The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.

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Sulfate is important for growth and development, and is supplied from mother to fetus throughout pregnancy. We used NaS1 sulfate transporter null (Nas1^-/-) mice to investigate the role of NaS1 in maintaining sulfate homeostasis during pregnancy and to determine the physiological consequences of maternal hyposulfataemia on fetal, placental and postnatal growth. We show that maternal serum (≤0.5 mM), fetal serum (<0.1 mM) and amniotic fluid (≤0.5 mM) sulfate levels were significantly lower in pregnant Nas1^-/- mice when compared with maternal serum (≈2.0 mM), fetal serum (≈1.5 mM) and amniotic fluid (≈1.7 mM) sulfate levels in pregnant Nas1^+/+ mice. After 12 days of pregnancy, fetal reabsorptions led to markedly reduced (by ≥50%) fetal numbers in Nas1^-/- mice. Placental labyrinth and spongiotrophoblast layers were increased (by ≈140%) in pregnant Nas1^-/- mice when compared to pregnant Nas1^+/+ mice. Birth weights of progeny from female Nas1^-/- mice were increased (by ≈7%) when compared to progeny of Nas1^+/+ mice. These findings show that NaS1 is essential to maintain high maternal and fetal sulfate levels, which is important for maintaining pregnancy, placental development and normal birth weight.

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In the present study, we investigated the relationship between the temperature-humidity index (THI) and the conception rate of lactating dairy cows in southwestern Japan, one of the hottest areas of the country. We also investigated the relationship between measurement of the vaginal temperature of lactating dairy cows as their core body temperature at one-hour intervals for 25 consecutive days in hot (August-September, n=6) and cool (January-February, n=5) periods and their THI. Furthermore, we discussed the above relationship using these vaginal temperatures, the conception rates and the THI. As a result, when the conception rates from day 2 to 0 before AI were classified into day 2, 1 and 0 groups by the six maximum THI values in each group (mTHI; <61, 61-65, 66-70, 71-75, 76-80, >80), only the conception rate for the mTHI over 80 at 1 day before AI group was significantly lower (P<0.05) than the other groups. The conception rate for days 15 to 17, but not days 19 to 22 and 30 to 35, after AI in the cows that experienced average mTHI over 80 (amTHI>80) was significantly lower (P<0.05) than that of the cows that did not experience amTHI>80. There was a significant positive correlation (P<0.01) between the mTHI and the mean daily vaginal temperature, but not during the cool period. When the mTHI reached 69, the vaginal temperature started to increase. As for the relationship between the conception rates and vaginal temperatures for all mTHI classes, in the mTHI>80 at 1 day before AI group, the vaginal temperature increased by 0.6 C from 38.7 C, resulting in a reduction of 11.6% in the conception rate from 40.5%. In conclusion, these results suggest that one of the causes of the fall in conception rate of lactating dairy cows during the summer season in southwestern Japan may be an increase in their core body temperature with a higher mTHI than the critical mTHI of 69 at 1 day before AI.

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In our previous study (Kawashima et al., Biol Reprod 2009; 80: 1293-1304), we suggested that the first cycle of spermatogenesis recovered from busulfan-induced temporary arrest was a good model system to analyze the proteins expressed at the specific stages of spermatogenesis in the mouse, and this has been confirmed in the present paper. Namely, six-week-old mice were injected with busulfan at 20 mg/kg body weight. The germ cells except for the undifferentiated spermatogonia disappeared by 32 days after injection. The surviving spermatogonia started to proliferate, and spermatogenesis was entirely recovered about 77 days after injection. By proteome analysis of the busulfan-treated testis during the process of recovery of spermatogenesis, we identified a protein that was expected to be expressed in the spermatogenic cells as Superkiller viralicidic activity-2-like-2 (SKIV2L2). Skiv2l2 mRNA was found in the kidney, epididymis and heart as well as the testis. In the testis, Skiv2l2 mRNA was shown to be highly expressed in the spermatocytes at stages I to VI. On the other hand, SKIV2L2 protein was found to be predominantly localized in the nuclei of round spermatids. In accordance with the fact that SKIV2L2 belongs to the Ski2 family within the superfamily 2 of RNA helicases, it has been shown that SKIV2L2 has both the RNA-binding and ATPase activities.

