In the present study, changes in localization of nerve growth factor (NGF) and its receptors, trkA and p75 in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization of NGF, trkA and p75 in the ovary was also investigated. NGF and its receptors trkA and p75 were localized in oocytes, granulosa cells and theca cells of various stages of follicles throughout the estrous cycle. NGF and its two receptors were also present in numerous interstitial cells and luteal cells. The number of interstitial cells staining positively for NGF and its two receptors was greater in ovaries of day 1 (day 1=day of ovulation) than the other days during the estrous cycle. Treatment with the antiserum against luteinizing hormone releasing hormone (LHRH-AS) at 1100 h on day 4 completely blocked ovulation. There were few positive reactions for NGF and its two receptors in interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. The distinct widespread distribution of NGF and its two receptors in the ovary of golden hamsters suggest that NGF may be an important growth factor for regulation of ovarian function. Furthermore, the LH surge may be an important factor for inducing production of NGF and its two receptors in interstitial cells of the cyclic golden hamster.
Renal tubular dysplasia is an autosomal recessively inherited disorder in Japanese black cattle that is due to deletion mutations in the claudin-16 gene and causes chronic renal failure and death of affected animals. Here, we report a multiplex-PCR procedure to determine the genotype for claudin-16 deficiency in preimplantation embryos. The presence or absence of the wild-type and mutant allele(s) was precisely detected with the multiplex-PCR using as little as 5 pg of genomic DNA from leukocytes. When biopsied embryo cells were examined for claudin-16 deficiency, 97.2% of genotypes were consistent with the PCR results obtained for the corresponding embryos. In addition, sexing of embryos by PCR was performed using an aliquot of DNA extracted from biopsied embryo cells, and determination of claudin-16 genotype and sex was successfully achieved with an efficiency of 91.7% for claudin-16 genotyping and 83.3% for sexing. The production of a 100-day fetus that was male and homozygous for claudin-16 deficiency, as expected from the analysis of biopsied embryo cells, gave evidence of the reliability and applicability of this procedure for preventing the transmission of this disease and for enabling advances in animal breeding.
Local angiogenesis and angiolysis in the corpus luteum (CL) relate to the luteal function. Recent studies indicate that angiopoietins (ANPT) and their receptors Tie regulate remodeling of microvasculature. We therefore examined 1) the relative changes in the expression of mRNA for ANPT-1, ANPT-2, Tie1 and Tie2 in bovine CL by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) during the estrous cycle and prostaglandin F2α (PGF2α)-induced luteolysis, and 2) the effect of ANPT-2 on progesterone (P4) release from CL at the late stage of the estrous cycle by an in vitro microdialysis system (MDS). The CLs were classified into 4 stages (early: Day 2-5, n=7, mid: Day 8-12, n=15, late: Day 15-17, n=9, regressing: Day >18, n=19). The levels of ANPT-1 mRNA in early and regressing CL were lower than those in mid and late CL, whereas ANPT-2 mRNA expression did not change during the estrous cycle. The Tie2 mRNA expression decreased as the CL aged. During PGF2α-induced luteolysis, ANPT-2 mRNA expression was acutely and temporally increased at 2 h after PGF2α injection. The expression of ANPT-1 mRNA was decreased from 4 h after PGF2α injection and kept low levels. In the experiment with the in vitro MDS, an infusion of ANPT-2 (100 ng/ml) acutely inhibited P4 release from late CL. Overall, results suggest that decrease of ANPT-1 mRNA is a basic mechanism of vascular remodeling in CL. In addition, ANPT-2 might play a role in regulation of P4 secretion in CL during luteolysis.
To determine the feasibility of preserving oocytes without freezing, we stored mouse oocytes in several media at different temperatures for one day. Confocal microscopy of the metaphase-II spindle in these stored oocytes revealed gross abnormalities in both the spindle and the arrangement of chromosomes. The abnormal spindles could not be rescued by transplanting the aged spindle-chromosome complex into a fresh enucleated oocyte. A diploid parthenogenetic development showed that some of the oocytes stored at room temperature could still develop into blastocysts (10-57%). However, oocytes stored in a refrigerator (5%) or incubator (0%) lost the potential almost entirely. Fertilization of room-temperature-preserved oocytes with fresh spermatozoa by ICSI or IVF resulted in, respectively, 4 and 10%, full-term births. These results suggest that when oocytes are stored at room temperature for one day, most have irreversible damage not only to their cytoplasm but also to the spindle. However, since at least a few percent of stored oocytes retained the potential for full-term development, it may be possible to overcome these problems and develop a simple method for preserving mammalian oocytes without freezing.
