Journal of Reproduction and Development最新文献

For the intracytoplasmic sperm injection (ICSI) procedure in pigs, an electrical pulse (EP) has been used as an effective method for oocyte stimulation, but unlike sperm, EP is unable to induce Ca²⁺ oscillations. In this study, we investigated the effects of generating artificial Ca²⁺ oscillations with phospholipase Cζ (PLCζ) mRNA, a candidate sperm factor, on fertilization, embryonic development, and gene expression after ICSI. Firstly, the concentration of PLCζ mRNA of a fixed volume (1.0 pl) that would induce a pattern of Ca²⁺ rise similar to that of in vitro fertilized (IVF) sperm was examined and determined to be 300 ng/μl. Secondly, the effects of oocyte stimulation methods on fertilization and embryonic development were investigated. ICSI-oocytes were activated by EP (EP group) or by PLCζ mRNA (PLCζ group). Furthermore, IVF-oocytes (IVF group) and ICSI-oocytes with and without an injection of buffer (buffer and untreated groups, respectively) were used as controls. It was found that the rates of normal fertilization in the PLCζ and EP groups were significantly higher than those in the buffer and untreated groups. The blastocyst formation rates did not differ among the groups. The embryo quality in the EP group was inferior to those in the PLCζ and IVF groups. Additionally, the expression level of a proapoptosis-related gene (Caspase-3) in the EP group was significantly higher than those in the PLCζ and IVF groups. Our data suggest that oocyte activation by PLCζ mRNA has the effect of improving embryo quality.

Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda epididymal and testicular sperm in Wistar (WI) and Brown-Norway (BN) rats. The transfer of ICSI oocytes with cryopreserved epididymal and testicular WI sperm resulted in offspring production of 26.2% and 3.7%-4.7%, respectively (P < 0.05). Treatments for artificial oocyte activation (AOA) and acrosome removal improved pronuclear formation in BN-ICSI oocytes; however, only AOA treatment was effective in producing offspring (3.7%-6.5%). In the case of ICSI with testicular sperm (TESE-ICSI), one offspring (0.6%) was derived from the BN-TESE-ICSI oocytes. The application of AOA or a hypo-osmotic sperm suspension did not improve the production of TESE-ICSI offspring. Thus, outbred WI rat offspring can be produced by using ICSI and less efficiently by using TESE-ICSI. Challenges in producing offspring by using ICSI/TESE-ICSI in inbred BN strain require further investigation.

This study aimed to develop a method to evaluate the quality of bovine in vitro fertilized (IVF) embryos based on gene expression profiling via whole-transcriptome amplification. The expression of 11 developmentally important genes in individual bovine in vivo-derived (IVD) and IVF embryos were examined. Gene expression profiling was conducted by classifying the expression level of each gene in individual embryos as low, medium, or high. The IVF group had a higher (P < 0.01) proportion of embryos with low expression of SOX2, NANOG, and FGF4. In addition, a correlation analysis between the expression levels of each gene in individual embryos demonstrated that the relationship between gene expression differed with respect to IVD and IVF embryos. Our results suggest that the expression profiling of developmentally important genes using IVD embryos as normal controls could be a useful indicator for evaluating the quality of bovine IVF embryos.

The present study examined whether male resveratrol intake affected mitochondrial DNA copy number (mt-cn) and telomere length (TL) in blastocysts fathered by young and aged male mice. C57BL/6N male mice supplied with water or water containing 0.1 mM resveratrol were used for embryo production at 14-23 and 48-58 weeks of age. Two-cell-stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 3 days until the blastocyst stage. Mt-cn and TL levels were measured by real-time polymerase chain reaction. Resveratrol intake did not affect body weight or water consumption. Resveratrol intake increased the expression levels of SIRT1 in the liver, the antioxidative ability of serum, and extended TL in the heart, whereas there was no significant difference in mt-cn in the heart or TL in sperm. The rate of blastocyst development was significantly lower in aged male mice than in younger mice, and resveratrol intake increased the total number of blastocysts derived from both young and aged males. Resveratrol intake did not affect mt-cn or TL in blastomeres of blastocyst-stage embryos derived from young mice, but significantly increased both mt-cn and TL in blastomeres of blastocysts derived from aged fathers. In conclusion, resveratrol intake increased mt-cn and TL levels in blastocysts derived from aged male mice.

Zinc is an essential trace element for various physiological functions, including reproduction. The influx/efflux of zinc ions is regulated by zinc transporters (Zip1–14 and ZnT1–8, 10). However, the precise roles of zinc transporters and zinc dynamics in reproductive functions are unknown. In this study, ZnT3/Slc30a3 gene knockout (KO) mice were used to analyze the role of ZnT3. In ZnT3 KO mice, intracellular zinc ions in oocytes/zygotes were significantly reduced compared to those in controls, and free zinc ions did not accumulate in the oocyte cytoplasm. However, fertilization of these oocytes and the average litter size were comparable to those of control mice. Our results suggest that ZnT3 plays an important role in the accumulation of zinc ions in oocytes but not in the developmental ability of mice. ZnT3 KO mice will be useful for examining zinc dynamics in oocytes and other tissues.

