Implantation, a critical step for mammals in establishing pregnancy, requires successful completion of sequential events such as maternal uterine development, conceptus development and attachment, and placental formation. To reach the stage of placental formation, synchronized development of the conceptus and uterus throughout the implantation period is absolutely required. A number of factors expressed at the uterine endometrium and/or conceptus, which are associated with peri-implantation development, have been identified. In addition to a temporal and spatial expression of these factors, their roles in intra- and inter-cellular interactions make it difficult to fully understand physiological roles played during the critical period. This paper focuses on early conceptus development, maternal preparation for implantation and uterine-conceptus communication during the pre-implantation period, rather than the subsequent events such as conceptus attachment to the maternal endometrium. New aspects of pre-implantation processes are evaluated through simultaneous expressions of transcription factors as they possibly regulate the complex processes of implantation events in murine species and ruminant ungulates.

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Developmental potential of bovine embryos that are not artificially activated after intracytoplasmic sperm injection (ICSI) is generally very low. In this study, we investigated effects of artificial activation with ethanol on kinetics of maturation promoting factor (MPF) activity (p34^cdc2 kinase activity) and development of bovine oocytes following ICSI. Treatment of oocytes with ethanol at 4 h after ICSI improved their first cleavage and further preimplantation development (51% vs. 13%, 14% vs. 4%: treatment with vs. without ethanol, respectively). MPF activity of oocytes was lowered until at least 2 h after ICSI. In oocytes without activation after ICSI, MPF activity temporarily elevated at 6 h after ICSI, whereas this phenomena was not observed in the oocytes treated with ethanol. Furthermore, MPF activity was elevated 20 h after ICSI in oocytes activated with ethanol, whereas this elevation of MPF activity was not shown in oocytes without activation. These results indicate that the stimulus of sperm was sufficient to lower MPF activity of oocytes following ICSI, and moreover the activation treatment of bovine oocytes with ethanol after ICSI served to maintain the low levels of MPF activity until the next cell cycle started.

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Most studies of oocyte apoptosis have been performed in vitro and have employed the method of artificial induction of apoptosis by an anti-cancer agent. However, the process of oocyte death in vivo has not been clearly identified. To investigate the death process in unfertilized oocytes in vivo, we examined the cytochemical change of oocytes collected by oviduct flushing at various intervals after hCG injection. At each collection time, the collected oocytes were phenotypically classified under the microscope into four groups: single-cell oocytes (non-activated and without a nucleus and cytokinesis), activated oocytes (single-, 2- or 4-cell with a nucleus), fragmented oocytes, and dead oocytes. The number of single oocytes decreased and dead oocytes increased with the lapse of time, but the number of activated oocytes or fragmented oocytes did not. Also, most of the dead oocytes observed were single cell. At each time point, single oocytes were stained with anti-tubulin antibody to examine their spindle status. At 24 h after hCG injection, all ovulated oocytes had a normal bipolar spindle, while at 64 h all single-cell oocytes had no spindle. From these observations, we concluded that most oocyte deaths in vivo occur in the single oocyte stage, not in activated or fragmented oocytes.

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Stress interferes with reproduction, adversely influencing implantation and fetal growth, and sometimes even leading to abortion. Here, we attempted to evaluate the early gestational effects of uncomfortable sound on pregnant mice and their offspring. Ten-week-old pregnant Jcl:ICR mice were exposed to sound (100 dB, random frequency between 9-34 kHz) for 8 hours on the 3^rd, 5^th and 7^th gestational days (GD). The effects of general anesthesia were also investigated, with or without acoustic stress. All groups were examined on the 18^th GD for fetal growth. Fetal weight, number of ossified sacrococcygeal vertebrae and placental weight were all significantly reduced (P<0.0001) when stress was induced on the 7^th GD, but not on the 3^rd or 5^th GD. This intra-uterine growth retardation (IUGR) was significantly inhibited by general anesthesia (P<0.0001), although general anesthesia alone induced significant IUGR (P<0.0001) when compared with control mice. This suggests that acoustic exposure indirectly exerts an effect on fetal growth, possibly via a psycho-maternal pathway. We also found that analysis of the number of ossified sacrococcygeal vertebrae is the most sensitive tool for the study of IUGR.

