Bovine cumulus-oocyte complexes (COCs) and mural granulosa cells express the mRNA coding for the micro-opioid receptor. The addition of beta-endorphin (beta-end) to oocytes cultured in hormonally-supplemented in vitro maturation (IVM) medium had no effect on the rates of oocytes reaching the metaphase II (MII) stage, but significantly decreased the maturation rate (P < 0.05) and arrested oocytes at metaphase I (MI) after culture in hormone-free medium (P < 0.001). Naloxone (Nx) reverted this inhibitory effect of beta-end. Moreover, Nx "per se" showed a dose-dependent dual effect. When added at high concentration (10 x (-3) M), it significantly reduced the rate of oocytes in MII (P < 0.001), thus increasing the rate of oocytes arrested in MI. However, Nx added at low concentration (10 x (-8) M) significantly increased oocyte maturation (P < 0.001). High concentration of Nx induced an increase in both intracellular calcium concentration ([Ca(2+)](i)) and in the activity of the mitogen-activated protein kinase (MAPK) also called extracellular-regulated kinase (ERK) in cumulus cells of bovine COCs. Blocking the rise in [Ca(2+)](i) with the calcium chelator acetoxymethylester-derived form of bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) reversed the Nx-dependent inhibition of meiotic maturation observed at high Nx concentrations. Whereas blocking ERK with the MAPK/ERK kinase (MEK) inhibitor, PD98059, had no effect on this process. Therefore, we concluded that the mocro-opioid receptor, by inducing [Ca(2+)](i) increase, participates in the cumulus-oocyte coupled signaling associated with oocyte maturation.

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The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) using frozen-thawed spermatozoa. Follicular fluid did not increase the maturation of oocytes to metaphase II stage compared to control. After IVF, there was no difference in fertilization rates between FF-supplemented oocytes and controls (7/87, 8.4% of oocytes showing two pronuclei with FF versus 7/116, 6% with EMS; not significant). However, after ICSI, FF-supplemented oocytes showed significantly increased normal fertilization (32/85, 37.6% of two-pronuclear oocytes) and developmental potential (15/31, 48% cleavage) compared to the control oocytes (7/47, 14.9%, P < 0.01; and 2/48, 4%, P < 0.01, respectively). Overall, ICSI resulted in increased fertilization rates compared to IVF, regardless of the presence or absence of FF (39/132, 29.5% with ICSI versus 14/203, 6.9%). These results suggest that follicular fluid supplementation may improve the maturity of equine cumulus-enclosed oocytes sufficiently for the successful use of ICSI, but not sufficiently for normal sperm-egg interaction occurring during IVF.

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Follicle atresia and granulosa cell apoptosis may be related to oocyte meiotic and developmental competence. We analyzed the relationships among granulosa cell apoptosis, initial cumulus morphology, oocyte nuclear maturation in vitro, and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the horse. For each follicle, the size was measured and granulosa cells were used for DNA laddering analysis. Oocytes were evaluated for cumulus morphology, cultured for in vitro maturation, and submitted to ICSI. Apoptosis was categorized as absent, intermediate, or advanced according to the relative concentrations of two DNA fragments at 900 and 360 base pairs (bp). In 98 oocyte-follicle pairs, 52 oocytes were classified as expanded (Exp), 39 as compact (Cp), and 7 as having a partial (P) cumulus. Advanced apoptosis was detected in 55% (54/98) of follicles; 37% (36/98) of follicles showed an intermediate level of apoptosis; and 8 follicles (8%) were nonapoptotic. Follicle size was not significantly correlated with granulosa cell apoptosis (P > 0.05). Significantly more Exp than Cp oocytes originated from follicles with advanced apoptosis (P < 0.001). The proportion of oocytes maturing in vitro was significantly higher in oocytes issuing from apoptotic follicles than in oocytes issuing from healthy follicles (P < 0.05). The proportion of normally (two pronuclei) or abnormally fertilized oocytes (one or greater than two pronuclei, or partially decondensed sperm) did not differ in relation to granulosa cell apoptosis. We conclude that, in the mare, granulosa cell apoptosis is related to cumulus expansion and an increase in oocyte meiotic competence but has no effect on the proportion of meiotically competent oocytes that activate after ICSI. These results provide selection criteria for horse oocytes used in assisted reproductive techniques so that embryo production may be maximized.

Background: The mycotoxin zearalenone (ZEA) and its derivatives, alpha and beta-zearalenol (alpha and beta-ZOL), synthesized by genera Fusarium, often occur as contaminants in cereal grains and animal feeds. The importance of ZEA on reproductive disorders is well known in domestic animals species, particularly in swine and cattle. In the horse, limited data are available to date on the influence of dietary exposure to ZEA on reproductive health and on its in vitro effects on reproductive cells. The aim of this study was to evaluate the effects of ZEA and its derivatives, alpha and beta-ZOL, on granulosa cells (GCs) from the ovaries of cycling mares.

The micro-opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT-PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cytoplasm of oocytes and cumulus cells. In vitro oocyte maturation rate (MR), analyzed by confocal microscopy for nuclear chromatin, microfilaments and microtubules, was reduced after the addition of 3 x 10(-8) M beta-endorphin in medium without additional hormones. Inhibitory effects of 10(-3) M Naloxone in oocytes collected in anestrous and spring transition were observed, both in presence and absence of hormones added to culture medium. Increased MRs were observed in oocytes collected in anestrous and cultured in presence of 10(-8) M Naloxone. The exposure to 10(-3) M Naloxone induced significant intracellular calcium increases in cumulus cells recovered all over the year. beta-Endorphin 3 x 10(-8) M induced significant calcium increases only in cumulus cells recovered in fall transition and anestrous. Naloxone 10(-8) M did not induce intracellular calcium modifications. We conclude that the MOR is differentially expressed in equine cumulus-oocyte complexes in the different seasons of the year and plays a role in the seasonal regulation of meiotic competence of equine oocytes.

The development of fertilizing ability in sperm cells is associated with changes in the plasma membrane. However, to date the exact nature of sequentially activated primary receptors and channels and the signal transduction pathways derived from these remains elusive. We analyzed the expression and localization of the m-opioid receptor in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds m agonists was amplified, sequenced and compared with the known sequences in humans, rats and cattle. The amplification product showed a high degree of nucleotide conservation. By immunofluorescence, m-opioid receptor labeling was found on the sperm head and on the tail and disappeared in the acrosomal region of acrosome-reacted sperm cells. Immunoblotting revealed two bands of 50 and 65 kDa. Effects of the opioid antagonist naloxone on motility and on viability and capacitation/acrosome reaction were investigated by computerassisted sperm analysis and Hoechst 33258/chlortetracycline (H258/CTC) staining. Progressive motility was significantly reduced after 3 h incubation in 10 23 M naloxone (P < 0.05), whereas it increased significantly after 5 h in 10 28 M naloxone (P < 0.05). Sperm velocity at 5 h was significantly reduced by the addition of 10 23 M naloxone (P < 0.05), but increased significantly in the presence of 10 28 M (P < 0.001). Curvilinear velocity and amplitude of lateral head displacement in spermatozoa incubated in the presence of naloxone were not indicative of hyperactivation. H258/CTC staining showed that 10 28 M naloxone significantly stimulated capacitation (P < 0.01) after 3 h. However, it had no effect on sperm cell viability and acrosomal status. Overall, this study provides the first evidence that the m-opioid receptor is expressed in equine spermatozoa and that naloxone significantly affects motility and capacitation.