Reproduction

Yonezawa, N.; Hatanaka, Y.; Takeyama, H.; Nakano, M.

doi: 10.1530/jrf.0.1030001pmid: 7707284

Pig zona pellucida (ZP) contains three families of glycoproteins: PZP2, PZP3α and PZP3β. PZP3α mediates the binding of the ZP to spermatozoa. In this study, the binding site of pig ZP on boar spermatozoa and the zona-binding proteins of boar spermatozoa were studied using chemically modified zona glycoproteins or anti-pig ZP antiserum. Endo-β-galactosidase-digested PZP3α (EβG-PZP3α), which is deficient in sulfated N-acetylpolylactosamine, as well as solubilized ZP, bound to the acrosomal region of acrosome-damaged or partially acrosome-reacted spermatozoa. However, they did not bind to acrosome-intact or fully acrosome-reacted spermatozoa. Solubilized ZP did bind to the acrosomal cap released upon acrosome reaction. In western blot analyses, EβG-PZP3α bound to the sperm proteins with molecular masses similar to proacrosin–acrosin and the binding was inhibited by fucoidan and anti-pig acrosin antiserum. These results suggest that the binding site of solubilized pig ZP and EβG-PZP3α on spermatozoa is located mainly in the acrosomal matrix and on the membranous compartments in the acrosome and suggest that EβG-PZP3α binds to proacrosin–acrosin. The binding of EβG-PZP3α to proacrosin–acrosin may be involved in the binding of the ZP to the acrosome of partially acrosome-reacted spermatozoa.

Brandon, J. M.

doi: 10.1530/jrf.0.1030009pmid: 7707305

Antibodies to differentiation markers have made it possible to identify macrophages in murine tissues. Macrophages are potent mediators of immunological reactions and it has been proposed that they are pivotal cells at the maternal–fetal interface. Studies of macrophage distribution in murine decidual tissue have provided conflicting evidence for and against the presence of significant numbers of macrophages at the maternal–fetal interface. The study described here used three independent macrophage differentiation antigens to examine the macrophage distribution in decidual tissue from day 6 to day 19 of pregnancy. Macrophage distribution was defined initially using a polyclonal antiserum to the plasma membrane differentiation antigen F4/80. Macrophages were virtually absent from antimesometrial decidual tissue until degenerating tissue was invaded by macrophages from about day 15. The resident population of macrophages in the mesometrial stroma was retained when this area decidualized but these cells did not survive beyond day 13. Mesometrial decidual tissue was virtually devoid of macrophages after this time. The metrial gland contained many macrophages until degeneration set in, but few were seen by day 15 of pregnancy. These distributions were confirmed using monoclonal antibodies (mAb) to F4/80, macrosialin, a monocyte- and macrophage-specific membrane sialoprotein (mAb FA/11), and the leucocyte β2-integrin CR3 (CD11b/CD18; Mac-1), which is expressed on neutrophils as well as monocytes and some tissue macrophages. CR3+, F4/80− and FA/11− neutrophils were found to be more widely distributed in decidual tissue than were F4/80+ or FA/11+ macrophages. Consideration of these observations led to the conclusion that decidual tissue does not support a resident or elicited population of macrophages and that macrophages are not normally found at the maternal–fetal interface in mice.

Griveau, J. F.; Dumont, E.; Renard, P.; Callegari, J. P.; Le Lannou, D.

doi: 10.1530/jrf.0.1030017pmid: 7707295

The reactive oxygen species, hydrogen peroxide (H2O2) and superoxide anion (O2°−), were generated with a xanthine–xanthine oxidase system and their effect on human sperm function was studied. The action of reactive oxygen species on selected human spermatozoa resulted in a decreased capacity for ionophore-induced acrosome reaction, a decrease in sperm motility, an increase in the concentration of lipid hydroperoxides and a loss of membrane polyunsaturated fatty acids. H2O2 was the key intermediate of the deleterious effects exerted by the xanthine and xanthine oxidase. Among these parameters, the acrosome reaction appeared most susceptible to the reactive oxygen species generated by the xanthine–xanthine oxidase system, and was decreased without sperm motility being affected. Treatment with H2O2 was shown to inactivate several enzymatic activities involved in the antioxidant defence of spermatozoa : glutathione peroxidase, Superoxide dismutase and glucose-6-phosphate dehydrogenase. H2O2 and O2°− were shown to be involved in the lipid alterations triggered by the xanthine–xanthine oxidase system. Singlet oxygen is proposed to intervene in the lipoperoxidation process. The inefficacy of mannitol in protecting spermatozoa suggests that hydroxyl radicals were not produced in the extracellular medium.

