Growth factors synthesized by ovarian somatic cells directly affect oocyte growth and function, but it is unclear whether oocyte-secreted factors play a reciprocal role in modulating somatic cell functions in vivo. Granulin (grn) and its precursor are polypeptides that possess growth-regulatory activities principally toward epithelial cells. To determine the localization of grn in rat ovary, we produced rabbit polyclonal antibodies that identified grn precursor protein using synthetic peptides. Four kinds of peptide on the basis of rat grn precursor protein were synthesized and immunized with a total of eight rabbits. All eight antisera were found to bind the original synthetic peptides in solid phase by ELISA method using synthetic peptide. Based on Western analysis of ovarian extracts using one antiserum raised against the synthetic peptides, an immunoreactive band at 67 kDa was detected. Immunohistochemical analysis in both neonatal and adult ovaries indicated that grn-like signals were obtained exclusively in oocytes and not granulosa or theca cells, suggesting the involvement of grn in regulating follicular functions.
The computer-assisted sperm motion analysis (CASA) system has been used as one of the useful means for reproductive toxicity studies in male animals. Since various parameters of sperm motility are measured by the CASA system, we investigated the effects of a herbicide dinoseb, a testicular toxicant, and which parameter was the most appropriate for detecting changes. Crj:CD(SD)IGS mature male rats were administered dinoseb at a dose of 0 (corn oil), 5 or 7.5 mg/kg for 3 consecutive days, and were necropsied for cauda epididymal sperm analysis after a 12-day (Day15) or 19-day (Day 22) withdrawal period (Experiment I). No sperm motility parameters of dinoseb-treated rats were changed on Day 15, but on Day 22, the percentage of motile sperm (MOT) was significantly decreased in rats treated with dinoseb at 7.5 mg/kg. Other parameters, such as path velocity (VAP), curvilinear velocity (VCL), and amplitude of lateral head displacement (ALH) were significantly decreased as well. In Experiment II, no parameters of sperm motility showed any change in rats treated with dinoseb at 7.5 mg/kg three times per week when evaluated on Day 21. It was concluded that MOT, VAP, VCL, and ALH were more sensitive to dinoseb effects on sperm in the CASA system.
In pelagic egg-spawning marine fish, sequential processing of a hepatically derived vitellogenin occurs concomitant with oocyte growth within the follicles. This process consists of yolk formation during early oocyte growth and yolk degradation during late oocyte growth. Since ovarian cathepsins B, D and L have been suspected of being involved in both events, the present study determines their different expression during oocyte growth in round scad Decapterus maruadsi. By adopting specific substrates and inhibitors in newly established assays, the activities of scad ovarian cathepsins B, D and L had been assessed as CA074-sensitive Z-Arg-Arg-MCA hydrolysis, pepstatin-sensitive MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-Arg-NH2 hydrolysis and Z-Phe-Phe-CHN2-sensitive Z-Phe-Arg-MCA hydrolysis, respectively. All ovarian cathepsins were expressed at the highest level in early growing oocytes. With oocyte growth, the expression of cathepsin L decreased significantly, while that of cathepsins B and D showed only marginal variation. The results indicate that highly expressed ovarian cathepsins B and D play roles in yolk degradation during late oocyte growth in scad.
The experiment was conducted to elucidate ovarian localization of immunoglobulin G (Ig G) and inhibin α-subunit and follicular development in guinea pigs after passive immunization against the inhibin α-subunit. Estrus was synchronized in laboratory guinea pigs by implanting progesterone containing-tubes. After removal of the progesterone implants, the guinea pigs were injected i.v. with 1.0 ml inhibin antiserum or normal goat serum (NGS). The results showed that Ig G mainly entered the follicular fluid of antral follicles, indicating that possibly inhibin antibodies could be a local regulator affecting follicular development in guinea pigs. The results also showed that passive immunization against inhibin increased the number of follicles and plasma concentrations of estradiol before ovulation, but did not change the plasma concentrations of follicle-stimulating hormone (FSH) or the numbers of follicles stained with inhibin antiserum. The present study first reports that the distribution of Ig G after injections of inhibin antiserum and confirms our previous hypothesis that only dominant follicles could be stained for inhibin α-subunit. The results also imply that only one injection of inhibin antiserum could not increase the ovulation rate in guinea pigs.
The concentrations of progesterone (P4), estradiol-17 β (E2), luteinizing hormone (LH) and prolactin (PRL) in serum obtained from 9 captive female Japanese black bears, Ursus thibetanus japonicus, (7 lactating and 2 non-lactating) were obtained by a heterologous radioimmunoassay to clarify hormonal profiles during the pre- and post-parturition and lactation periods. The present study provides the first evidence to validate LH and PRL radioimmunoassay in bear sera by using the canine LH and PRL and the antibody to these hormones. In 7 bears which gave birth and lactated, serum P4 concentrations were high during prepartum phases in December and January but fell rapidly after parturition in February (p<0.05; comparison of pre- and post-parturient phases). Serum E2 and LH concentrations did not change significantly throughout pre- and post-parturition. Serum PRL concentrations increased significantly after parturition in February (p<0.05; comparison of pre- and post-parturient phases) and exceeded 10 ng/ml in all lactating bears from Days 41~60 after parturition in March. On the other hand, in 2 bears which did not produce cubs, serum P4 concentrations were high in December and January and decreased after February. Serum E2 concentrations were relatively low between December and February but increased in March. Serum LH concentrations were relatively high in December and remained low throughout January to April, whereas PRL concentrations increased gradually after February, but were below 10 ng/ml. These results suggest that a rapid decline in P4 and a marked increase in PRL occurred in association with events such as parturition and lactation, but there is no clear evidence that changes in serum E2 and LH concentrations are associated with parturition and lactation.
