The widespread use of pesticides in agriculture increases the risk of chronic dietary exposure in poultry. This study investigated the effects of low-dose exposure to tebuconazole (TEB), imidacloprid (IMI), and glyphosate (GLP), administered individually or in combination at concentrations not exceeding maximum residue limits (MRLs), on male reproductive performance in Gallus gallus. Roosters were assigned to eight groups and exposed for six weeks (Phase I), followed by a four-week pesticide-free recovery period (Phase II).Sub-MRL pesticide exposure impaired male reproductive function, with the most pronounced effects observed following combined treatments. During Phase I, exposure resulted in reduced semen quality, decreased fertility and hatchability, and increased embryo mortality, particularly in groups receiving IMI alone or in combination. These functional impairments were accompanied by detectable pesticide residues in reproductive tissues and body fluids, as well as modulation of local and systemic immune parameters.During Phase II, semen parameters showed partial recovery toward control levels; however, fertility and hatchability remained reduced in several groups, coinciding with the persistence of pesticide residues in semen and selected tissues. Combined pesticide exposure consistently produced stronger and more persistent reproductive effects than individual compounds, indicating mixture-specific toxicity and incomplete reversibility.Collectively, these findings demonstrate that chronic exposure to MRL-compliant pesticide doses can compromise avian reproductive performance, particularly under combined exposure scenarios. The persistence of residues in reproductive compartments and excreta further highlights potential environmental and biological risks, supporting the need to consider reproductive endpoints and chronic mixture exposure in pesticide risk assessment frameworks.

Mitochondria are key organelles that regulate energy production, oxidative stress, and steroidogenesis. They are highly dynamic, continuously undergoing fusion and fission. While disruptions in these processes affect follicular development and fertility in mammals, little is known about their role in avian species. This study investigated the expression and functional roles of mitochondrial fusion in hen granulosa and theca cells isolated from dominant (F1) and smaller (F3/F4) follicles. Gene expression analysis revealed that fusion-related genes (MFN1, MFN2, and OMA1) are more highly expressed in theca cells than in granulosa cells. Furthermore, MFN1 and MFN2 levels significantly increase as follicles mature from the F3/F4 to the F1 stage, suggesting that mitochondrial fusion is closely linked to follicular development. To explore the functional impact, cells were treated with Mdivi-1 and leflunomide; both compounds successfully promoted mitochondrial fusion, as evidenced by elongated mitochondrial morphologies. However, their effects on mitochondrial homeostasis differed: leflunomide, but not Mdivi-1, significantly increased fusion gene expression and reduced mitochondrial DNA (mtDNA) copy number (ND4, ND6, ATP6). Inducing mitochondrial hyperfusion generally impaired cell viability and proliferation. High doses of both inhibitors suppressed proliferation across both cell types, molecularly confirmed by the downregulation of the pro-proliferative gene CCND1 and the upregulation of the cell cycle inhibitor P21. Regarding steroidogenesis, mitochondrial fusion exerted cell-specific effects. In granulosa cells, both treatments enhanced progesterone secretion, supported by increased expression of STAR, 3BHSD, and CYP11A1. Conversely, in theca cells, fusion led to decreased testosterone secretion and reduced expression of CYP11A1 and CYP19A1. Finally, while Mdivi-1 did not alter reactive oxygen species (ROS) or ATP levels, leflunomide significantly reduced oxidative stress and increased ATP production. In conclusion, mitochondrial dynamics play a critical, cell-specific role in regulating hen ovarian physiology, influencing the balance between proliferation, metabolism, and steroidogenesis.