Miyoshi, K.; Abeydeera, L. R.; Okuda, K.; Niwa, K.

doi: 10.1530/jrf.0.1030027pmid: 7707298

Rat one-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (R1ECM) under different experimental conditions. When the osmolarity of the medium with a reduced concentration (63.8 mmol l−1) of NaCl was varied by adding different amounts of d-sorbitol, more (79–91%) of the one-cell embryos developed to the four-cell stage at 212–278 mosmol than at 306 mosmol (13%). The greatest proportions of morulae (74%) and blastocysts (60%) were obtained at 246 mosmol. When the medium was supplemented with amino acids in various combinations and the osmolarity adjusted to about 246 mosmol, more (80–98%) of the embryos developed to the morula stage. More blastocysts were obtained in medium supplemented with glutamine (Gln: 80%), minimal essential medium (MEM) essential amino acids (EAA) (90%), Gln + EAA (83%), EAA + MEM nonessential amino acids (NEAA) (83%) or EAA + Gln + NEAA (90%) than in medium without amino acids (59%). Few (3–10%) hatching or hatched blastocysts were observed 120 h after the start of culture in the medium with EAA plus Gin or NEAA. The mean number of cells in blastocysts developed in the medium with EAA + Gin + NEAA was 46.7 ± 7.2. When a total of 82 morulae or early blastocysts that had developed in culture were transferred to eight pseudopregnant rats on day 4, six recipients into which 62 embryos were transferred maintained their pregnancies beyond day 23, although no deliveries had occurred by day 25 or 26. When the rats were killed, 42 (68%) implantation sites and eight (13%) full-term fetuses with no gross abnormality were observed in the uterine horns.

Mahmoudzadeh, A. R.; Van Soom, A.; Bols, P.; Ysebaert, M. T.; de Kruif, A.

doi: 10.1530/jrf.0.1030033pmid: 7707299

Experiments were designed to determine optimal conditions for the cryopreservation of bovine embryos produced in vitro. In Expt 1, embryos were exposed for 1, 3 or 5 min to a vitrification solution consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll and 10.26% (w/v) sucrose (EFS) and were subsequently vitrified. After warming in water at room temperature and diluting in a solution of 0.25 mol sucrose l−1, the in vitro survival rate in Ménézo-B2 medium was highest after exposure to EFS for 1 min. In Expt 2, embryos at day 7 and day 8 were vitrified after exposure to EFS for 1 min. The survival rate of embryos at day 7 was significantly improved, especially at the blastocyst and expanded blastocyst stage, when the Ménézo-B2 medium was supplemented with bovine oviduct epithelial cells (BOEC). Embryos at day 8 exhibited a significantly lower survival rate than did embryos at day 7 in both culture media. In Expt 3, one-step exposure of embryos to EFS for 1 min was compared with two-step exposure to 20% ethylene glycol for 3 min and EFS for 30–45 s. Embryos exhibited significantly higher survival and hatching rates after two-step vitrification, especially at the expanded blastocyst (89% and 69%, respectively) and the blastocyst stage (75% and 38%, respectively). In Expt 4, embryos were diluted in solutions of 0, 0.25 or 0.5 mol sucrose l−1 after two-step vitrification. There were no significant differences in the survival rates between the three dilution treatments. It can be concluded that (i) the optimal exposure time to EFS for one-step vitrification is 1 min; (ii) embryonic survival depends on the developmental stage; (iii) the addition of BOEC to culture medium after warming is beneficial for culture of vitrified embryos in vitro; (iv) two-step addition of EFS improves the survival rate and (v) vitrified embryos can be diluted from EFS in a single step without the use of sucrose as an osmotic buffer.

Hochereau-de Reviers, M. T.; Perreau, C.; Pisselet, C.; Locatelli, A.; Bosc, M.

