of offspring. Despite the accumulation of Gly and its metabolite aminomethylphosphonic acid (AMPA) in the hen blood plasma, the body weight and laying rate were similar in GBH and CT animals. We observed from the 4th day of exposure an accumulation of Gly (but not AMPA) only in the yolk of the eggs produced by the exposed hens. After artificial insemination of the hens followed by eggs incubation, we showed a strong significant early embryonic mortality level in GBH compared to CT animals (78 ± 2 % vs 2.5 ± 0.3 %, p < 0.0001) with embryo death mainly occurring on the third day of incubation. By using computed tomography (CT) and magnetic resonance imaging (MRI) tools, we noted a significant delay in the embryo development of GBH survivors at 15 days with a reduction by half of the embryo volume and some disturbances in the calculated volumes of the embryonic annexes. At 20 days of incubation, we showed a reduction in the length of the tibia and in the volume of the soft tissues whereas the skeleton volume was increased in GBH chicks. The vast majority of these phenotypes disappeared two weeks after an arrest of the GBH maternal dietary exposure. Taken together, the dietary chronic exposure of broiler hens to GBH at a Gly equivalent concentration lower than NOAEL induces an accumulation of Gly in the egg yolk resulting in severe early embryonic mortality and a delayed embryonic development in survivors that were abolished two weeks after the end of GBH exposure.

Endothelial cell-derived fibroblast growth factor-18 regulates ovarian function in sheep.

Increasing the efficiency of farm animal reproduction is necessary to reduce the environmental impact of food production systems. One approach is to increase the number of healthy eggs (oocytes) produced per female for fertilization, thus it is important to understand factors that decrease oocyte health. One paracrine factor that decreases ovarian follicle growth is fibroblast growth factor 18 (FGF18) secreted by cells in the theca layer of the ovarian follicle, however the factors that regulate FGF18 secretion are unknown. In this study we hypothesized that FGF18 secretion is controled by intrafollicular factors and is linked to fertility, which we tested by using cell culture and sheep genetic models in vivo. Separation of theca cell populations revealed that FGF18 messenger RNA (mRNA) is located mainly in thecal endothelial rather than endocrine cells, and immunohistochemistry localized FGF18 protein to microvessels in the theca layer in situ. Culture of ovine theca-derived endothelial cells was used to demonstrate stimulation of FGF18 mRNA and protein abundance by bone morphogenetic protein 4 (BMP4), a growth factor derived from theca endocrine cells. Taking advantage of a sheep genetic model, we demonstrate reduced ovarian and peripheral FGF18 concentrations in the hyperprolific Booroola ewe harboring the FecBB mutation in BMPR1B. These data suggest a novel control of fertility by follicular endothelial cells, in which theca endocrine cells secrete BMP4 that stimulates the secretion of FGF18 from thecal endothelial cells, which in turn diffuses into the granulosa cell layer and promotes apoptosis.

The Hepatokine FGF21 Increases the Human Spermatozoa Motility.

Lifestyle, environment and excess body weight are not only associated with an increased risk of metabolic disorders, such as type 2 diabetes, but also to other pathological processes, such as infertility. A hormone produced mainly by the liver called fibroblast growth factor 21 (FGF21) is closely linked to the energy status and is increased in patients suffering from obesity or insulin resistance. Recently, FGF21 has been shown to be associated with female fertility disorders, but no or few data about the role of FGF21 on human male fertility has been described. In the present study, FGF21 was measured in the seminal fluid at a lower level in comparison to the blood level. Thus, in the present in vitro study, we aimed to decipher the FGF21 system in human semen. To evaluate the putative role of FGF21 on spermatozoa function, we incubated human spermatozoa with increasing concentrations of recombinant human FGF21. The FGF21 in seminal fluid is potentially produced by male reproductive tract tissues. In spermatozoa, the FGF21 signal was transduced by the two main receptors FGFR1-c and FGFR3 and the cofactor ß-klotho, which are colocalized in the middle piece of spermatozoa and stimulated the PI3K/Akt and MAPK pathways. Finally, in vitro treatment by FGF21 significantly increased sperm motility and ATP levels. Concomitantly, exposure to FGF21 improved the oxidative stress, as a lower ROS level was observed. Overall, these results seem to indicate that the metabolic factor, FGF21, positively modifies the activity and quality of the parameters of human spermatozoa.