This study evaluated the effects of oral supplementation with Scabiosa atropurpurea aqueous extract (AES) and/or its inclusion in the semen extender on the post-thaw semen quality and oxidative status of ram semen. Ten "Queue Fine de l'Ouest" rams were assigned to either a control (n = 5) or a treated group (n = 5) receiving AES orally (50 mL/day, 2 mg/kg). Semenwas collected, pooled, and divided into five aliquots, then diluted with extenders containing 0-1 g/L AES, resulting in 10 experimental conditions. After thawing, several parameters were assessed: sperm motility, kinetics, viability, morphology, membrane integrity, oxidative stress markers (TAS, TOS, OSI, MDA), ATP and Ca & sup2;(+) concentrations, DNA fragmentation, and HSP70levels. Oral AES supplementation did not significantly affect total motility (P > 0.05); however, itimproved antioxidant status by increasing TAS (22.0 +/- 0.5 vs. 20.3 +/- 0.3 mmol/L; P < 0.05) and reducing MDA (5.2 +/- 0.3 vs 3.5 +/- 0.3 & micro;mol/L; p < 0.05) and OSI (1.1 +/- 0.0 vs 0.7 +/- 0.0; P < 0.05). The inclusion of AES in the extender reduced the proportion of damaged spermatozoa (24.0 +/- 1.1% vs. 37.7 +/- 1.2%; P < 0.05) and improved both the ATP ratio (2.1 +/- 0.0 vs. 0.9 +/- 0.0mg/L per g protein) and Ca & sup2;(+) concentration (32.9 +/- 1.0 vs. 22.6 +/- 1.1 mg/L per g protein; P < 0.05). The combined supplementation protocol (2 mg/kg oral AES + 1 g/L extender AES) provided the most effective protection against oxidative stress and significantly enhanced membrane integrity, ATP production, and Ca & sup2;(+) levels in post-thaw sperm. In conclusion, AES supplementation improves the biochemical stability and cellular integrity of cryopreserved ram semen.

This study investigates the endocrine-disrupting effects of the fungicide carbendazim (CBZ) on human and murine reproductive health. Mass spectrometry confirmed systemic human exposure, detecting CBZ in the plasma and follicular fluid of 15-18% of the women sampled. In vitro, CBZ did not affect viability in human KGN and primary granulosa cells (hGCs) but significantly inhibited KGN proliferation. Furthermore, CBZ reduced steroidogenesis stimulated by FSH, LH, or IGF-1 in both cell types. This dysfunction correlated with a dose-dependent increase in the Oxidative Stress Index, driven by elevated total oxidant status and reactive oxygen species. Corroborating these findings, in vivo murine models exposed to CBZ (100 μg/kg/day) exhibited a prolonged diestrus phase, alongside reduced numbers of corpora lutea and preovulatory follicles, and decreased gonadotropin and E2 plasma levels. Collectively, these results identify CBZ as a potent reproductive toxicant that impairs ovarian function via oxidative stress-mediated hormonal disruption.

Glyphosate-based herbicides (GBHs) are widely used, yet their effects on rooster spermatozoa and epigenetic regulation remain poorly understood. This study evaluated the in vitro effects of glyphosate (GLP), a commercial GBH formulation, and the model surfactant polyethoxylated tallow amine (POEA) on rooster sperm function and global methylation. Pooled rooster semen samples were exposed to increasing concentrations of GLP or GBHs for 1 h and 3 h to assess dose- and time-dependent effects (Experiments 1 and 2). Based on these findings, a third experiment compared GLP, Roundup®, and POEA at the highest concentration after 1 h of incubation. Sperm motility was evaluated by CASA, whereas functional parameters were assessed by flow cytometry. Global DNA methylation was measured using a colorimetric assay. GLP had limited effects on motility and did not impair plasma membrane integrity or mitochondrial membrane potential, but altered DNA fragmentation, intracellular Ca²⁺ levels, caspase activity, and lipid peroxidation. In contrast, GBH induced broader alterations, including reduced motility by approximately 30-50%, impaired membrane integrity, increased DNA fragmentation, altered apoptotic responses, decreased intracellular Ca²⁺ and lipid peroxidation, and reduced mitochondrial activity under the most severe conditions. POEA alone affected selected functional endpoints, including DNA fragmentation, caspase activity, intracellular Ca²⁺ levels, lipid peroxidation, and acrosome status. Global DNA methylation decreased by approximately 70-80% compared with the control, with the strongest reduction observed after POEA exposure. These findings demonstrate that the GBH formulation exerts stronger adverse effects on rooster spermatozoa than GLP and POEA alone and underscore the importance of evaluating complete herbicide formulations, rather than active ingredients, in reproductive risk assessment.

To investigate pesticide impacts on declining French pheasant populations, unhatched wild eggs were screened for over 600 compounds using mass spectrometry, identifying a mixture of six pesticides. Pheasant ovarian cells (granulosa (GCs) and theca interstitial (TICs) were exposed in vitro to this mixture at environmentally relevant concentrations in basal state and in response to exogenous hormones (IGF-1 and gonadotropins). Effects were evaluated using CCK8 (cellular metabolic activity), BrdU (proliferation), ELISA (hormone production), and RTqPCR (gene expression). While cellular metabolic activity mainly increased, proliferation significantly decreased. Basal and stimulated progesterone and testosterone production increased in GCs and TICs, respectively. Furthermore, TOS, TAC, and OSI analyses revealed marked oxidative stress in both cell types, accompanied by the upregulation of genes involved in mitochondrial fusion including MFN1 in GCs. Overall, these endocrine and cellular disruptions could contribute to the ongoing decline of bird populations across agricultural landscapes.