doi: 10.1530/jrf.0.1030041pmid: 7707300

Testicular development of sheep fetuses was studied between day 42 of gestation and birth. Testis mass and the total number of testicular cells increased curvilinearly with fetal age and a positive linear relationship was established between the logarithmic values of age and testis mass, sex cord total length, total number of Sertoli cells, germ cells and Leydig cells per testis. The mean number of gonocytes per unit length of sex cord, the Sertoli cell nuclear cross-sectional area and the Leydig cell cross-sectional area decreased linearly with age between day 42 of gestation and birth. Hypophysectomy and hemicastration were performed to study the regulation of testicular cell divisions during fetal life and to determine whether they were under pituitary control and whether a feedback mechanism was present. Hypophysectomy at day 100 or 110 of gestation nonsignificantly decreased (0.05 < P < 0.01) the testis mass, total length of sex cords and total number of Sertoli cells and significantly decreased (P < 0.05) the cross-sectional area of Leydig cells and nuclei of Sertoli cells. Sex cord diameter and total number of gonocytes were unaltered. Hemicastration at day 110 of gestation significantly increased (P < 0.05) the total number of Leydig cells per testis without changing any other testicular parameter. In male sheep fetuses, the proliferation of testicular somatic and germ cells occurs throughout testicular fetal growth at a higher rate before day 100 of gestation than later, but without any differentiation. Mitotic divisions of Sertoli cells are more numerous before birth than afterwards. Before birth, the proliferation of gonocytes is not under pituitary control.

Lederer, K. J.; Luciano, A. M.; Pappalardo, A.; Peluso, J. J.

doi: 10.1530/jrf.0.1030047pmid: 7707301

Equine chorionic gonadotrophin stimulates both rat granulosa cell mitoses and oestradiol secretion. However, the mitotic potential of oestradiol-secreting granulosa cells is not known. In the first study, granulosa cells of different sizes were isolated and their ability to secrete oestradiol and proliferate in vitro was determined. Granulosa cells were harvested from equine chorionic gonadotrophin-primed immature rats, separated on a 15–45% Percoll gradient, and collected in 12 fractions. An enriched population of small granulosa cells (44 ± 1 μm2) was collected in fractions 3 and 4 and an enriched population of large granulosa cells (97 ± 2 μm2) in fractions 6–8 When granulosa cells from each fraction were cultured for 24 h in the presence of testosterone, the large cells secreted 50% more oestradiol than did the small cells (P <0.05). Aromatase was shown, by immunocytochemistry, to be expressed mainly by granulosa cells larger than 73 μm2, with the relative amount of aromatase expressed per cell increasing with increasing cell size. However, not all large granulosa cells expressed aromatase. To test proliferative capacity, cells from each fraction were cultured with testosterone and the mitogen, insulin. This study showed that only small cells were able to undergo insulin-induced mitosis. In a second study, follicles of different sizes were isolated from immature and equine chorionic gonadotrophin-primed immature rats and the granulosa cell size distribution determined for each follicle size. This study confirmed that equine chorionic gonadotrophin altered the size distribution from principally small mitotically competent cells to large oestradiol-secreting cells. Studies in vitro further demonstrated that FSH in the presence of 8-bromo-cAMP stimulated small granulosa cells to differentiate into large cells. It is proposed that changes in the population of granulosa cells could account for both the slower growth rate of large antral follicles compared with small antral follicles and the inverse relationship between follicular oestradiol secretion and DNA synthesis of granulosa cells.

Crankshaw, D. J.; Gaspar, V.

doi: 10.1530/jrf.0.1030055pmid: 7535849

Prostanoid receptors regulating the contractility of strips of myometrium obtained from nonpregnant ewes during the breeding season were classified pharmacologically. Natural prostanoids, receptor-type selective synthetic analogues, and selective antagonists were used where available. The natural prostanoids PGD2, PGE2, and PGF2α were equipotent in causing contractions (pD2 values of 6.9, 6.7, and 6.9, respectively) but were 100 times less potent than oxytocin (pD2 = 9.2). The synthetic prostanoids iloprost (pD2 = 8.3), GR63799x (pD2 = 7.0), cloprostenol (pD2 = 6.8), and U46619 (pD2 = 6.2) also caused contractions. The effects of iloprost, but not of GR63799x, were blocked by the selective EP1 antagonist AH6809. This suggests the presence of both EP1 and EP3 receptors. The similar potencies of cloprostenol and PGF2α suggest the presence of FP receptors. Although the potency of the TP agonist U46619 was relatively low, its effects were blocked by the selective TP antagonist L670596 (pKB = 8.4), an observation consistent with the presence of TP receptors. Thus, all currently recognized excitatory prostanoid receptors (EP1, EP3, FP and TP) appeared to be present. Contractions induced by cloprostenol and KCl could be inhibited by the β-adrenoceptor agonist isoprenaline (pD2 = 8.S against cloprostenol) and the Ca2+-channel blocker, D600 (pD2 = 6.3 against cloprostenol), but a number of relaxant prostanoids, BW245c, ZK110841, AH13205 and cicaprost, could not produce inhibition. These results suggest that DP, EP2 and IP receptors do not regulate contractility under these conditions.

