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.

Porcine oocyte preincubation in oviductal fluid flush before in vitro fertilization in the presence of oviductal epithelial cells improves monospermic zygote production.

The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.

Intraoviductal concentrations of steroid hormones during in vitro culture changed phospholipid profiles and cryotolerance of bovine embryos.

The objective of this study was to evaluate the effect of progesterone (P4), estradiol (E2), and cortisol (CO) at intraoviductal concentrations on bovine embryo development and quality in vitro. After fertilization of in vitro matured oocytes, zygotes were cultured for 8 days in synthetic oviductal fluid, supplemented with 55 ng/ml P4, 120 pg/ml E2, 40 ng/ml CO, or their combination (ALL). Control embryos were cultured with vehicle (0.1% ethanol). Exposure to steroids did not affect the embryo developmental rate nor the mean number of cells per blastocyst. However, at 24 hr after vitrification-warming, exposure to P4 improved the proportion of embryos that re-expanded and were viable while exposure to CO decreased the proportion of viable embryos. By intact cell MALDI-TOF mass spectrometry, a total of 242 phospholipid masses of 400-1000 m/z were detected from individual fresh blastocysts. Exposure to ALL induced the highest and most specific changes in embryo phospholipids, followed by P4, E2, and CO. In particular, the m/z 546.3 and 546.4 attributed to lysophosphatidylcholines were found less abundant after exposure to P4. In conclusion, exposure of bovine embryos to intraoviductal concentrations of steroid hormones did not affect in vitro development but changed blastocyst quality in terms of cryotolerance and phospholipid profiles.

Docosahexaenoic acid mechanisms of action on the bovine oocyte-cumulus complex.

BACKGROUND: Supplementation of bovine oocyte-cumulus complexes during in vitro maturation (IVM) with 1 µM of docosahexaenoic acid (DHA), C22:6 n-3 polyunsaturated fatty acid, was reported to improve in vitro embryo development. The objective of this paper was to decipher the mechanisms of DHA action. RESULTS: Transcriptomic analysis of 1 µM DHA-treated and control cumulus cells after 4 h IVM showed no significant difference in gene expression. MALDI-TOF mass spectrometry analysis of lipid profiles in DHA-treated and control oocytes and cumulus cells after IVM showed variations of only 3 out of 700 molecular species in oocytes and 7 out of 698 species in cumulus cells (p < 0.01). We showed expression of free fatty acid receptor FFAR4 in both oocytes and cumulus cells, this receptor is known to be activated by binding to DHA. FFAR4 protein was localized close to the cellular membrane by immunofluorescence. Functional studies demonstrated that supplementation with FFAR4 agonist TUG-891 (1 µM or 5 µM) during IVM led to an increased blastocyst rate (39.5% ± 4.1%, 41.3% ± 4.1%), similar to DHA 1 µM treatment (39.2% ± 4.1%) as compared to control (25.2% ± 3.6%). FFAR4 activation via TUG-891 led to beneficial effect on oocyte developmental competence and might explain in part similar effects of DHA. CONCLUSIONS: In conclusion, we suggested that low dose of DHA (1 µM) during IVM might activate regulatory mechanisms without evident effect on gene expression and lipid content in oocyte-cumulus complexes, likely through signaling pathways which need to be elucidated in further studies.

Steroid hormones regulate sperm-oviduct interactions in the bovine.

After insemination in the cow, a sperm reservoir is formed within the oviducts, allowing the storage and then progressive release of spermatozoa toward the ovulated oocyte. In order to investigate the hormonal regulation of these events in vitro, the ovarian steroids 17ß-estradiol (E2) and progesterone (P4) were added at various concentrations to monolayers of bovine oviduct epithelial cells (BOEC) before or during co-incubation with spermatozoa. Main findings demonstrate that (1) a 18-h pretreatment of BOEC with 100 pg/mL and 100 ng/mL of E2 decreased by 25% the ability of BOEC to bind spermatozoa after 10 min, and for the highest dose of E2, 60 min of co-incubation; (2) P4 at concentrations of 10, 100 and 1000 ng/mL induced the release within 60 min of 32-47% of bound spermatozoa from BOEC; this sperm-releasing effect was maintained after a 18-h pretreatment of BOEC with 100 pg/mL of E2; (3) E2 in concentrations above 100 pg/mL inhibited the releasing effect of P4 on bound sperm in a dose-dependent manner; (4) spermatozoa bound to BOEC, then released from BOEC by the action of P4-induced higher cleavage and blastocyst rates after in vitro fertilization than the control group. These results support the hypothesis that the dynamic changes in steroid hormones around the time of ovulation regulate the formation of the sperm reservoir and the timed delivery of capacitated spermatozoa to the site of fertilization.

Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk.

Successful pregnancy requires an appropriate communication between the mother and the embryo. Recently, exosomes and microvesicles, both membrane-bound extracellular vesicles (EVs) present in the oviduct fluid have been proposed as key modulators of this unique cross-talk. However, little is known about their content and their role during oviduct-embryo dialog. Given the known differences in secretions by in vivo and in vitro oviduct epithelial cells (OEC), we aimed at deciphering the oviduct EVs protein content from both sources. Moreover, we analyzed their functional effect on embryo development. Our study demonstrated for the first time the substantial differences between in vivo and in vitro oviduct EVs secretion/content. Mass spectrometry analysis identified 319 proteins in EVs, from which 186 were differentially expressed when in vivo and in vitro EVs were compared (P < 0.01). Interestingly, 97 were exclusively expressed in in vivo EVs, 47 were present only in in vitro and 175 were common. Functional analysis revealed key proteins involved in sperm-oocyte binding, fertilization and embryo development, some of them lacking in in vitro EVs. Moreover, we showed that in vitro-produced embryos were able to internalize in vivo EVs during culture with a functional effect in the embryo development. In vivo EVs increased blastocyst rate, extended embryo survival over time and improved embryo quality. Our study provides the first characterization of oviduct EVs, increasing our understanding of the role of oviduct EVs as modulators of gamete/embryo-oviduct interactions. Moreover, our results point them as promising tools to improve embryo development and survival under in vitro conditions.

Intrinsic quality of goat oocytes already found denuded at collection for in vitro embryo production.

Although cumulus cells are essential for efficient oocyte maturation, the establishment of protocols that support IVD of embryos obtained from denuded oocytes (DOCs) is important for optimizing the use of reproductive biotechnologies. Thus, this study aimed to establish a protocol for IVD of goat DOC using different strategies of IVM and methods of oocyte activation. Four experiments were performed. Similar developmental competence of slaughterhouse DOC was obtained, regardless of maturation media (complex, semidefined or simplified). However, the ability to reach the blastocyst stage was affected by the activation method. Denuded oocytes subjected to parthenogenetic activation had greater (P < 0.05) development capacity, compared with those undergoing IVF with average cleavage rate of 83% and 75%, blastocyst rate of 49% and 28%, and blastocysts in relation to the cleaved embryos of 59% and 38, respectively. In addition, the quality of embryos evaluated after vitrification/warming was similar between parthenogenetic activation and IVF. Finally, we demonstrated that the coculture of cumulus-oocyte complex (COC) with DOC increased the competence of DOC at a ratio of 1:1 and 1:9 (DOC:COC). We believe that presence of cumulus cells (CCs) is not essential to the meiotic maturation, if at the time of removal of the oocyte from follicular environment, they already acquired competence to development. However, when the oocytes still need to acquire competence, the presence of CC may significantly contribute in their developmental capacity acquisition during IVM. Thus, regardless of the source, these oocytes will require longer time in IVM, contrary to what happens in the absence of CC. In conclusion, although DOC had a lower developmental potential, especially after IVF, they were able to produce blastocysts and the coculture of DOC with COC increased this developmental capacity.

Combination of oviduct fluid and heparin to improve monospermic zygotes production during porcine in vitro fertilization.

In vivo, the oviduct provides appropriate microenvironment conditions for monospermic fertilization and early embryo development. In addition, glycosaminoglycans such as heparin are present in the oviduct and have been shown to modulate the activity of oviduct-secreted proteins on the regulation of sperms parameters. Thus, the present study was designed to evaluate the effect of porcine oocytes exposure to oviduct fluid (OF) before in vitro fertilization (IVF; incubation of oocytes in OF for 30 minutes before IVF), during IVF (supplementation of IVF medium with 10% OF), and during IVF in combination with heparin (10% OF + 10-µg/mL heparin) on IVF parameters. Regardless of sperm concentration used (0.5, 1.5, or 4.5 × 10(5) cells/mL), exposure of oocytes to OF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control group. This resulted in a general increase (P < 0.05) in the final output of the IVF system in terms of zygotes with two pronuclei in OF-exposed groups: 56 ± 9% (OF before) and 60 ± 7% (10% OF during IVF), compared with control (21 ± 8%), when IVF was performed with 4.5 × 10(5) cells/mL. The combination of 10% OF with heparin during IVF induced a decrease (P < 0.05) of the penetration rate, with no effect (P > 0.05) on the monospermy rate in comparison with 10% OF alone. This resulted in a general reduction (P < 0.05) in the final output of the IVF system (%), which was 33 ± 6% and 52 ± 8%, for 10% OF + heparin and 10% OF, respectively. In conclusion, the OF, used in porcine IVF, exerted a beneficial effect on oocytes by reducing the incidence of polyspermy without decreasing the penetration rate. However, the association of the OF with heparin reduced the efficiency of monospermic zygotes' production.