This study investigated the protective role of Scabiosa atropurpurea aqueous extract (AES) against natural heat stress in rams. Sixteen mature rams (4-6 years old, 61.2 +/- 2.6 to 63.5 +/- 0.3 kg) were monitored for 90 days (August-October 2022). Animals were divided into two groups: controls (C; n = 8) received 50 mL water daily, while the treated group (T; n = 8) received 50 mL AES (1 mg/kg body weight/day). The severity of heat stress was assessed using the temperature-humidity index (THI). Semen was collected every 10 days to assess sperm quality, oxidative status, ATP and calcium levels, and gene expression related to steroidogenesis. Plasma testosterone and LH concentrations were also determined. Under hot summer conditions, AES supplementation significantly enhanced individual sperm motility (93 +/- 1.1 % vs. 89 +/- 0.9 %; p = 0.006) and sperm concentration (3.77 +/- 0.06 vs. 3.34 +/- 0.05 X 10(9) spz/mL; p = 0.02), while reducing abnormal forms (7.5 +/- 0.4 % vs. 12.9 +/- 0.2 %; p = 0.06) and DNA damage (15.2 +/- 3.4 % vs. 26.7 +/- 2.4 %; p = 0.008). Treated rams showed higher seminal antioxidant activity (6.4 +/- 0.2 vs. 3.3 +/- 0.08 mmol Trolox equiv/L; p < 0.0001), lower ROS levels (12.9 +/- 0.29 vs. 21.3 +/- 0.11 mmol H2O2; p = 0.03), and increased plasma testosterone. AES also improved LH secretion and upregulated key steroidogenic genes, including 3(3-HSD, aromatase, StAR, LH, and FSH(3 in testis. In conclusion, AES supplementation mitigates heat stress by improving sperm traits, antioxidant defenses, and endocrine activity in rams.

IntroductionThis study investigated the physiological, biochemical, and molecular shifts in male Mule ducks during an assisted-feeding period and the subsequent recovery phase following the cessation of overfeeding. While assisted-feeding is known to induce significant hepatic changes, the timeline and extent of the liver's capacity to return to a basal state remain critical areas of inquiry.MethodsMale Mule ducks were subjected to a period of assisted-feeding followed by a recovery phase where they returned to an ad libitum diet. We monitored body weight and liver mass, analyzed plasma metabolic markers (including lipids and liver enzymes), and evaluated hepatic composition. Molecular analysis was conducted to assess gene expression related to lipogenesis, inflammation, and apoptosis, while antioxidant enzyme activities and hypoxia markers were measured to determine cellular stress levels.ResultsAssisted-feeding significantly increased body weight, liver mass, and hepatic steatosis, accompanied by a sharp rise in plasma markers such as triglycerides, cholesterol, and liver enzymes (ALAT, LDH, ALP). At the molecular level, there was a marked upregulation of genes involved in lipogenesis (scd1, dgat2), inflammation (TNFα, IL8), and apoptosis. Furthermore, increased activities of antioxidant enzymes (SOD, Cat, GPX1) and markers of hypoxia (HIF-1α, HIF-2α) indicated significant metabolic load and cellular stress. Following the cessation of overfeeding, most physiological and biochemical parameters, including liver mass and enzyme levels, returned to control values within 20 to 29 days. Notably, while hepatic alterations were fully abolished, abdominal fat remained significantly higher in ex-force-fed ducks compared to the control group.ConclusionThe study demonstrates that hepatic steatosis induced by assisted-feeding in Mule ducks is a highly reversible process. Despite triggering significant oxidative stress, hypoxia, and inflammatory responses, the avian liver exhibits a remarkable regenerative capacity, returning to its basal physiological and molecular state within 29 days of returning to a standard diet.