Price, C. A.; Carrière, P. D.; Bhatia, B.; Groome, N. P.

doi: 10.1530/jrf.0.1030063pmid: 7707302

The aim of the study was to compare histological and endocrinological indices of ovarian follicle health in cattle with monitoring of follicle growth and regression by ultrasound imaging in vivo. Ultrasound scanning was performed daily. Follicles were obtained at ovariectomy; follicular fluid was collected for assay, and the degree of atresia was assessed histologically. Histological atresia was correlated with growth patterns when anovulatory growing and regressing follicles were compared (P < 0.05), but was not different between growing and static follicles. Oestradiol concentrations were lower in static than in growing follicles, although the difference was not significant (35 ± 7 versus 260 ± 120 ng ml−1; P < 0.08), and were significantly lower in regressing follicles (7 ± 5 ng ml−1; P < 0.05). Oestradiol concentrations were significantly lower in histologically atretic than in nonatretic follicles (16 ± 8 versus 282 ± 132 ng ml−1; P < 0.05), but were not different between nonatretic and early atretic follicles (P > 0.05). There was a significant negative correlation between oestradiol concentration and the number of days the follicle was visible by ultrasound (r = −0.71; P < 0.001). Concentrations of progesterone in follicular fluid were correlated with the number of days the follicles were detected (r = 0.61; P < 0.01) and were higher in regressing than in growing follicles (122 ± 71 versus 48 ± 13 ng ml−1; P < 0.05) but not significantly higher in atretic compared with nonatretic follicles (129 ± 102 versus 53 ± 15 ng ml−1). The progesterone:oestradiol ratio was significantly correlated with the number of days a follicle was detected by ultrasound (r = 0.8; P < 0.001) and was significantly higher in regressing than in growing follicles, and higher in atretic than in nonatretic follicles (P < 0.05). Concentrations of dimeric inhibin in follicular fluid were not significantly correlated with number of days that a follicle was detected by ultrasound (P > 0.05), but were significantly (P < 0.05) higher in regressing (3.0 ± 0.7 μg ml−1) than in growing or static follicles (1.2 ± 0.2 versus 1.1 ± 0.2 μg ml−1). Inhibin concentrations were not significantly affected by degree of atresia. Oestradiol concentrations of preovulatory follicles were significantly higher than those of regressing follicles (1131 ± 382 versus 35 ± 7 ng ml−1; P < 0.05) and concentrations of dimeric inhibin were lower (1.0 ± 0.2 versus 3.9 ± 0.4 μg ml−1; P < 0.05). Oestradiol and inhibin concentrations were negatively correlated (r = −0.65; P < 0.05). Dimeric inhibin concentrations were similar between growing nonovulatory and presumptive preovulatory follicles (P > 0.05). These results demonstrate that growing follicles contain high oestradiol and low progesterone and dimeric inhibin concentrations and that, as the rate of growth of the follicle slows, oestradiol concentrations decrease, but progesterone and dimeric inhibin concentrations do not increase until the follicle starts to regress. Histological indices of atresia did not closely correlate with either morphological or endocrinological measures of follicular growth.

Lamming, G. E.; Mann, G. E.

doi: 10.1530/jrf.0.1030069pmid: 7707303

We have investigated changes in endometrial oxytocin receptor concentrations and prostaglandin F2α release in response to exogenous oxytocin treatment in ovariectomized cows treated with progesterone and oestradiol, and made comparisons with similar treatment in cyclic cows. In long-term ovariectomized cows, endometrial oxytocin receptors were present (300 fmol mg−1 protein), but no prostaglandin F2α was released in response to oxytocin treatment until after the administration of progesterone. Subsequent administration of a concentration of oestradiol sufficient to induce oestrus resulted in the downregulation of these receptors and the loss of oxytocin responsiveness, which did not reappear within 20 days in the absence of further hormone treatment. When induced oestrus was followed by further treatment with luteal phase concentrations of progesterone and oestradiol, both oxytocin receptors and oxytocin-stimulated release of prostaglandin F2α reappeared by day 16 after oestrus, in a pattern similar to that seen during the luteal phase of cyclic cows. These results demonstrate how progesterone and oestradiol control the development and responsiveness of endometrial oxytocin receptors in cows, and provide a valuable model in which to investigate further the precise control of the oxytocin receptor in this species.