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The cellular localization of nerve growth factor (NGF) and its receptors (TrkA, p75) was investigated during the estrous cycle in gilts. Also, the levels of expression of these factors in walls of tertiary follicles and corpora lutea (CLs) were determined using Western blot. The ovaries from days 3, 7, 16 and 20 of the cycle revealed the presence of NGF and its receptors in oocytes of secondary and tertiary follicles, follicular cells of primary and secondary follicles, thecal and granulosa cells of tertiary follicles and steroidogenic cells of CLs. In wall cells of primary follicles, NGF, TrkA and p75 staining was strongest on day 16, while in secondary follicles, only p75 was more intensely stained on day 16 and 20. In walls of small (to 3 mm in diameter) and medium (4-6 mm in diameter) follicles, NGF staining was lower on day 16, and the p75 reaction was strongest on day 20. On day 20, NGF staining in large follicles (7-10 mm in diameter) was higher than in smaller follicles. The levels of NGF and p75 in small and medium follicles were highest on day 20. The contents of NGF and TrkA in large follicles on day 20 were higher than in smaller follicles. NGF and TrkA contents in CLs were highest on day 7. Our study demonstrates that NGF, TrkA and p75 are expressed in the ovary during the estrous cycle in gilts. These results suggest that NGF and its receptors may be important for ovarian function in cycling gilts.

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The objective of the present study was to elucidate the involvement of FOXO3 in the activation of bovine primordial follicles. In immunohistochemistry, FOXO3 was detected in all of the oocytes in primordial and primary follicles. The FOXO3 decreased after treatment with FOXO3 small interfering RNAs (siRNAs). Ovarian tissues containing dominantly primordial follicles were treated with FOXO3 siRNAs and then xenografted to severe combined immune deficiency (SCID) mice. Two months after xenografting, some primordial follicles developed to the secondary and tertiary stages, and the total percentage of these developing follicles (secondary and tertiary follicles: 18 ± 7%) was higher than in the control grafts treated with control siRNA (7 ± 1%). It is thought that bovine primordial follicle activation is regulated by the FOXO3-dependent mechanism and that knockdown of FOXO3 induces the release of primordial follicles from FOXO3 suppression, initiating their growth.

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To determine if reductions in developmental competence related to heat stress exposure were correlated with perturbations in certain RNA populations, poly(A) RNA, total RNA, RNA size distribution, and the abundance of transcripts (cyclin B1, GDF9, BMP15, poly(A) polymerase, HSP70, 18S & 28S rRNA) were examined in oocytes matured at 38.5 or 41 C. Performing in vitro fertilization resulted in embryos for examining RNA. Relative to germinal vesicle-stage oocytes, total amount of poly(A) RNA decreased similarly in oocytes matured at 38.5 or 41 C. Total RNA did not change during meiotic maturation or up through the 4 to 8-cell stage of embryonic development. Blastocyst-stage embryos had more total RNA; those originating from heat-stressed oocytes had more than those from nonheat-stressed oocytes. Oocytes and 4 to 8-cell embryos had similar RIN values and ratios for rRNA, 18S/fast region, and 18S/inter region. Values obtained for blastocyst-stage embryos were similar to those obtained for cumulus cell RNA, which did not change during maturation. Culture at 41 C for the first 12 h of meiotic maturation had no impact on RNA size distribution or transcripts examined from oocytes, surrounding cumulus or resultant 4 to 8-cell embryos. Interestingly, however, RNA from blastocysts originating from heat-stressed oocytes had lower 18S/fast region and 18S/inter region ratios compared to other developmental stages and cumulus cells. Although biological significance of these RNA changes is unclear, differences at the molecular level in embryos from heat-stressed oocytes emphasize the importance of minimizing stress exposure during meiotic maturation, if the intent is to obtain developmentally-competent embryos.