Scabiosa artropurperea, a member of the Dipsacaceae family and Scabiosa genus, is renowned for its medicinal properties. In the present study, we investigated the impact of Scabiosa artropurperea aqueous extract (AES) on the in vivo reproductive functions in Queue Fine de l’Ouest ewes, and on in vitro ovine granulosa cells. Ewes were synchronized for 14 days with intra-vagina progesterone (P4) devices (FGA, 20 mg) and divided into four groups receiving daily oral doses of 0, 1, 2, and 4mg of AES/kg Live Body Weight (LBW), respectively. After sponge removal, all ewes received an intramuscular injection of 400 IU of eCG. Estrous behavior parameters as latency and duration, and prolificacy rates, and plasma hormone levels (estradiol and progesterone) were assessed. Estrus latency was reduced and the duration of estrus was increased in ewes that received 1 mg and 2mg/kg LBW compared to the control. Prolificacy rates were also significantly improved in 1 mg or 2 mg/kg groups compared to the control. Plasma levels of E2 were also higher on 2mg/kg LBW treated group. Moreover, ovine granulosa cells were cultured and treated with various concentrations of AES (ranging from 0 to 5 mg/ml). While AES did not affect cell viability and proliferation whatever the conditions, it significantly increased basal steroidogenesis (P4 and E2 concentrations) at the concentration of 0.5 and 0.05 mg/ml and in response to IGF-1 but not FSH at the 0.05 mg/ml concentration. These latter data were associated to an increase in the expression of CYP19A1 and STAR genes but not those of CYP11A1 and HSD3B and to an increase in cellular lactate concentration. Taken together, AES extracts enhanced in vivo reproductive performance in ewe and this was associated to an increase in in vitro granulosa cell steroidogenesis.

Overconsumption of fructose is linked to metabolic diseases, which are often associated with reduced fertility. GLUT5 is the most specific fructose transporter. To investigate its role in the testes, we analyzed the male reproductive phenotype of transgenic male mice deficient in GLUT5 (GLUT5-/- or GLUT5 knockout [KO] mice). Glut5 expression was shown in Leydig cells and germ cells, from primary spermatocytes to spermatozoa. We found reduced intratesticular fructose and pyruvate concentrations in GLUT5-/- mice. These mice exhibited 30% lower litter sizes compared with control mice. Histological analysis of the testes revealed some seminiferous tubules with a "Sertoli cell-only" phenotype, although spermatogenesis occurred normally in most tubules. Reduced fertility in GLUT5 KO mice was linked to lower sperm production and impaired sperm quality. Spermatozoa from these mice displayed reduced motility, head abnormalities, and a diminished acrosome reaction, which was associated with reduced cyclic adenosine monophosphate content and impaired phosphorylation of protein kinase A substrates in the acrosome. Unexpectedly, androgen production in GLUT5 KO mice was 3-fold higher than in controls, despite unchanged luteinizing hormone levels. Electron microscopy of Leydig cells revealed a highly developed smooth endoplasmic reticulum, increased lipid droplets, and abnormal mitochondrial structures, suggesting disrupted mitochondrial dynamics. Proteomic analysis identified 155 deregulated proteins in the testicular tissue of GLUT5 KO mice, nearly half of which were associated with sperm motility, germ cell morphology, glycolysis, mitochondrial dynamics, and oxidative stress. In conclusion, the absence of the specific fructose transporter GLUT5 reduced testicular fructose content and led to an asthenozoospermia phenotype accompanied by hyperandrogenism.

Background:Infertility is a significant public health issue that can be influenced by environmental pollutants. As a radioactive heavy metal and environmental contaminant, uranium has the potential to impact fertility. Objective:This study assesses the multigenerational reproductive effects of chronic, non-nephrotoxic uranium exposure across three generations of male rats. Methods:In this study, a non-nephrotoxic uranium solution (40 mg/L) was chronically administered via drinking water to male and female F0 rats (n = 20 per group) throughout their lifespan. The objective was to evaluate the potential reprotoxic effects of uranium on males across three generations (F0, F1, F2), with a focus on spermatogenesis, steroidogenesis, and testicular homeostasis, including oxidative stress, inflammation, apoptosis, and vitamin D metabolism. Results:Steroidogenesis was modulated in all generation, with dysregulation of sex and pituitary hormones (testosterone, estradiol, gonadotropins, Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH). Morphological and histological changes in the testes were observed in both the F1 and F2 generations. Spermatogenesis was dysregulated by an increased proportion of seminiferous tubules at stage I-VI and reduced expression of TH2B and eppin mRNA. Interestingly, gene expression analysis of several markers involved in the regulation and protection of testicular homeostasis revealed significant effects only on the F2 generation. In this generation, uranium exposure also disrupted vitamin D metabolism in the testes. Conclusion:Uranium may impair testicular function, with more pronounced effects observed in the F2 generation. These findings highlight its potential for multigenerational toxicity and underscore the need for further research into its impact on human reproductive health.