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The present study was conducted to examine the developmental potential to offspring of rat embryos cultured from 1-cell to morula/blastocyst stage. Pronuclear zygotes from Wistar × Wistar or (SD × DA) × Wistar strains were cultured in modified rat 1-cell embryo culture medium (mR1ECM) for 96 h in 5% CO₂ in air at 37 C. The proportion of the 3-way cross hybrid zygotes developing into morula/blastocyst stage (74%) was higher than that of the Wistar zygotes (66%). Day-5 morulae/blastocysts developed in vitro were transferred into Day-3 or -4 pseudopregnant recipients of Wistar or SD × DA strain. The transfer of cultured embryos resulted in the birth of offspring at 13-59%, while that of non-cultured control blastocysts showed birth rates of 35-65%. The best offspring rate of cultured embryos (59%) was obtained when the hybrid 1-cell zygotes were cultured in mR1ECM medium and transferred into the 2-days earlier uteri of SD × DA recipients. These results suggest that genetic background of recipients as well as donors is a possible factor affecting full-term development of rat morulae/blastocysts derived from 1-cell stage zygotes cultured in vitro.

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It has been shown in various species that the onset of puberty is closely associated with body growth and nutritional state rather than age. The present study was conducted to determine the timing of puberty and to clarify body growth and metabolic changes around the pubertal period in female Shiba goats. Blood samples were collected between 10 to 38 weeks of age from 12 female goats, and plasma concentrations of progesterone, metabolites (glucose, nonesterified fatty acid, ketone body and acetic acid) and metabolic hormones (insulin and insulin-like growth factor-I (IGF-I)) were analyzed. Physical parameters (body weight, withers height and body length) were also measured at the blood sampling. The week when plasma progesterone concentrations first exceeded 1.0 ng/ml was designated as the onset of puberty. The results showed that the average age of the onset of puberty was 27.0 ± 0.9 (mean ± SEM) weeks in female Shiba goats. When the goats reached puberty, the average values of body weight and goat body mass index ((body weight (kg)/withers height (cm)/body length (cm)) × 10³) were 12.2 ± 0.5 kg and 5.7 ± 0.2, respectively. No particular change associated with puberty was apparent for plasma concentrations of the metabolites examined. Plasma insulin concentrations were maintained at lower levels until the onset of puberty, and then they began to gradually increase. Plasma IGF-I concentrations began to gradually increase 1 to 4 weeks before the onset of puberty and this increase continued throughout the peripubertal period. These results imply that IGF-I acts as a peripheral nutritional signal to trigger the onset of puberty in Shiba goats.

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We have previously identified a testis-specific poly(A) polymerase, TPAP (PAPβ), involved in poly(A) tail extension of specific mRNAs in the cytoplasm of round spermatids. Targeted disruption of the mouse TPAP gene resulted in the arrest of spermiogenesis due to reduced expression of haploid-specific genes required for morphogenesis of germ cells. To further elucidate the role(s) of TPAP in spermatogenesis, transgenic mice expressing an exogenous TPAP transgene on wild-type and TPAP-deficient backgrounds were generated and characterized. The transgenic mice overexpressing TPAP exhibited normal spermatogenesis and fertility. The sizes of some transcription factor mRNAs as the substrates of TPAP were also unaffected. Transgenic expression of the TPAP gene in the TPAP-deficient mice complemented both the incomplete elongation of the poly(A) tails of specific transcription factor mRNAs, and reduced expression of haploid-specific genes, resulting in the resumption of normal spermiogenesis. These data conclusively show that spermatogenesis requires the cytoplasmic elongation of the mRNA poly(A) tails catalyzed by TPAP, and imply the presence of a regulatory mechanism(s) defining the extent of the cytoplasmic mRNA polyadenylation.