N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle.

The positive effect of n-3 polyunsaturated fatty acids (FAs) on fertility in ruminants seems to be partly mediated through direct effects on the oocyte developmental potential. We aimed to investigate whether supplementation with physiological levels of docosahexaenoic acid (DHA, C22:6 n-3 polyunsaturated fatty acids) during IVM has an effect on oocyte maturation and in vitro embryo development in cattle. We reported that DHA (0, 1, 10, or 100 µM) had no effect on oocyte viability or maturation rate after 22-hour IVM. Incubation of oocyte-cumulus complexes with 1-µM DHA during IVM significantly increased (P < 0.05) oocyte cleavage rate as compared with control (86.1% vs. 78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (39.1% vs. 29.7%, respectively). Supplementation with 1 µM DHA during IVM also induced a significant increase in the blastocyst rate at Day 7 after IVF as compared with control (30.6% vs. 17.6%, respectively) and tended to increase the number of cells in the blastocysts (97.1 ± 4.9 vs. 81.2 ± 5.3, respectively; P = 0.08). On the contrary, 10-µM DHA had no effects, whereas 100-µM DHA significantly decreased the cleavage rate compared with control (69.5% vs.78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (19.5% vs. 29.7%). As was shown by real-time polymerase chain reaction, negative effects of 100-µM DHA were associated with significant increase of progesterone synthesis by oocyte-cumulus complexes, a three-fold increase in expression level of FA transporter CD36 and a two-fold decrease of FA synthase FASN genes in cumulus cells (CCs) of corresponding oocytes. Docosahexaenoic acid at 1 and 10 µM had no effect on expression of those and other key lipid metabolism-related genes in CC. In conclusion, administration of a low physiological dose of DHA (1 µM) during IVM may have beneficial effects on oocyte developmental competence in vitro without affecting lipid metabolism gene expression in surrounding CCs, contrarily to 100 µM DHA which diminished oocyte quality associated with perturbation of lipid and steroid metabolism in CC.

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up-derived oocytes have different kinetics and requirements regarding maturation media.

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.

Influence of heparin or the presence of cumulus cells during fertilization on the in vitro production of goat embryos.

Considerable research has been focused on in vitro production (IVP) of goat embryos to improve its efficiency. In Experiment 1, the effect of the cumulus cells by comparing slaughterhouse-oocytes denuded on purpose (DOP) prior to IVF to intact COC, and the effect of heparin during IVF were assessed. In Experiment 2, oocytes that were already denuded at collection (DOC), DOP and intact COC were studied. Three treatments used oocytes denuded at collection: DOC oocytes were cultured alone for both IVM and IVF; DOC and COC were cultured together for both IVM and IVF or DOC were IVM alone and then mixed with COC for IVF. In other treatments, COC were allocated to four IVF treatments: Intact COC; COC were denuded prior to IVF; COC were denuded and IVF with added cumulus cells; COC were denuded and IVF mixed with intact COC giving two sub-treatments: Denuded oocytes that were IVF with COC; and COC that were IVF with denuded oocytes. After fertilization, all presumptive zygotes were cultured for 8 days. In Experiment 1, the yield of blastocysts as a proportion of total oocytes was greater (P<0.05) for COC that were IVF in the presence of heparin (54%) than without heparin (42%) or oocytes already denuded at collection that were IVF with or without heparin (41%; 38%; respectively). In Experiment 2, the developmental potential of oocytes denuded at collection was reduced (cleavage and blastocyst rates calculated from total oocytes: 34%; 11%, respectively) as compared to COC (77%; 59%, P<0.05). However, when equal numbers of both were mixed at the start of IVM, the rates were not significantly different to COC alone (68%; 45%), but when both were mixed equally only for IVF, the rates were reduced (57%; 40%, P<0.05). Denuded oocytes co-cultured with cumulus cells were not significantly different to intact COC (76%; 55%). The effect of adding COC during IVF to oocytes denuded after IVM was similar to adding cumulus cells to the same type of oocytes. In conclusion, both the use of heparin and the association of oocytes with cumulus cells, either detached or in intimate contact, during IVM and/or IVF significantly improve IVP of goat embryos. Furthermore, some oocytes that are already denuded at collection will develop satisfactorily to blastocysts when matured and fertilized with intact COC.