Tebuconazole (TEB), a fungicide that inhibits 14α-demethylase (CYP51) and disrupts ergosterol synthesis, poses environmental and health risks due to its persistence and low biodegradability. This study examined TEB in vitro effects on rooster spermatozoa. In Experiment 1, semen from 10 Green-legged Partridge roosters was incubated with TEB (0, 0.1, 1, 10, 100 µM) at 36°C for 3 hours. Sperm motility was analyzed with Computer-Aided Sperm Analysis (CASA) system, while flow cytometry assessed membrane integrity, mitochondrial function, acrosome status, chromatin structure, intracellular calcium, apoptosis, caspase activity, and lipid peroxidation after 1 and 3 hours of exposure. Malondialdehyde (MDA) concentration and total antioxidant capacity (T-OAC) were measured by spectrophotometer. In Experiment 2, calcium channel blockers (SNX 325, MRS-1845, Nifedipine, HC-056456) were tested under the same conditions, focusing on motility, membrane integrity, calcium levels, apoptosis, caspase activity, and lipid peroxidation. Results in experiment 1 have shown that TEB (0.1, 1, 10 µM) reduced sperm velocity (VAP) after 3 hours (P < 0.01) without altering other motility parameters. Acrosome status, intracellular calcium level, and lipid peroxidation decreased significantly at all TEB concentrations (P < 0.01). Early apoptosis declined at 1 µM TEB (P < 0.01), while mitochondrial activity and membrane integrity remained stable. MDA levels were reduced (P < 0.01), with no effect on T-OAC. In Experiment 2, calcium channel blockers decreased motility parameters (VAP, VCL, VSL, MOT, PROG) and intracellular calcium levels (P < 0.01), but did not affect membrane integrity. Lipid peroxidation and caspase activity declined (P < 0.01), with no impact on early apoptosis. These findings underscore TEB's role in inhibiting calcium channels, reducing ion influx, blocking calcium-driven pore formation, thereby preserving membrane integrity. This mechanism mitigates early apoptosis and lipid peroxidation in chicken sperm, shedding light on TEB's impact on motility, calcium balance, and cell function.

Female infertility, which affects 10-20% of couples worldwide, is a growing health concern in developing countries. It can be caused by multiple factors, including reproductive disorders, hormonal dysfunctions, congenital malformations and infections. In vitro and in vivo studies have shown that plant extracts regulate gonadotropin-releasing hormone, kisspeptin, and gonadotropin expression and/or secretion at the hypothalamic-pituitary level and modulate somatic and germ cells, such as steroidogenesis, proliferation, apoptosis, and oxidative stress at the ovarian level. In this review, we report evidence for the role of certain plant extracts or plant bioactive compounds in the regulation of the hypothalamic-pituitary-ovary axis and, consequently, for the treatment of female infertility. We will also summarize their possible involvement in ovarian disorders such as polycystic ovary syndrome (PCOS), premature ovarian failure (POF) and ovarian cancers.

Are human ovarian cells exposed to carbemdazin and what are the consequences on their viability, proliferation and steroid production? Carbendazim is detected in human follicular fluid mainly in PCOS patients and it increases steroid secretion and decreases cell proliferation in primary human granulosa cells. Carbendazim (Methyl 2-benzimidazolecarbamate) is a fungicide which was banned last year by the european union but is still present in the french groundwaters and soils. It exhibits antimitotic activity in fungal and mammalian cells. Peri-fertilization exposure to carbendazim induces infertility and early pregnancy loss in female hamsters and in male rat, carbendazim has adverse effects on spermatogenesis, also resulting in a reduced fertility. However, no study until now has been performed experiments in human ovarian fluid and cells. A single centre cohort study involving 62 patients was performed from 2021-2023 for the analysis of follicular fluid. The effect of carbendazim on human granulosa cells has been determined on granulosa cells from 30 patients with male factor infertility and in the human granulosa cell line, KGN from 2024 and still ongoing. From each patient, only one cycle is included in the study. Sixty-two patients including 16 PCOS was used from 2021-2023 for the follicular fluid analysis and 30 patients with male factor infertility for cell culture. The study was carried out in accordance with the Declaration of Helsinki principles and free informed consent was obtained from all participants. Carbendazim concentrations were assessed by mass spectrophotometry, while steroid concentrations were measured by ELISA assays. Cell viability and proliferation was dertermined by using CCK8 and BrDU assays, respectively. Carbendazim (CBZ) was detected in follicular fluid in PCOS patients (9 out 16), with a maximum concentration of 76 ng/mL but not in patients with male factor infertility. We next investigated the impact of CBZ on the cell viability, proliferation, and steroidogenesis, in both granulosa tumor cell line (KGN) and primary human granulosa cells. Using a CBZ concentration range from 0 to 1 µg/mL, including the highest CBZ dose found in follicular fluid, we demonstrated that CBZ does not affect cell viability whereas it reduced cell proliferation from the concentrationsof 10 ng/mL in KGN cells. At the opposite, we showed that CBZ increased in a concentration dependent manner progesterone and oestradiol secretion from 1 ng/ml in both KGN and primary granulosa cells. Furthermore, in these cells, CBZ (100 ng/ml) significantly improved IGF-1-induced steroid secretion whereas it did not affect FSH or LH effect. The CBZ effect on the mRNA expression of the cholesterol carrier, StAR and the steroidogenesis enzymes and on the oxidative stress is in progress. In addition, we next compare PCOS and normal granulosa cell sensitivity to CBZ. Taken together, carbendazim exposure could alter human folliculogenesis and could contribute to explain some ovarian disorders. The number of PCOS and normal patients is limited. For primary granulosa cell culture, cells are from different diameter of follicles. Environmental pollution is more and more important and human infertility with or without known reasons increases. The use of some phytochemical products could explain some ovarian disorders in women. No

