Oviduct fluid extracellular vesicles regulate polyspermy during porcine in vitro fertilisation.

High polyspermy is one of the major limitations of porcine invitro fertilisation (IVF). The addition of oviductal fluid (OF) during IVF reduces polyspermy without decreasing the fertilisation rate. Because extracellular vesicles (EVs) have been described as important OF components, the aim of this study was to evaluate the effect of porcine oviductal EVs (poEVs) on IVF efficiency compared with porcine OF (fresh and lyophilised). OF was collected from abattoir oviducts by phosphate-buffered saline flush, and poEVs were isolated by serial ultracentrifugation. Four IVF treatments were conducted: poEVs (0.2mgmL-1), OF (10%), lyophilized and reconstituted pure OF (LOF; 1%) and IVF without supplementation (control). Penetration, monospermy and IVF efficiency were evaluated. Transmission electron microscopy showed an EVs population primarily composed of exosomes (83%; 30-150nm). Supplementation with poEVs during IVF increased monospermy compared with control (44% vs 17%) while maintaining an acceptable penetration rate (61% vs 78% respectively) in a similar way to OF and LOF. Western blotting revealed poEVs proteins involved in early reproductive events, including zona pellucida hardening. In conclusion, our finding show that poEVs are key components of porcine OF and may play roles in porcine fertilisation and polyspermy regulation, suggesting that supplementation with poEVs is a reliable strategy to decrease porcine polyspermy and improve invitro embryo production outcomes.

Expression of CD44 in sheep oocytes and preimplantation embryos.

The CD44 family belongs to a larger group of hyaluronic acid-binding proteins and plays important roles in oocyte maturation, fertilization and preimplantational embryo development. We analyzed the CD44 receptor in sheep oocytes and embryos. Immature oocytes (N = 66) were obtained from a local abattoir; mature oocytes (N = 35) and embryos (N = 41) were obtained by laparotomy from adult hair ewes submitted to ovarian stimulation treatment. The CD44 mRNA was detected by hemi-nested PCR, after reverse transcription, while proteins were located by indirect immunofluorescence, using anti-human CD44 monoclonal antibody. Human lymphocytes and immature bovine oocytes were used as positive and negative controls, respectively. Assessment of the oocyte nuclear stages as well as classification of the embryonic development stage were made with Hoechst 33342 staining. Indirect immunofluorescence detected CD44 expression on the surface of mature oocytes and embryos; immature oocytes did not take up the stain. These findings were supported by the RT-PCR data, which showed no mRNA templates for CD44, even after two consecutive amplifications, in material from immature oocytes and cumulus cells. The CD44 amplicons were detected after a second hemi-nested PCR in mature oocytes and embryos. The finding of CD44 in mature oocytes and preimplantational embryos could reflect the expression profile of hyaluronic acid during terminal folliculogenesis and preimplantational embryo development in sheep.

Goat oocyte production by standard or one-shot FSH treatments and quantitative analysis of transcripts for EGF ligands and its receptor after in vitro maturation.

Hormonal ovarian stimulation may affect the success of embryo production by regulating transcripts in recovered cumulus-oocyte complexes (COCs). Here, in parallel to morphological classification and in vitro maturation (IVM) rate analysis, we investigated the expression of epidermal growth factor (EGF) and its receptor (EGFR) in oocytes and cumulus cells from goat COCs recovered by laparoscopy after standard [multi-dose follicle-stimulating hormone (FSH)] or one-shot (single dose FSH plus eCG) treatments. No differences were observed among the number of recovered and morphologically graded COCs or the IVM rates for both gonadotropic treatments. However, the standard protocol produced COCs with higher EGFR expression in the cumulus cells than the one-shot treatment. Additionally, EGF mRNA levels were less than EGFR mRNA levels, and they did not differ among COCs from both treatments. However, during maturation, the EGF transcripts increased in oocytes derived only from the standard protocol. Interestingly, IVM strikingly increased EGFR expression in oocytes and cumulus cells but not in oocytes that fail in first polar body extrusion, irrespective of hormonal treatment. These results appear to be related to the resumption of meiosis and suggest that EGF may act through the cumulus cells or directly on the oocyte receptor.