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Mammalian preimplantation embryos enter the S phase immediately after the end of the M phase; their cell cycle lacks a substantial G1 phase. Previously, we suggested that the absence of the G1 phase was attributable to a loss of retinoblastoma protein (RB) function, which is required for suppression of S phase entrance and that this loss of RB function in turn was attributable to the low RB expression level during preimplantation development in mouse embryos. The present study aimed to examine whether or not RB inhibition by CDK4/6-cyclin D-dependent phosphorylation is involved in the loss of RB function in preimplantation mouse embryos by the expression of p16^INK4a, a potent endogenous inhibitor of CDK4/6-cyclin D. First, the decrease in RB expression between the four-cell and morula stages was confirmed in in vivo-derived mouse embryos. We then examined the efficiency of the p16^INK4a expression vector in inhibiting RB phosphorylation and cell cycle progression using NIH-3T3 cells and obtained gradual RB dephosphorylation and a significantly lower proliferation rate in p16^INK4a-transfected cells than in control cells. This indicated the successful p16^INK4a effects on cell-cycle progression by the vector used. On the other hand, the development rate of mouse embryos injected with the p16^INK4a expression vector was the same as that of the control embryos, although p16^INK4a expression was detected at mRNA and protein levels in the former group but not in the control group. These results suggest that RB phosphorylation is not involved in RB dysfunction or in the lack of a G1 phase in mouse embryos and that the decrease in RB expression is important for preimplantation-embryo-specific cell-cycle regulation. Moreover, the present study indicates the similarity between preimplantation embryos and cancer cells, which p16^INK4a expression does not arrest at the G1 phase.

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Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16- to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16- to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16- to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.

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Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4¢,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.

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Numerous transcription factors that regulate trophoblast developmental processes have been identified; however, the regulation of trophoblast-specific gene expression has not been definitively characterized. While a new role of Gata3 in trophoblast development was being demonstrated in mice, we examined effects of GATA transcription factors on conceptus interferon tau (IFNT), a major trophectoderm factor in ruminants. In this study, expression patterns of trophoblast ASCL2, CDX2, CSH1, ELF5, HAND1, IFNT, and TKDP1 mRNAs were initially examined, from which ASCL2, CDX2, IFNT, and TKDP1 mRNAs were found to be similar to those of GATA2 and GATA3 in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses. A chromatin immunoprecipitation (ChIP) assay revealed that endogenous GATA2 and GATA3 occupied GATA binding sites on the upstream regions of CSH1, IFNT, and TKDP1 genes and on the intron 1 region of CDX2 gene in bovine trophoblast CT-1 cells. In transient transfection analyses of the upstream region of bovine CSH1, and IFNT or the intron 1 region of CDX2 gene, over-expression of GATA2 induced transactivation of these trophoblast-specific genes in bovine non-trophoblast ear fibroblast EF cells, but over-expression of GATA3 did not substantially affect their transactivation. In CT-1 cells, endogenous CDX2 and IFNT mRNAs were down-regulated by GATA2 siRNA, while endogenous ASCL2 and CDX2 mRNAs were down-regulated by GATA3 siRNA. Our results indicate that in addition to trophectoderm lineage specification, GATA2 and/or GATA3 are involved in the regulation of trophoblast-specific gene transcription in bovine trophoblast CT-1 cells.

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The Gsg2 (Haspin) gene encodes a serine/threonine protein kinase and is predominantly expressed in haploid germ cells. In proliferating somatic cells, Gsg2 is shown to be expressed weakly but plays an essential role in mitosis. Although the Gsg2 minimal promoter recognized by the spermatogenic cell-specific nuclear factor(s) has been found, to date, the molecular mechanism that differentially controls Gsg2 expression levels in germ and somatic cells remains to be sufficiently clarified. In this study, we analyzed the DNA methylation status of the upstream region containing the Gsg2 promoter. We found a tissue-dependent and differentially methylated region (T-DMR) upstream (-641 to -517) of the authentic promoter that is hypomethylated in germ cells but hypermethylated in other somatic tissues. Profiling of Gsg2 expression and DNA methylation status at the T-DMR in spermatogenic cells indicated that the hypomethylation of the T-DMR is maintained during spermatogenesis. Using the reporter assay, we also demonstrated that DNA methylation at the T-DMR of Gsg2 reduced the promoter activity by 60-80%, but did not fully suppress it. Therefore, the T-DMR functions as a modulator in a DNA methylation-dependent manner. In conclusion, Gsg2 is under epigenetic control.