Visfatin regulates energy homeostasis, metabolism, inflammation, and reproduction via the hypothalamus-pituitary-ovary axis. Our previous study showed the visfatin gene and protein expression in the human placenta. This study aimed to investigate the in vitro effect of visfatin on the proliferation and apoptosis of placental JEG-3 and BeWo cells but also in villous explants collected from normal pregnancies and complicated by intrauterine growth restriction (IUGR), preeclampsia (PE), and gestational diabetes mellitus (GDM). We studied placenta cells viability, proliferation, cell cycle, proliferation/apoptotic factors and insulin receptor (INSR) expression, DNA fragmentation, CASP3/7 activity, and phosphorylation of ERK1/2, AKT, AMPKα, STAT3 with their involvement after pharmacological inhibition in visfatin action on proliferation and apoptosis. Visfatin (1, 10, 100 ng/mL) decreased the viability and proliferation of JEG-3 after 48 h, and a similar effect was observed via co-administration of visfatin (10 ng/mL) and insulin (10 ng/mL) in JEG-3 and BeWo after 48 h and 72 h, respectively. Visfatin reduced the transition from the G2/M phase, and expression of PCNA or cyclins D, E, A, and B in JEG-3 and PCNA in normal, IUGR, PE, and GDM placentas. It increased DNA fragmentation, CASP3/7 activity, P53, BAX/BCL2, CASP9, CASP 8, CASP3 levels in BeWo, and CASP3 expression in tested placentas. Furthermore, visfatin modulated INSR, ERK1/2, AKT, AMPKα, and STAT3 expression in JEG-3 and BeWo, and its anti-proliferative and pro-apoptotic effects occurred via mentioned factors. In conclusion, visfatin, by affecting the proliferation and apoptosis of human placenta cells, may be an important factor in the development and function of the organ.

how human exosomes from PCOS ovarian cells could change the activity of granulosa cells ? miRNA and proteins presented in exosomes from human follicular fluid change cell proliferation and steroid secretion of granulosa cells by activating the STAT3 pathway. Main follicular fluid (FF) factors include proteins, metabolites, as well as extracellular vesicles (exosomes). All these secretory products participate to intertalk between different cell types in the ovary that contribute to follicular development and oocyte maturation. Recent studies have demonstrated that FF-derived exosomes contents, especially miRNAs, play a role in Polycystic Ovary Syndrome (PCOS) etiology and can be used as diagnostic and therapeutic biomarkers. However, no study until now has been analysed on same patients proteomic and miRNA sequencing of exosomes purified from blood and follicular fluid from control and PCOS patients. A cohort study involving 22 PCOS patients and 98 control patients was performed from 2021-2023 for the analysis of exosomes purified from follicular fluid and in 10 patients from blood. EVs were analysed for proteomic and miRNA sequencing. The biological effect of exosomes was performed on human granulosa cells lineage KGN from 2023 and still ongoing. 22 PCOS patients and 98 control patients was used from 2021-2023 for the exosomes purification from follicular fluid and blood. The study was carried out in accordance with the Declaration of Helsinki principles and free informed consent was obtained from all participants. exosomes were assessed by mass spectrophotometry and sequencing with a NextSeq500. steroid concentrations measured in medium was performed by ELISA. Cell viability and proliferation was determined by using CCK8 and BrDU assays, respectively. To better understand the importance of exosome in ovary, the objective of our project was to obtain a “global” view of the content and activity of exosomes from ovarian fluid (follicular fluid) surrounded the germ cells purified from PCOS patients versus control patients by using transcriptomic and proteomic approaches. First results have observed the presence of approximately 1195 proteins in exosomes purified from human follicular fluid and 183 miRNA. Furthermore, in all patients and whatever the status, exosomes present similar morphology and diameter. Our study found that the protein STAT3 is overexpressed in exosomes from PCOS. STAT3 interacts with many proteins associated with the PCOS phenotype observed in granulosa cells. When granulosa cells were exposed to PCOS-derived exosomes along with STAT3 inhibitors, cell proliferation, migration, and steroid production returned to normal levels. In addition, the miR30c-5p is highly expressed and inhibition with antagomir induced a return to a control phenotype. In summary, the study showed that the content of follicular fluid-derived exosomes are specific and can directly regulate granulosa cell activity. The miRNA and proteins content of these exosomes differs in PCOS including inflammation, steroidogenesis, and cytoskeleton architecture. The number of PCOS and normal patients is limited. Exosomes allow cells to communicate with each other by transporting molecules from one cell to another. This transfer can alter the behavior of cells. Exosomes could present a different activity in function of pathology or could be modified to present a positive effect during IVF or cumulus maturation. No