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The present study was undertaken to investigate whether withdrawal of estrogen and progesterone (EP-withdrawal) stimulates prostaglandin F2α (PGF2α) production through oxygen radical (ROS)-induced NF-κB activation in human endometrial stromal cells (ESC). To study the EP-withdrawal, ESC that had been treated with estradiol (E, 10^-8 M) and medroxyprogesterone acetate (MPA, 10^-6 M) for 12 days were then incubated with or without E+MPA for a further 11 days. PGF2α concentrations in the medium and cyclooxygenase-2 (COX-2) mRNA levels were significantly increased after EP-withdrawal, while they were unchanged by the continuous treatment with E+MPA. When ESC were incubated with N-acetyl-L-cysteine (Nac, 50 mM), an antioxidant, during EP-withdrawal, Nac blocked the increases in PGF2α production and COX-2 mRNA expression caused by EP-withdrawal. Next, we examined whether ROS generated in response to EP-withdrawal acted through NF-κB activation. Electrophoretic mobility shift assay revealed that EP-withdrawal caused marked increases in NF-κB DNA binding activity, which was completely suppressed by Nac. Furthermore, when ESC were incubated with MG132 (3 μM), which inhibits NF-κB activation, during EP-withdrawal, MG132 blocked the increases in PGF2α production and COX-2 mRNA expression caused by EP-withdrawal. In conclusion, EP-withdrawal stimulates COX-2 expression and PGF2α production through ROS-induced NF-κB activation, suggesting a possible mechanism for menstruation.

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Sry, Sox9 and M33 are thought to act as architectural transcription factors or as a chromatin regulator in gonadal development. However, the direct relationship between chromatin structure and sex determination has not yet been revealed. To clarify the effect of chromatin structural change on gonadal development, we examined the effects of trichostatin A, a histone deacetylase inhibitor, on mouse gonadal development in vitro. In the 0.1 μM treated testicular explants, the size of the gonad was significantly decreased, although the testicular cord formation occurred normally. In the 1.0 μM treated explants, the gonads revealed one or two large testicular cords. Sox9 and MIS expressions suggest that Sertoli cell differentiation is induced normally within the testicular cord, while Dnmt3b expression suggests that several immature Sertoli cells are located on the outside of the testicular cord. The 3β-hsd expression indicates that Leydig cell differentiation occurs normally. On the other hand, germ cell loss was observed in the treated testicular explants. In the treated ovarian explants, the number of premeiotic germ cells was reduced without gonadal size change. Thus, trichostatin A affects the development of germ cells, but does not affect sex determination.

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Pigs serve as a better research model for human beings than other species. The Chinese laboratory miniature pig is a new laboratory animal and is expected to be applicable in many medical research fields. This study was to establish effective technologies to isolate and culture ES cells in Chinese miniature pigs. For isolation of the inner cell mass from blastocysts, an enzyme-digestive method was compared with the traditional immunosurgery. Isolated ICM were cultured in three feeder cell layers: mouse embryonic fibroblasts (MEF), porcine embryonic fibroblasts (PEF) and a continuous cell line of mouse embryonic fibroblasts (STO). Microtubule activity of the three feeder cells was further examined by immunofluorescence. ICM were successfully isolated from 85% of blastocysts by the enzyme-digestive method, compared to only 40% by immunosurgery. When ICM were cultured in three feeder layers for two to three days, 75%, 65% and 20% of ICMs formed primary cell colonies in MEF, PEF and STO, respectively. Colonies were also formed during subcultures after 9, 5 and 1 passage in MEF, PEF and STO, respectively. Microtubules in STO cells were significantly fewer than those in MEF and PEF. When the ES-like cells were cultured in a differentiation medium, they differentiated to neuron-like cells and other types of cells. These results indicate that healthier ICM can be obtained with the enzyme-digestive method. Successful culture of ICM to ES-like cells has been achieved not only in MEF, but also in homologous (pig) feeder layer. The ES cells obtained in the present study were pluripotent.