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For fertilization using round spermatid injection (ROSI) in mice, oocytes need to be artificially preactivated because of the lack of oocyte-activating capacity in round spermatids of this species. However, when round spermatids were frozen-thawed before microinjection, 11-71% of injected oocytes developed into 2-cell embryos without any artificial activation. After being transferred into recipient females, 5-27% of these embryos reached term. At least some of the injected oocytes showed intracellular Ca²⁺ oscillations, which normally occur after fertilization by mature spermatozoa. Thus, these round spermatids could transmit a sperm-borne oocyte-activating factor, which might have been released from spermatozoa and elongated spermatids in the same suspension by freezing and thawing. This possibility was further supported by activation of intact oocytes following transplantation of the pronuclei from ROSI-generated embryos. Thus, one-step ROSI can be achieved in mice simply by injecting frozen-thawed round spermatids into intact oocytes. Clearly, there is a need for careful interpretation of microinjection experiments when assessing the oocyte-activating capacity of spermatogenic cells, especially when they are derived from frozen-thawed stocks.

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Widely used bovine sexing primers were compared in terms of suitability in determining the sex of bovine embryos. Under optimized multiplex PCR conditions, the ConBV/ConEY couple primers did not show accurate results when combined together in multiplex PCR, but worked well when the couple primers were used separately. The S4BF/S4BR primers showed accurate results; however, some unexpected bands were detected. When the BY/BSP couple primers were used to determine one-cell, two-cell, four-cell and eight-cell stage embryos of known sexed SCNT-derived embryos, the results showed 100% accuracy. The BY/BSP couple primers were also able to identify the sex of one-cell and two-cell IVF-derived embryos.

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Embryos of good, fair and poor quality were collected from superovulated cows and subjected to zona cutting (ZC) treatment using a needle under either an inverted microscope or a stereomicroscope. One (single transfer) or 2 (twin transfer) embryos with or without prior ZC treatment were transferred nonsurgically to recipients. Without the ZC treatment, lower embryonic quality resulted in lower pregnancy success rates. However, the ZC treatment increased the pregnancy success rate following transfer of poor-quality embryos, but not the pregnancy rate after transfer of good- or fair-quality embryos. No differences were observed between the pregnancy success rates after the transfer of embryos treated under the inverted microscope and those after transfer of embryos treated under the stereomicroscope, and this was the same after single and twin transfer. Moreover, ZC treatment of embryos prior to transfer did not result in an increased abortion rate, irrespective of the number of transferred embryos. In conclusion, ZC treatment improves pregnancy success rates following transfer of poor-quality embryos. Moreover, the results indicate that ZC treatment by using a stereomicroscope is practical for on-farm application.

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The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZEN-containing (0-1000 μg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 μg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 μg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 μg/l may not affect the fertility of the oocytes.

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Elevated CO₂ is required for in vitro embryo culture to maintain proper media pH and to supply embryo metabolic pathways. As an alternative to current approaches using gas cylinders, we examined use of a chemical reaction to supply CO₂. A closed culture system was constructed and chemicals added to generate CO₂, which was then supplied to developing embryos. This system was shown to provide a stable pH (7.2-7.4) over 4 days of use. One-cell mouse embryos were cultured in the device and no difference in blastocyst formation or cell number was apparent between embryos grown in a closed system with CO₂ supplied by a chemical reaction or positive controls grown in a an open system in a CO₂ incubator. This approach provides a highly purified, inexpensive, and easily obtainable gas source and offers potential for development of new, self-contained culture platforms.

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