To determine the effect of Pueraria mirifica (PM) on serum parathyroid hormone (PTH) and calcium levels on aged menopausal monkeys (Macaca fascicularis), subjects were treated with 10, 100, or 1,000 mg/day of PM. Blood samples were collected every 5 days for 30, 90, and 60 days during pre-treatment, treatment, and post-treatment periods, respectively. Sera were assayed for PTH, estradiol, and calcium levels. PM-1,000 had the strongest effect on the decrease in PTH (0.001<P≤0.05) and calcium levels (0.001<P≤0.03) during the treatment period. PTH levels remained low for the first 15 days of the post-treatment period (0.01≤P ≤0.05). PM-10 induced a significant decrease in PTH level on day 80 (P=0.02) during the treatment period and a significant decrease in calcium level on day 75 (P<0.01). There were no changes in serum PTH and calcium levels throughout the study period in the PM-100 group. Estradiol levels decreased significantly during the treatment period in all treatment groups. The results suggest that long-term treatment with 1,000 mg/day of PM decreases serum PTH and calcium levels in aged menopausal monkeys, indicating that PM ameliorates bone loss caused by estrogen deficiency.
Taxol and vinblastine have been widely used in cancer chemotherapy as anti-microtubule agents. However, there are on-going efforts to find new anti-microtubule agents with fewer of the side effects associated with these drugs, such as toxicity or the development of resistance. The standard method used to identify anti-microtubule agents has been the in vitro microtubule polymerization assay. One limitation of this system is that the only compounds selected are those that act on tubulin. Novel compounds whose targets are upstream or are related unknown molecules are not detected. Therefore, many researchers have recently tried to develop novel, phenotype-based drug screening systems. In this study, we developed an oocyte-based screening system for anti-microtubule agents. Dramatic phenotypic changes in microtubules can easily be observed in ovulated oocytes treated with microtubule-stabilizing or -destabilizing agents, such as taxol or vinblastine. After culturing with test samples for 5 h, oocytes were analyzed with fluorescence microscopy after immunostaining. In the oocyte-based screening system, the effective dose (ED50) of taxol for microtubule polymerization is ~5 nM, and the ED50 of vinblastine for microtubule depolymerization is ~2.5 nM. In addition, taxol-like and vinblastine-like compounds can be evaluated simultaneously in a single assay using this system.
Mongolian gerbil 2-cell embryos were cultured in modified M16. When osmolarity of the medium with 5.0 mmol glucose l-1 was varied by adjusting the amount of NaCl added, 2-cell embryos at 280, 290, 300 and 310 mOsmol developed to the 8- and 16-cell stages. The incorporation and oxidation of 14C-Methionine were compared between fresh recovered and cultured embryos at the 1-cell to 16-cell stages. Development beyond the 8-cell stage of fresh recovered embryos showed an enhanced rate of total protein synthesis, indicating activation of the transcription process of the embryonic genome. However, we found that the lowest incorporation and oxidation of 14C-Methionine was observed in cultured embryos of the 16-cell stage at 115 h after hCG injection. In the medium without phosphate, glucose promoted development of 2-cell embryos to the 8-cell stage, and low concentrations of glucose were necessary for the development of the 2-cell to 8-cell stages. These results suggest that Mongolian gerbil preimplantation embryos can be cultured in vitro in a chemically defined medium with a low concentration of glucose.
The developmental ability and the nucleus and microtubule dynamics of nuclear transplanted goat embryos derived from in vitro matured oocytes were studied while controlling cell-cycle coordination of donor embryonic nuclei and recipient cytoplasts. Three groups of transfers were studied: G0/G1 (after the fibroblast cells grew to 100% confluence) and G2/M (nocodazole treated) phase fibroblasts transferred to MII cytoplasts (G0/G1→MII and G2/M→MII group, respectively), and G0/G1 phase fibroblasts transferred to preactivated cytoplasts, mostly at S-phase, (G0/G1→Pre group) by electrical fusion. The results showed that fusion and developmental ability did not differ between G0/G1→MII and G0/G1→Pre groups. However the developmental rate of embryos in the G0/G1→MII group was significantly higher than that of the G2/M→MII group. Most fibroblast nuclei (G0/G1 and G2/M) transferred into MII oocytes underwent premature chromosome condensation (PCC). Normal spindle were only detected in the G0/G1→MII group. In contract, fibroblast nuclei in pre-activated oocytes rarely underwent PCC, but formed a swollen nuclear structure. The data suggest that in vitro matured goat oocytes can support the development of somatic fibroblasts after nuclear transfer, G0/G1 →MII and G0/G1→S nuclear transfer might be effective ways for improving the developmental competence of the reconstituted embryos, and that G2/M→MII nuclear transfer by electrical fusion (even in Ca2+-free fusion medium) induces abnormal chromosome ploidy.