Seasonal vascular plasticity in the mediobasal hypothalamus of the adult ewe.

Sheep, like most seasonal mammals, exhibit a cyclic adaptive reproductive physiology that allows ewes to give birth to their progeny during the spring when environmental conditions are favorable to their survival. This process relies on the detection of day length (or photoperiod) and is associated with profound changes in cellular plasticity and gene expression in the hypothalamic-pituitary-gonadal axis, mechanisms that are suggested to participate in the seasonal adaptation of neuroendocrine circuits. Recently, pituitary vascular growth has been proposed as a seasonally regulated process in which the vascular endothelial growth factor A (VEGFA), a well-known angiogenic cytokine, is suspected to play a crucial role. However, whether this mechanism is restricted to the pituitary gland or also occurs in the mediobasal hypothalamus (MBH), a crucial contributor to the control of the reproductive function, remains unexplored. Using newly developed image analysis tools, we showed that the arcuate nucleus (ARH) of the MBH exhibits an enhanced vascular density during the long photoperiod or non-breeding season, associated with higher expression of VEGFA. In the median eminence (ME), a structure connecting the MBH to the pituitary gland, higher VEGFA, kinase insert domain receptor (KDR/VEGFR2) and plasmalemma vesicle-associated protein (PLVAP) gene expressions were detected during the long photoperiod. We also found that VEGFA and its receptor, VEGFR2, are expressed by neurons and tanycytes in both the ARH and ME. Altogether, these data show variations in the MBH vasculature according to seasons potentially through a VEGFA-dependent pathway, paving the way for future studies aiming to decipher the role of these changes in the hypothalamic control of seasonal reproduction.

Chronic Dietary Exposure of Roosters to a Glyphosate-Based Herbicide Increases Seminal Plasma Glyphosate and AMPA Concentrations, Alters Sperm Parameters, and Induces Metabolic Disorders in the Progeny.

The effects of chronic dietary Roundup (RU) exposure on rooster sperm parameters, fertility, and offspring are unknown. We investigated the effects of chronic RU dietary exposure (46.8 mg kg-1 day-1 glyphosate) for 5 weeks in 32-week-old roosters (n = 5 RU-exposed and n = 5 control (CT)). Although the concentrations of glyphosate and its main metabolite AMPA (aminomethylphosphonic acid) increased in blood plasma and seminal fluid during exposure, no significant differences in testis weight and sperm concentrations were observed between RU and CT roosters. However, sperm motility was significantly reduced, associated with decreased calcium and ATP concentrations in RU spermatozoa. Plasma testosterone and oestradiol concentrations increased in RU roosters. These negative effects ceased 14 days after RU removal from the diet. Epigenetic analysis showed a global DNA hypomethylation in RU roosters. After artificial insemination of hens (n = 40) with sperm from CT or RU roosters, eggs were collected and artificially incubated. Embryo viability did not differ, but chicks from RU roosters (n = 118) had a higher food consumption, body weight and subcutaneous adipose tissue content. Chronic dietary RU exposure in roosters reduces sperm motility and increases plasma testosterone levels, growth performance, and fattening in offspring.

Review: Mechanisms of Glyphosate and Glyphosate-Based Herbicides Action in Female and Male Fertility in Humans and Animal Models.

Glyphosate (G), also known as N-(phosphonomethyl)glycine is the declared active ingredient of glyphosate-based herbicides (GBHs) such as Roundup largely used in conventional agriculture. It is always used mixed with formulants. G acts in particular on the shikimate pathway, which exists in bacteria, for aromatic amino acids synthesis, but this pathway does not exist in vertebrates. In recent decades, researchers have shown by using various animal models that GBHs are endocrine disruptors that might alter reproductive functions. Our review describes the effects of exposure to G or GBHs on the hypothalamic-pituitary-gonadal (HPG) axis in males and females in terms of endocrine disruption, cell viability, and proliferation. Most of the main regulators of the reproductive axis (GPR54, GnRH, LH, FSH, estradiol, testosterone) are altered at all levels of the HPG axis (hypothalamus, pituitary, ovaries, testis, placenta, uterus) by exposure to GBHs which are considered more toxic than G alone due to the presence of formulants such as polyoxyethylene tallow amine (POEA)." In addition, we report intergenerational impacts of exposure to G or GBHs and, finally, we discuss different strategies to reduce the negative effects of GBHs on fertility.