Expression and localization of the mu-opioid receptor (MOR) in the equine cumulus-oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence.

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.

Localization and quantitative expression of the calcium-sensing receptor protein in human oocytes.

OBJECTIVE: To investigate the expression of the calcium-sensing receptor (CaSR) protein in human oocytes at the germinal vesicle (GV) and metaphase I (MI) and II (MII) stages. DESIGN: Prospective study. SETTING: Academic basic research laboratory and hospital-based fertility center. PATIENT(S): Immature and supernumerary mature oocytes (n = 118) excluded from intracytoplasmic sperm injection treatment. INTERVENTION(S): Immunofluorescence and Western blot with a primary antibody against human CaSR. Confocal laser scanning microscopy (CLSM) together with quantitative image analysis used to measure the fluorescence intensity variations in oocytes at GV, MI, and MII stages. MAIN OUTCOME MEASURE(S): The CaSR expression pattern as evaluated by immunostaining in denuded oocytes and cumulus cells, CLSM, and three-dimensional image reconstructions; quantitative analysis at the equatorial plane of the oocyte. RESULT(S): We identified CaSR in human oocytes and cumulus cells. The fluorescence intensity within the oocyte varied with the developmental stage, with the greatest increase at the MI stage. CONCLUSION(S): The present study demonstrates for the first time the expression and localization of CaSR protein in human oocytes. Increased CaSR protein expression in the MI stage suggests that it may be involved in the regulation of human oocyte development and maturation.

Expression and subcellular localization of the mu-opioid receptor in equine spermatozoa: evidence for its functional role.

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 mu-opioid receptor in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds mu 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, mu-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 computer-assisted sperm analysis and Hoechst 33258/chlortetracycline (H258/CTC) staining. Progressive motility was significantly reduced after 3 h incubation in 10(-3) M naloxone (P <0.05), whereas it increased significantly after 5 h in 10(-8) M naloxone (P <0.05). Sperm velocity at 5 h was significantly reduced by the addition of 10(-3) M naloxone (P <0.05), but increased significantly in the presence of 10(-8) 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(-8) 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 mu-opioid receptor is expressed in equine spermatozoa and that naloxone significantly affects motility and capacitation.

Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis.

The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was increased with 1 microgram/ml eFSH, and was the highest with 20% FCS. It was not influenced by precursors for hyaluronic acid synthesis or glutamine. The expression of transcripts related to cumulus expansion was analyzed in equine cumulus cells before maturation, and after in vivo and in vitro maturation, by using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Connexin 43, cyclooxygenase-2 (COX-2) and FSH receptor (FSHr) mRNA were detected in equine cumulus cells before and after maturation. Their level did not vary during in vivo or in vitro maturation and was influenced neither by FSH nor by precursors for hyaluronic acid synthesis. Results indicate that previously reported regulation of connexin 43 and COX-2 proteins during equine COC maturation may involve post-transcriptional mechanisms.

Meiotic competence of equine oocytes and pronucleus formation after intracytoplasmic sperm injection (ICSI) as related to granulosa cell apoptosis.

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.