Mammalian spermatozoa must undergo acrosomal exocytosis prior to penetration of the oocyte at fertilization. The mechanisms underlying acrosomal exocytosis have not yet been fully elucidated. This study explored the possible involvement of ceramide in exocytosis of the boar sperm acrosome. Ejaculated boar spermatozoa, stored with the Beltsville TS extender at 17C for up to 3 days, were washed and preincubated for 10 min with C2-ceramide, an analogue of endogenous ceramide, C2-dihydroceramide (C2-DH-ceramide), a negative control to C2-ceramide, or with (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP), an inhibitor of alkaline ceramidase, followed by incubation and stimulation with 3 mM Ca2+ and 0.3 μM A23187 (Ca2+/A23187) at 37C in air in a water bath. Spermatozoa fixed at specific intervals were examined, and the % of acrosomal exocytosis was monitored. Stimulation of spermatozoa with Ca2+/A23187 resulted in a time-dependent increase. There were no obvious changes at 5 min, but this was followed by a rapid increase at 10 min, reaching nearly a maximum level after 15 min or more of incubation. Preincubation with C2-ceramide or D-erythro-MAPP enhanced acrosomal exocytosis triggered by Ca2+/A23187 in a dose-dependent manner, whereas C2-DH-ceramide was without effect. These results suggest the possibility that ceramide may be involved in the mechanisms underlying acrosomal exocytosis.
Congenital hypothyroid mutant male rdw rats have enlarged testes in adulthood with dwarfism accompanied by infertility. To explain how rdw rats acquire enlarged testes in adulthood, we compared age-matched normal (N) rats at various developmental stages for blood levels of hormones, thyroxine (T4), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T), and investigated whether T4 therapy (rdw+T4) from 3 weeks of age (w) until adulthood could induce recovery of fertility in rdw rats, as well as how rdw+T4 affected hormonal patterns. Testes weights of rdw rats were higher than those of N rats at 19 w in adulthood though it was low during development. Serum T4 values in rdw rats were markedly lower than those in N rats but steadily increased up to 19 w. The serum FSH values in rdw rats were lower than those in N rats at all ages, and neither serum LH nor T value was significantly different at any age. The testes weight of rdw+T4 rats was significantly higher than that of N rats at 13 w with recovered growth, and was higher than that of rdw rats at 19 w. When they were mated with proestrous females after 16 w, all females became pregnant and gave birth to a normal number of pups. The T4 and FSH values of rdw+T4 rats were significantly higher than those in rdw rats, but similar to those in N rats in adulthood. The results suggest that even low levels of circulating thyroid hormone (TH) in rdw rats stimulate the development of their testes, probably through Sertoli cells, resulting in the enlarged adult testes without fertility, and that a sufficient circulating TH level from the immature stage plays a pivotal role in restoring mating activity, probably through FSH-mediated action towards adulthood.
Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology (ART). Vitrification allows glasslike solidification of a solution without ice crystal formation in the living cells. We have attempted to improve the survival rates of oocytes by a vitrification technique using bovine models. In vitro matured oocytes with or without cumulus cells were vitrified with either 15.0% (v/v) ethylene glycol (EG) + 15% (v/v) dimethylsulfoxide (DMSO) + 0.5 M sucrose or 15% (v/v) EG + 15% (v/v) 1,2-propanediol (PROH) + 0.5 M sucrose, using `Cryotop' or `thin plastic sticker', respectively. The oocyte survival rates after vitrifying-warming, and the capacity for fertilization and embryonic development were examined in vitro. The rate of embryonic development to blastocyst was significantly higher (P<0.05) in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose than in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) DMSO + 0.5 M sucrose (7.4% ± 4.1 vs. 1.7% ± 3.0, respectively). Oocytes vitrified without cumulus cells had a higher survival rate after thawing and a superior embryonic developmental capacity compared with oocytes vitrified with cumulus cells. Prolonged pre-incubation time after thawing adversely affected the rates of embryonic cleavage and development. These results indicate that in vitro matured bovine oocytes can be vitrified successfully with the mixture of the cryoprotectants, EG + PROH, the absence of cumulus cells for vitrification does not affect oocyte survival rate after warming, and vitrified and warmed oocytes do not require pre-incubation before in vitro fertilization.