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Pyrethroid insecticides are among the most commonly used classes of insecticides worldwide, but their endocrine disrupting activities remain unclear. Therefore, in the present study, we examined the estrogenic activities of pyrethroid insecticides in E-screen and competition binding assays. In addition, we measured estrogen receptor (ER) protein and pS2 mRNA levels in human breast cancer cells (MCF-7 BUS) to clarify the mechanism of their estrogenicity. Seven pyrethroid insecticides (bioallethrine, cypermethrin, deltamethrin, fenvalerate, permethrin, sumithrin, and tetramethrin) were tested because of their worldwide usage. In addition, 17β-estradiol was tested as a positive control. As expected, 17β-estradiol significantly increased MCF-7 BUS cell proliferation at concentrations of 10^-11 M and above. Of the pyrethroid insecticides tested, only sumithrin increased MCF-7 BUS cell proliferation in a dose-dependent manner; the maximum induction of cell proliferation was observed at a dose of 10^-5 M. In the anti-estrogenic activity test, bioallethrin, fenvalerate, and permethrin significantly inhibited 17β-estradiol-induced MCF-7 BUS cell proliferation at 10^-6 M, a concentration comparable to the effective dose (10^-9 M) of ICI 182,780, a pure ER antagonist. However, none of the pyrethroid insecticides competitively inhibited the binding of [³H]estradiol to rat uterus ERs in competition binding assays. Both 17β-estradiol (10^-10 M) and sumithrin (10^-5 M) decreased the levels of cytosolic ERα and ERβ protein expression significantly as compared with the vehicle control. In addition, 17β-estradiol (10^-10 M) increased pS2 mRNA expression markedly, and sumithrin significantly increased pS2 mRNA levels in a dose-dependent manner. The other six compounds tested in the present study did not affect ER protein levels or pS2 mRNA levels. These results suggest that certain pyrethroid insecticides may be considered to be estrogen-like chemicals that act through pathways other than direct ER binding, and may function as endocrine modulators in both wildlife and humans.

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Luteinizing hormone (LH)-stimulated steroidogenesis in luteal cells is known to be mediated through the activation of cyclic AMP (cAMP)-dependent protein kinase, and to be also modulated by calcium-dependent mechanisms. In the present study, we tested the hypothesis that LH stimulates progesterone (P4) production in bovine luteal cells through activation of phospholipase (PL) C by using a cell culture system. Bovine mid-luteal cells (Days 8-12 of the estrous cycle) were cultured for 24 h and then exposed to a PLC inhibitor (U-73122; 10 μM) with or without LH (10 ng/ml) for 4 h. U-73122 blocked LH-stimulated P4 production without affecting cAMP accumulation. Moreover, exposure of luteal cells to PLC increased P4 production in a dose-dependent manner. These results support the hypothesis that the luteotropic action of LH in bovine luteal cells is mediated not only by activation of adenylate cyclase but also by activation of PLC.

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Enucleated oocytes receiving mouse embryonic stem (ES) cells develop into fertile young. The developmental potential to young is low, however, and the rate of postnatal death is high. We examined the effect of various nuclear transfer procedures on the in vitro and in vivo developmental potential of nuclear-transferred oocytes. The potential of oocytes receiving ES cells at M phase to develop into blastocysts after fusion by Sendai virus was high compared with that after direct injection (67% vs. 30%). The developmental potential of oocytes receiving ES cells at the M phase is higher than that of oocytes receiving ES cells at the G₁ phase (30-67% vs. 2-5%). Developmental ability to live young was low in all groups (0-4%). Different activation protocols affected the potential to develop into blastocysts to a different extent (27-62%), but did not affect the potential to develop into live young (0-3%). The present study demonstrated that the various conditions examined did not affect the potential of nuclear-transferred oocytes receiving ES cells to develop into live young or the incidence of postnatal death.

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