Gellan gum (GG) is a soft, tractable,　and natural polysaccharide substrate used for cell incubation. In this study, we examined the effects of GG on porcine oocyte maturation. Cumulus cells and oocyte complexes (COCs) were collected from slaughterhouse-derived porcine ovaries and cultured on plastic plates containing 0.05% or 0.1% GG gels. The 0.1% GG gel improved the maturation rate and quality of blastocysts, as determined by the total cell number and the rate of abnormally condensed nuclei. GG gels have antioxidant abilities and oocytes cultured on GG gels (0.05% and 0.1%) have reduced reactive oxygen species (ROS) content. Furthermore, GG gels (0.05% and 0.1%) increased F-actin formation, whereas treatment of oocytes with H₂O₂ reduced F-actin levels. GG gels increased the ATP content in oocytes but did not affect the mitochondrial DNA copy number or mitochondrial membrane potential. In addition, the medium cultured on 0.05% GG increased the glucose consumption of COCs. In conclusion, GG gel reduced ROS content, increased energy content, and improved subsequent embryonic development in pigs.

Cryopreservation adversely affects embryo quality and viability in vitro.We investigated the effects of cryopreservation solutions supplemented with the antioxidant carnosine on frozen-thawed bovine embryo viability. Bovine blastocysts were produced in vitro and cryopreserved using slow freezing. The rates of re-expanded and hatched blastocysts in the 50 μg/ml carnosine-supplemented group at 4, 24, and 48 h after thawing were higher than those in the control (P< 0.05) group. In frozen-thawed embryos, cryopreservation solution supplemented with carnosine (50 μg/ml) significantly reduced reactive oxygen species (ROS) production(P < 0.05), decreased TUNEL-positive apoptotic cells (P< 0.05), and increased the mRNA expression of BCL2 (P< 0.05), an apoptosis suppressor gene. The expression of translocase of outer mitochondrial membrane 20 (TOMM20), which is involved in protein mitochondrial transport, in the carnosine (50 μg/ml)-treated embryos was significantly higher than that in the control group (P < 0.05). ATP production in frozen-thawed embryos in the 50 μg/ml carnosine-supplemented group was significantly higher than that in the control group (P< 0.05), however no significant difference in the total number of cells per embryo among the groups was observed. These results suggest that supplementing the cryopreservation solution with carnosine can improve the viability of frozen-thawed bovine embryos by reducing oxidative damage.

The number of cows in estrus often influences estrus behavior; however, the effects of social order are not well documented. This study examined the effects of social order on the expression of behaviorally-scored and pedometer-detected estrus, combined with the effects of the number of cows in estrus. In a herd comprising 13 or 15 beef cattle, cows with orders 1st-7th were defined as dominant and the remaining cows as subordinate. Sole or simultaneous estrus was induced by prostaglandin F_2α analog injection and/or intravaginal progesterone treatment. Ovulation timing was determined using ultrasonography at 6-hour intervals. Estrous signs and steps of the cows were recorded 49 h before ovulation using video monitoring and a pedometer, respectively. Among the 59 treated cows, 56 behaviorally-scored estruses (27 sole and 29 simultaneous) were detected. In the sole estrus, 61.5% of the dominant-rank cows had no zero-point period; however, 35.7% of the subordinate-rank cows had that period. The dominant-rank cows in estrus alone had a significantly shorter duration of scored estrus than those in simultaneous estrus (P < 0.05). Among the 50 pedometer-detected estruses (24 sole and 26 simultaneous), the subordinate-rank cows in sole estrus had a shorter interval from estrus onset to ovulation than the dominant-rank cows in simultaneous estrus (P < 0.05). The effects of social order varied in response to the number of cows in estrus, which might have influenced determining the optimal time for artificial insemination.

Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.

Mammalian reproduction is more inefficient than expected and embryo/conceptus implantation into the maternal endometrium is considered to be a rate-limiting process. Although extensive physiological and structural diversity exists among mammalian species, the basic molecular mechanisms underlying successful implantation are conserved. The extensive use of genetically engineered mouse models has provided considerable information on uterine receptivity for embryo implantation. The molecular mechanisms and cellular processes identified thus far require further validation in other mammalian species. In this review, representative ovarian steroid hormone-induced signaling pathways controlling uterine adaptation are presented based on the results of rodent studies. Selected examples of functional conservation in mammals, such as humans and cattle, are briefly described. To date, molecular therapeutic trials for fertility improvement have not been conducted. Considerable efforts are required to provide further understanding of these molecular mechanisms. Such understanding will contribute to the development of reliable clinical diagnostics and therapeutics for implantation failure, leading to reproductive success in a wide variety of mammals in the future.