To date, a practical method for inducing superovulation in the Mongolian gerbil has not been defined; therefore, in this study, we attempted to develop a flexible superovulation protocol for this species. Superovulation can be induced in the Mongolian gerbils by using PMSG with or without hCG. The injection schedule for PMSG (and hCG) has a high degree of flexibility, but the best protocol for embryo collection for reproductive biology is 20 IU PMSG followed by 20 IU hCG 54 hours later.
In porcine growing oocytes, chromatin remains diffuse. As the oocytes approach their final size, 120 μm, the chromatin becomes partly condensed and forms a perinucleolar sheath. These changes occur simultaneously with a decrease in transcriptional activity. In many other cell types, it has been shown that the state of acetylation of nucleosome core histones is essential in chromatin remodeling and transcription so that partial chromatin condensation in oocytes may involve the recruitment of histone deacetylases. In order to test this hypothesis, porcine oocyte-cumulus cell complexes were treated with a specific inhibitor of histone deacetylases, trichostatin A (TSA). The perinucleolar sheath loosened or disappeared after 24 hr culture with 100 nM TSA, but after further culture in TSA-free medium, about 40% developed the perinucleolar sheath again. In the presence of 4 mM hypoxanthine, the decondensation induced by TSA progressed rather slowly, but continuously, for 72 hr. When oocytes were denuded before culture, spontaneous maturation occurred in the presence of TSA. Thus, the inhibitor-induced decondensation is not attributed to the inhibition of the maturation-promoting factor. These results suggest that deacetylation of histones may be involved in chromatin remodeling in oocytes near the end of the growth phase.
Vitrification has been focused on as a promising approach for human blastocyst cryopreservation, but few reports are available on the effect of assisted hatching (AH) in conjunction with human vitrified blastocyst transfers. Therefore, in this study, AH with acidic Tyrode was performed at the time of warming of vitrified blastocysts before transfer in order to improve the implantation and pregnancy rates. In the AH group, 13 clinical pregnancies (54.2%) and 15 implantations (36.6%) out of 41 blastocysts transferred were obtained. In the non-AH group, 1 clinical pregnancy (12.5%) and 1 implantation (6.7%) out of 15 blastocysts transferred were obtained. AH on the vitrified blastocysts after warming improved the implantation rate significantly (P<0.03). The pregnancy rate was also increased statistically in the AH group (P<0.05). The results suggest that the vitrification procedure may cause hardening of the zona pellucida and AH of vitrified blastocysts would be useful for clinical application.
In order to develop an in-vitro assay system for detection of cytogenetic toxicity of chemicals, we cultured mouse oocytes in vitro with two kinds of spindle poisons, carbendazim (MBC) and griseofulvin (GF). When cultured for 15 h with MBC (6 μg/ml), the majority of the oocytes arrested maturation at the metaphase in the first meiosis. This effect of MBC could be achieved with the latter half of the exposure during 15 h for the entire culture. In contrast, a significant proportion of the oocytes cultured with GF (10 μg/ml) could not continue meiosis from the germinal vesicle stage. Therefore, we characterized the difference in the effects of MBC and GF on meiotic progression of mouse oocytes by using this in-vitro assay system, demonstrating that the system would be useful for detection of cytogenetic toxicity of chemicals.
The proliferation and differentiation of most cells are regulated by cytokine signaling, but the mechanism that regulates pre-implantation development remains unclear. Recently, it has been shown that Jak2, which mediates various cytokine signaling pathways, is expressed in pre-implantation mouse embryos. In this study, we investigated the expression of the cytokine receptors that activate Jak2, i.e., the receptors for prolactin (PrlR), growth hormone (GHR), tumor necrosis factor (TNFR), interleukin-3 (IL-3R), interleukin-5 (IL-5R), and granulocyte-macrophage colony stimulating factor (GM-CSFR). RT-PCR analysis revealed that PrlR was expressed in MII stage oocytes at a relatively high level, and that the level of expression decreased between the 2-cell and 4-cell stages. The expression levels of GHR, TNFR, IL-3R, IL-5R and GM-CSFR were relatively low before the morula stage, but they increased thereafter until the hatched blastocyst stage. These results suggest that various cytokine signaling pathways mediated by Jak2 activation are involved in the regulation of pre-implantation development.