Polycyclic Aromatic Hydrocarbons (PAHs) exposure leads to disorders reported in female infertility patients. Our hypothesis is that PAHs accumulate in granulosa cells (Gc) according to body mass index (BMI) and directly affects its functions. All 16 high-priority PAHs were in human FF, Gc and blood plasma with the highest concentration in Gc (GC-MS/MS). Their highest concentration was in obese Gc, except for acenaphthene and acenaphthylene, and positively correlated with BMI. In FF, we noted only positive correlation between naphthalene and BMI, whereas in blood plasma positive correlation between naphthalene, acenaphthene, pyrene and BMI. Phenanthrene and naphthalene but not fluoranthene inhibited totally steroidogenesis (ELISA), CYP19A1 mRNA expression (real-time PCR) and increased oxidative stress index and catalase expression in Gc independently on BMI. While all studied PAHs decreased Gc proliferation (BrdU assay) and viability (Cell Count kit-8 assay). Thus, Gc PAHs concentrations are positively correlated with BMI and alter ovarian functions.

Over the past few decades, a significant change globally in sugar intake has coincided with a rising incidence of male infertility, which is now a major public health concern. Diets rich in fructose have been implicated in both male infertility and increased susceptibility to metabolic disorders, such as obesity, diabetes, and related heart problems. While fructose is known to be present in seminal fluid and crucial for sperm motility, the precise role of fructose in testicular function remains largely unknown. GLUT5 is an exclusive fructose transporter essential for dietary fructose uptake in the intestine. It is also expressed mainly in germ and Leydig cells. We recently revealed that disrupting the Glut5 gene in male mice impairs spermatogenesis and steroidogenesis. However, its specific role within Leydig cells remains unexplored. Therefore, we investigated its role by inhibiting GLUT5 in a murine Leydig cell line (mLTC-1) using a specific inhibitor of GLUT5, MSNBA, combined with a multi-omics approach. Exposing mLTC-1 cells to MSNBA reduced the intracellular fructose content, limited cell proliferation, and enhanced progesterone and androgens production (Δ4-androstenedione and testosterone). The latter was associated with the upregulation of two genes and proteins involved in steroidogenesis, such as Hsd3b and steroidogenic acute regulatory protein (StAR). GLUT5 inhibition in mLTC-1 cells also modified lipid and carbohydrate metabolism. Lipidomic analysis showed decreased cholesterol esters and a shift in the ratio of polyunsaturated fatty acids (PUFAs) to monounsaturated fatty acids (MUFAs). These lipid changes correlated with alterations in the expression of mRNA-encoding enzymes involved in lipogenesis, such as ELOVL6. Metabolomics analysis showed a reduction in most glycolysis metabolites, except for pyruvate and lactate. However, pyruvate could conserve its level by a production through an amino acid pathway using the higher branched-chain amino acid content. Nevertheless, the activity of mitochondria measured by seahorse was not altered. The transcriptomic analysis performed by BRB-seq approach revealed an upregulation of several androgen-sensitive genes, such as Akap5, Slc39a9, an androgen receptor or lactate dehydrogenase A (Ldha), which produces lactate, and downregulation of several genes associated with the insulin pathway such as Tsc2 or the hexokinase Hkdc1. In conclusion, GLUT5 supported fructose intake in the murine Leydig cell line mLTC-1, leading to a reduction in cell proliferation. The consequences of inhibition of GLUT5 led to an increase in fatty acids cell content, a perturbation in glycolysis and amino-acid metabolism but an enhanced androgen production. Since androgens regulate spermatogenesis, hyperandrogenism induced by a lower fructose content in Leydig cells may be a primary cause leading to the disruption of sperm production and quality, as well as sexual behavior, as described in the GLUT5 KO mouse model.