Real-time qRT-PCR analysis of EGF receptor in cumulus-oocyte complexes recovered by laparoscopy in hormonally treated goats.

Ovarian stimulation with exogenous follicle stimulating hormone (FSH) has been used to increase the number of viable oocytes for laparoscopic oocyte recovery (LOR) in goats. The aim of this study was to evaluate the effect of two FSH protocols for ovarian stimulation in goats on the expression pattern of epidermal growth factor (EGF) receptor (EGFR) in cumulus-oocyte complexes (COCs) recovered by LOR. After real-time qRT-PCR analysis, expression profiles of morphologically graded COCs were compared prior to and after in vitro maturation (IVM) on a FSH protocol basis. The use of a protocol with higher number of FSH injections at a shorter interval resulted in GI/GII COCs with a higher level of EGFR expression in cumulus cells, but not in the oocyte, which was correlated with an elevated meiotic competence following IVM. Based on the maturation profile and EGFR expression patterns observed between groups, the morphological selection of COCs prior to IVM was not a good predictor of oocyte meiotic competence. Therefore, EGFR may be a good candidate marker for indirect prediction of goat oocyte quality. The IVM process of goat COCs increased the EGFR expression in oocytes and cumulus cells, which seemed to be strongly associated with the resumption of meiosis. In summary, differential EGFR expression in goat cumulus cells was associated with the in vivo prematuration process, and in turn, the upregulation in the entire COC was associated with IVM. Cause-and-effect relationships between such increased expression levels, particularly in the oocyte, and oocyte competence itself still need to be further investigated.

Oviduct fluid during IVF moderately modulates polyspermy in in vitro-produced goat embryos during the non-breeding season.

The present study determined i) the presence of proteins (oviduct-specific glycoprotein, OVGP1; heat shock protein-70A, HSPA1A; heat shock protein-A8, HSPA8; annexin A1, ANXA1; annexin A5, ANXA5; and myosin-9, MYH9) known to be involved in early reproduction in the oviduct fluid (OF) of anestrous goats; and ii) the functional effect of during IVF on polyspermy modulation and embryonic development. In vitro-matured oocytes were co-cultured with spermatozoa (1.0, 2.0, or 4.0 x 106 cells/mL) for 18 h in SOF medium supplemented with 5 µg/mL of heparin, 4 µg/mL gentamicin, and 10% estrus sheep serum (CTRL1, CTRL2, and CTRL4 groups) or the same medium plus 10% OF (OF1, OF2, and OF4 groups) obtained from anestrus goats. The analysis of OF by western blotting confirmed the presence of the six proteins tested for. The increase in sperm concentration had no effect (P > 0.05) on the penetration rate in any group; however, monospermy rate decreased as sperm concentration was increased in both OF and CTRL. Regardless of the concentration used, when data were pooled, OF supplementation improved (P < 0.05) monospermy and tended (P = 0.057) to enhance IVF efficiency. Additionally, IVF efficiency was higher (P < 0.05) in OF1 than in OF4 [60 ± 13 vs 37 ± 5%). The development capacity was not affected (P > 0.05) by the sperm concentration and OF treatment, and the average values were cleavage (72 ± 2.6%), blastocyst (37 ± 3.0%), blastocyst in relation to the cleaved (51 ± 4.8%), hatched (62 ± 1.2%), and number of cells per blastocyst (174 ± 1.8%). In conclusion, the six proteins analyzed are present in the OF of anestrous goats, and the supplementation of this OF during IVF may modulate the polyspermy incidence and enhance IVF efficiency, especially when 1x106 sperm per mL is used.