This work was undertaken to investigate the activation of pig oocytes after microinjection of crude sperm extract prepared from ejaculated boar spermatozoa. The results were compared with those of electro-stimulated oocytes. When in vitro-matured pig oocytes were microinjected with sperm extract and others were electro-stimulated, 100% and 92%, respectively, of the oocytes were released from arrest at metaphase II (MII) and formed female pronuclei. To test their developmental ability, the injected oocytes were treated with cytochalasin B and then cultured in NCSU23 medium. After 168 h, 30% and 44% of the oocytes that had been microinjected with sperm extract and electro-stimulated, respectively, developed to the blastocyst stage. At 6-8 h after the microinjection of sperm extract or electro-stimulation, cortical granules were released from the oocytes. In addition, Cdc2 kinase activity declined to a low level in the treated oocytes. These results indicate that microinjection of crude sperm extract induces the release of in vitro-matured pig oocytes from MII-arrest and leads them into a series of events related to oocyte activation.
This study aimed to investigate the effects of metals on semen profiles. The concentrations of 50 elements in seminal plasma collected from 128 infertile men were measured. Eleven (Na, Mg, P, K, Ca, Fe, Cu, Zn, Se, Rb, Sr) elements were positively detected in all samples. Another eight elements (V, Mn, Co, As, Mo, Cd, Sn, Ba) were detected in over 75% of the samples. In these 19 elements, significant correlations were observed only between copper concentration and sperm motility. The presence of cadmium and zinc in seminal plasma was associated with a low total sperm number (p=0.067) and low sperm motility (p=0.052), respectively. Higher concentrations of cadmium were observed in the Brinkmann index under 100 than in that over 100 (p=0.055). Recovery of sperm motility after EDTA treatment was observed with in vitro exposure to 300 μg/ml of zinc sulfate. Declines in sperm motility after exposure to 50 μg/ml of copper sulfate were irreversible, even with EDTA treatment. It was suggested that excess copper and zinc in seminal plasma was detrimental for male reproductive capacity by reducing sperm motility. It also appeared that cadmium may exert toxic effects on spermatogenesis, after long-term exposure, as occurs with cigarette smoking.
The presence or absence of sperm-borne oocyte-activating factor (SOAF) in the Antarctic minke whale haploid spermatogenic cells was determined by assessing the meiosis resumption of microinseminated mouse oocytes. The relative capacity of mature spermatozoa from mouse, cattle and whale to resume the meiosis of BDF1 mouse oocytes was, respectively, 90.5, 84.6 and 76.5%, while nuclear changes in non-treated or buffer-injected oocytes did not occur after 90-min culture. In the whales, the late-stage elongating spermatids as well as the testicular spermatozoa triggered the meiosis resumption of mouse oocytes at similar rates (oocyte activation rates; 68.0 and 62.5%, respectively). The oocyte activating capacity of the early-stage elongating spermatids was significantly lower (25.0%), and the round spermatids did not activate mouse oocytes at all. This result suggests that the SOAF activity in the Antarctic minke whales is acquired during the early phase of spermiogenesis.
Xenografting of ovarian tissue into immuno-deficient mice is useful in studying the dynamics of follicular development. We have demonstrated that xenografted bovine secondary follicles develop to the antral stage in female severe combined immunodeficient (SCID) mice. We did this by examining the development of bovine secondary follicles (140-190 μm in diameter) that had been grafted into male and female SCID mice for 4 and 6 weeks. We then compared the results for the two groups. The rate of surviving follicles in the grafts was similar in male and female mice, but the survival rate of oocytes was lower in male mice in these follicles, especially the antral follicles. In addition, the basement membranes of relatively large follicles were thinner and torn in the male mice, and erythrocytes had invaded the follicular cavity. The mean diameters of surviving follicles and oocytes were significantly larger in both male and female mice than before grafting. In female mice, the diameter of antral follicles increased gradually as the grafting was prolonged, although the difference was not in significant. Surviving oocytes in the follicles increased in diameter. In contrast, development of antral follicles in male mice seemed to be accelerated, but, in contrast to female mice, the mean diameters of antral follicles and surviving oocytes showed no further increase after 4 weeks of grafting. These results suggest that bovine follicles can develop in male SCID mice, but oocyte degeneration together with the follicular degeneration occurs in large antral follicles at a higher rate in males than in the females.
To demonstrate delivery by using re-vitrified blastocysts derived from supernumerary embryos in the event of an unsuccessful pregnancy attempt. Nine early stage cleaved embryos were frozen by vitrification, and subsequently thawed. Four of the nine embryos developed to the blastocyst stage. Two of the four blastocysts were transferred, and two supernumerary good embryos remained unexpectedly, and they were refrozen by vitrification under consent. Subsequently, one of the re-vitrified embryos developed to 38 weeks and was delivered by cesarean section (a health female; 46, XX). The transfer of re-vitrified supernumerary blastocysts resulted in a successful pregnancy and delivery outcome. This study suggests that re-vitrification is one rescue procedure which allows the re-use of supernumerary embryos in patients who failed to have a pregnancy after frozen embryo transfer.