In brief:Adipolin (C1QTNF12) has been described as a regulator of metabolism and is linked with the pathophysiology of PCOS. In this study, for the first time, we show the expression of C1QTNF12 in granulosa cells and its positive effect on porcine granulosa cell proliferation and steroid synthesis. Abstract:Adipolin (C1QTNF12) is a recently discovered adipokine that plays an important role in glucose and insulin level regulation. Previous studies showed its reduced level in serum of women suffering from polycystic ovarian syndrome; however, whether C1QTNF12 regulates ovary function is still unknown. The aim of the study was first to determine the level of C1QTNF12 in the porcine ovarian follicles granulosa cells (Gc) and then its in vitro effect on proliferation and steroidogenesis as well as phosphorylation of several signalling pathways. Our results showed that the expression of C1QTNF12 was dependent on follicle size and was higher at the mRNA and protein level in Gc of small than large follicles from both prepubertal and mature animals. Similar pattern was observed for C1QTNF12 concentration in porcine follicular fluid. Additionally, we observed immunolocalisation of C1QTNF12 in Gc, theca cells and oocytes. We found that C1QTNF12 stimulated porcine Gc proliferation via the activation of protein kinase B (AKT). Moreover, C1QTNF12 enhanced progesterone, testosterone and oestradiol secretion by elevating STAR, CYP11A1, HSD3B and CYP19A1 mRNA expression and by activation of MAP3/1 pathway. Additionally, C1QTNF12 increased pMAP3/1-to-MAP3/1 protein expression ratio and enhanced IGF1-induced pTyr-IGF1Rβ-to-IGFR1β and pMAP3/1-to-MAP3/1 protein ratios. Taken together, C1QTNF12 could act directly on proliferation and steroid synthesis and serve as an important factor in in vivo ovarian follicle function, possibly regulating the course of folliculogenesis.

Beyond their link to metabolic issues like type 2 diabetes, factors like lifestyle, environment, and excess weight may also influence fertility. Fibroblast growth factor 21 (FGF21), a liver-derived hormone linked to energy balance, has recently emerged as a potential player in female mammalian reproduction. In male, only two studies have described potential effects of FGF21 on fertility. A recent study has described a negative correlation observed in obese patients presenting a low testosterone level associated with elevated FGF21 plasma levels. To investigate the role of FGF21 in steroidogenesis, we have studied the involvement of FGF21 in lipid and steroid activity by Leydig cells.Leydig cell model expressed all FGF21 receptors and β-Klotho cofactor as determined by RT-qPCR and by western-blot. Cultured mLTC-1 Leydig cell line exposed to increasing FGF21 concentration induced phosphorylation (Ser 473) of Akt and modified the CREB factor activity, suggesting the functionality of the FGF21 pathway.FGF21 consequences on mLTC-1 Leydig cells are inhibition of the lipid synthesis, leading to a reduction in the content of lipid droplets. The drop in lipid synthesis is associated with a reduction in the amount of lipids (mainly PUFA, cholesterol esterified, and triglycerides) as measured by lipidomic approach. The main consequence is to reduce the quantity of cholesterol, the steroid precursor, in mLTC-1 Leydig cells and is associated with a low production in testosterone. The decrease in androgens was also associated with a reduction in the steroid enzyme genes expression, which are under the control of CREB activity, and present a lower activity due to low cAMP intracellular levels.In vivo, steroid production was lowering after FGF21 administration in adult male mice associated to a decrease in progressive motility and velocity of sperm. In addition, these experimental data are reinforced by a data mining analysis focused on “gonad“ terms in 1,319,905 article references showing the link already described between FGF21 with the fatty acids pathways, cholesterol storage, and steroid production.In conclusion, we demonstrated that Leydig cells in the testes present a functional FGF21 pathway, which regulates lipid metabolism and steroid function. In mLTC-1 Leydig cells, FGF21 reduced cholesterol, PUFA content, and testosterone production. Finally, this work highlighted that the hepatokine FGF21 could have a negative impact on androgen synthesis and testicular activity.