Lectin binding patterns in the testes of the Java fruit bat, Pteropus vampyrus, and the Japanese lesser horseshoe bat, Rhinolophus cornutus, were investigated by light microscopy. Binding patterns were similar in both species, except for some slight differences. UEA-I, SBA, DBA and BSL-I revealed no reaction in the testes of either species. Con A and WGA gave a diffuse reaction all over the seminiferous epithelium in both species. In addition to this binding pattern, PHA-E exhibited a granular reaction within the cytoplasm of pachytene spermatocytes. PNA and RCA-I gave an intense reaction in the acrosomal region from Golgi to acrosome-phase spermatids in both species, but, in addition to this binding pattern, RCA-I reacted in the cytoplasm of spermatocytes and spermatids in the Java fruit bats. PSA revealed a granular reaction in the cytoplasm of spermatids in the Japanese lesser horseshoe bats. These binding patterns were similar to those of mammals studied before, except that a few specific bindings were detected in the bats.
The purpose of this study was to examine fertilities from mating and artificial insemination (AI) with frozen-thawed spermatozoa by indexing the luteinizing hormone (LH) surge with the Canine Ovulation Timing Test (ICG Status-LHTM) in beagle bitches. Eleven 2-8-year-old multiparous dogs were used for this experiment and blood was collected daily at 6-15 days after the onset of proestrus. Each serum sample was then used for detection of the LH surge. Five dogs were mated at 5 days after the LH surge. Whelping was successful in all dogs and litter size was 5.8 ± 1.9 (Mean ± SD). The remaining 6 dogs were artificially inseminated with frozen-thawed spermatozoa at 4, 5 and 6 days after the LH surge. Sperm motility after thawing was 49.6 ± 3.6%. All dogs whelped and litter size was 4.0 ± 3.0. These results suggest that fertilities from mating and AI with frozen-thawed spermatozoa are satisfactory by indexing the LH surge with the Canine Ovulation Timing Test in dogs and further demonstrate that the optimal period for insemination is around 5 days after the LH surge.
Effect of sperm selection according to the degree of motility after insemination on in-vitro penetration was examined by using a new in-vitro fertilization system designated as a climbing-over-a-wall (COW) IVF method. When the sperm penetration rate in the COW-IVF method was compared with a standard method at the same sperm concentration (5 × 10 5 cells/ml), the rates (95.1 ± 1.9 and 98.2 ± 1.0%, respectively) were similar, but the incidence of monospermic penetration was higher in the COW-IVF (25.5 ± 4.5%) than the standard method (10.4 ± 2.5%). When sperm concentration was changed from 0.5 × 10 5 to 10 × 105 cells/ml in the COW-IVF method, sperm penetration rate was higher at a higher concentration, whereas monospermic penetration rate was increased at a lower concentration. The proportion of monospermic oocytes in matured oocytes was similar among sperm concentrations, 0.5 × 105 to 5 × 105 cells/ml, at fertilization in the COW method. These results demonstrate that the COW-IVF method, selection according to the degree of sperm motility after insemination, can increase the normal penetration of frozen-thawed boar spermatozoa into IVM oocytes without any reduction in the sperm penetration rate.
Determination of the expression level and localization of cell adhesion molecules is crucial for understanding the mechanism of maintenance and remodeling of the ovarian follicle structure. We immunocytochemically investigated expression of the cell adhesion molecules, "classic" cadherins and β-catenin, in developing and/or atretic follicles of porcine ovaries. Healthy follicles showed strong staining for cadherin-8 and β-catenin in granulosa cells tightly attached to the basement membrane, and moderate/weak staining was seen on the inner surface of the granulosa cell layer. Strong VE-cadherin expression was seen in a single cell layer attached to the basement membrane in the theca interna layer of healthy follicles. The expression of cadherin-8, β-catenin and VE-cadherin decreased during follicular atresia. No positive staining was observed for R-cadherin, E-cadherin, T-cadherin, BR-cadherin or P-cadherin, and a weak positive reaction for N-cadherin was seen only in the granulosa cells of healthy and early atretic follicles. These findings indicate that cadherin-8, N-cadherin and VE-cadherin have important roles in follicular development and/or degeneration, and that decreases in the expression of these cadherins are involved in follicular atresia in porcine follicles.
Luteal cells utilize lipoproteins (LIPs) for progesterone (P) synthesis. A luteolysin prostaglandin F2α (PGF 2α) has shown to prevent the LIPs utilization for steroidogenesis in bovine midcycle luteal cells. However, a luteolytic injection of PGF2α before Day 5 (Day 0=estrus) can not induce the regression of the corpus luteum (CL). This suggests that the intraluteal mechanism of PGF2α action on the LIPs utilization in early CL is different from that of the mid luteal stage. Thus, this study aimed to investigate the interaction between PGF2 α and LIPs on the secretion of P and oxytocin (OT) from bovine early CL. An in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (Days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of LIPs (1 μg/ml) stimulated P release during infusion, but had no effect on OT release. The infusion of PGF2α (10-5 M) stimulated both P and OT release during infusion. When LIPs were infused after PGF2α exposure, P and OT release continued to be stimulated until 3 h thereafter. The results show that PGF2α is capable of enhancing the LIPs utilization in early CL, thus support the concept that luteal PGF2α acts as a luteotropic promoter in bovine early CL.