The time course of GnRH pulse generator activity and plasma concentrations of energy substrates and insulin were simultaneously observed in female goats during 4-day fasting and subsequent refeeding in the presence or absence of estrogen for a better understanding of the mechanism of energetic control of gonadotropin secretion in ruminants. The GnRH pulse generator activity was electrophysiologically assessed with the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In estradiol-treated ovariectomized (OVX+E2) goats, the MUA volley intervals increased as fasting progressed. Plasma concentrations of non-esterified fatty acid and ketone body increased, while those of acetic acid and insulin decreased during fasting. The MUA volley intervals and plasma concentrations of those metabolites and insulin were restored to pre-fasting levels after subsequent refeeding. In ovariectomized (OVX) goats, changes in plasma metabolites and insulin concentrations were similar to those in OVX+E2 goats, but the MUA volley intervals were not altered. The present results demonstrated that fasting suppressed GnRH pulse generator activity in an estrogen-dependent manner. Changes in plasma concentrations of energy substrates and insulin during fasting were associated with the GnRH pulse generator activity in the presence of estrogen, but not in the absence of the steroid in female goats.
We reported previously that passive immunization against inhibin enhances follicular growth and increases the ovulation rate. However, the ovulation rate was not comparable to the number of follicles. Therefore, the aim of this study was to attempt to increase the ovulation rate by increasing the interval between inhibin immunization and PGF2α injection. Five miniature Shiba goats were treated with 10 ml inhibin antiserum (inhibin-AS) developed against [Tyro30]-inhibin α (1-30). A control group (n=5) was treated with normal goat serum. All animals were injected intramuscularly with 125 μg PGF2α 72 h after treatment to induce estrus and ovulation. Blood samples were collected for hormonal assay and the ovulation rate was determined by laparotomy. In contrast to the control group, there was a significant increase in plasma concentrations of FSH in the immunized group. After luteolysis, plasma concentrations of estradiol-17β increased markedly to a preovulatory peak about 2 folds higher (P<0.01) than that of controls. In addition, the ovulation rate was greater in the immunized group (14.4 ± 2.2) than in the control group (2.2 ± 0.6), and the mean number of follicles ≥ 4 mm in diameter was 10.0 ± 0.8 in the inhibin-AS group compared with 2.4 ± 0.3 in control group. The present results demonstrate that immunoneutralization of endogenous inhibin increased FSH secretions in miniature shiba goats. The increased FSH secretion enhanced follicular growth and increased the ovulation rate. Additionally, increasing the interval between inhibin-AS and PGF2α injections (to 72 h) resulted in a greater ovulation rate compared with the previous protocol (48 h). Therefore, inhibin-AS treatment proved to be an effective alternative to exogenous gonadotropin methods for induction of superovulation in goats.
To improve the efficiency of transgenesis, we investigated the effects of a radical scavenger during microinjection on the development to blastocysts or pups of mouse pronuclear embryos, microinjected with the enhanced green fluorescent protein (EGFP) transgene. When embryos were microinjected in medium containing 0-1,000 units/ml catalase, the developmental rate to blastocysts was significantly higher (P<0.01) in 100-units/ml catalase (81%) than those in 0 and 1,000 units/ml (56 and 65%). To investigate the ontogenetic ability of DNA-injected embryos, EGFP-injected embryos manipulated under 0 or 100 units/ml catalase were transferred separately to recipient mice. The proportion of fetuses derived from EGFP-injected embryos manipulated under 100 units/ml catalase (29%) was significantly higher (P<0.05) than that manipulated under 0 units/ml catalase (19%). Furthermore, the numbers of transgenic pups were 17 in 100 units/ml catalase and 14 in 0 units/ml catalase. The results of the present study indicate that scavenging reactive oxygen species during in vitro micromanipulation is beneficial for the development of DNA-injected embryos.
The objective of this study was to generate antisera against recombinant bovine leptin and synthetic oligopeptides corresponding to the amino acid sequence 21-40 and 91-110 of bovine leptin. Recombinant bovine leptin was raised in the 293 cells and purified from 10 L of conditioned medium and utilized for immunization. The synthetic peptides were conjugated with keyhole limpet hemocyanin and inoculated into rabbits for antibody generation. Antibody titer was monitored by enzymeimmunoassay, immunoblotting and sandwich binding assay techniques. Each of the antisera, against three different antigens, was found to react with bovine leptin. The titers of anti-peptide antisera were lower than that of anti-recombinant leptin antiserum. Since anti-recombinant leptin antiserum was not neutralized by the leptin peptides 21-40 and 91-110, it is suggested that each antiserum recognizes a distinct epitope. In immunoblot analyses, all antisera exhibited cross-reactivity with human and mouse leptins. However, in the sandwich binding assay, the combination of anti-peptide antisera and anti-recombinant leptin antiserum, originated from bovine leptin, did not cross-react with either human or mouse leptin. The discrepancy of antigenic recognition between the immunoblot analyses and sandwich assay is thought to be dependent on the conformational status of leptin molecules between the species. The antisera generated in this study, which recognized distinct epitopes of bovine leptin, will provide a useful tool for studies of bovine leptin functions.