Meat pig breeds used commercially in North America lose significant numbers of genetically-normal fetuses in the peri-implantation (attachment) period and at mid-gestation (day 50 of the114 day gestation interval). Fetal demand that is in excess to the placental blood supply is thought to underlie these waves of fetal loss. In many species, the endometrium of early normal pregnancy is enriched in innate immune cells, particularly uterine natural killer (uNK) cells. In pigs, a species with epitheliochorial placentation, conceptuses mediate about a three-fold enrichment in uNK cells at attachment sites but the functions of these cells are unknown. In species with hemochorial placentation, uNK cells are highly enriched during the process of decidualization and promote endometrial angiogenesis. We have conducted molecular analyses using pure samples of endometrial lymphocytes or endothelium and trophoblast from healthy and arresting conceptus attachment sites in Yorkshire gilts immediately post-attachment [gestation day (GD) 20] and at mid pregnancy (GD50). In healthy sites, angiogenesis was more robustly promoted by lymphocytes than by trophoblasts. An early sign of impending fetal arrest was loss of vascular endothelial growth factor (VEGF) transcription from the lymphocytes and elevation in transcription of the pro-inflammatory gene Interferon (IFN)-γ. We have postulated that newly differentiated endometrial endothelial cells, not fetal trophoblasts, are damaged by the maternal withdrawal of vascular support and onset of inflammation and that this endometrial damage contributes significantly to peri-implantation fetal death.

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Expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) is mainly found in the Leydig cells from which steroid hormones are biosynthesized in the testes. To investigate whether endocrine disruptors affect the microenvironment of the testes, the mRNA expression of 3β-HSD as a molecular marker for androgen biosynthesis was analyzed in rat testes exposed to several endocrine disruptors using a reverse transcription-polymerase chain reaction technique. Testosterone [50, 200 and 1,000 μg/kg body weight (BW)], flutamide (1, 5 and 25 mg/kg BW), ketoconazole (0.2, 1, 5 and 25 mg/kg BW), diethylhexyl phthalate (10, 50 and 250 mg/kg BW), nonylphenol (10, 50, 100 and 250 mg/kg BW), octylphenol (10, 50 and 250 mg/kg BW), and diethylstilbestrol (10, 20 and 40 μg/kg BW) were orally administered to 4-week-old Sprague-Dawley rats for 3 weeks daily. Although testosterone at a low dose (50 μg/kg/day) increased the expression of 3β-HSD mRNA, it was significantly decreased in the rats treated with 200 or 1,000 μg/kg/day testosterone compared with the control group (P<0.05). Furthermore, ketoconazole, diethylhexyl phthalate, nonylphenol, octylphenol and diethylstilbestrol caused significant downregulation of 3β-HSD mRNA in the testes at all doses (P<0.05). However, flutamide remarkably increased the level of 3β-HSD mRNA in the testes (P<0.05). These results suggest that endocrine disruptors may influence androgen biosynthesis in the testes by alteration of 3β-HSD mRNA expression.

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To establish a storage system for isolated bovine luteal endothelial cells (LECs), we investigated the basal and tumor necrosis factor (TNF) α-stimulated production of endothelin-1 (ET-1) and prostaglandin (PG) F2α in unfrozen and frozen-thawed LECs until passage 10. LECs were obtained from developing corpora lutea (CL; days 5-7 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. The LECs were frozen at -80 C or further cultured and/or passaged until passage 10 in DMEM/Ham's F-12 supplemented with 10% calf serum. The hormonal productions of unfrozen and frozen/thawed LECs were compared through passages 2-10. When both the unfrozen and frozen/thawed cells reached confluence, the culture medium was replaced with fresh medium containing 0.1% bovine serum albumin (BSA), and the cells were incubated with TNFα (50 ng/ml) for 12 h. The basal productions of ET-1 and PGF2α by the unfrozen and frozen/thawed LECs were similar at passage 2. The basal production of PGF2α by LECs was not altered by passage and storage at -80 C, whereas the basal production of ET-1 decreased from passage 2 and 3 to passage 4 in the unfrozen LECs and from passage 2 to passage 3 in the frozen/thawed LECs. However, production of ET-1 by the unfrozen and frozen/thawed LECs was similar between passages 4-10 and passages 3-10, respectively. Exposure of LECs to TNFα increased (P<0.05) ET-1 and PGF2α production by the unfrozen and frozen-thawed LECs in all passages examined. Thus, LECs obtained from developing CLs and stored until passage 10 can be used for study of the physiology of LECs in vitro.

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More than 99% of follicles in mammalian ovaries undergo a degenerative process known as atresia, and only a few follicles actually ovulate during follicular growth and development. Follicular selection mostly depends on granulosa cell apoptosis, but the molecular mechanism behind the regulation of this selective atresia is still largely unknown. In the present study, to examine whether or not interleukin-6 (IL-6), a multifunctional cytokine, is involved in apoptosis during atresia in pig ovaries, the expression of IL-6 mRNA in granulosa cells was quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR). The level of mRNA decreased during atresia. Enzyme-linked immunosorbent assay (ELISA) showed that the level of IL-6 protein in follicular fluid also decreased during atresia. Moreover, recombinant human IL-6 upregulated the expression of an intracellular apoptosis inhibitor, cellular FLICE-like inhibitory protein long form (cFLIP_L), in cultured cells derived from human granulosa cells. It is possible that IL-6 is produced in the granulosa cells of healthy follicles, that it increases the cFLIP_L level, and cFLIP_L then prevents apoptotic cell death.

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The aim of the present study was to examine whether cumulus and fibroblast cell nuclear-transferred oocytes, which have high and low potential to develop into normal calves, respectively, are different in terms of in their patterns of timing of first cleavage and in their relationships between timing of first cleavage and in vitro developmental potential. The timing of first cleavage was similar in both types of nuclear-transferred and in vitro fertilized oocytes. More than 86% of the oocytes cleaved within 24 h after activation or in vitro fertilization; these oocytes contributed to more than 98% of the total number of blastocysts in all three groups. The potential of oocytes that cleaved at different intervals to develop into blastocysts differed among the groups. The developmental potential of the cumulus cell nuclear-transferred oocytes and in vitro fertilized oocytes decreased with the increase in time required for cleavage. Fibroblast cell nuclear-transferred oocytes that cleaved at 20 h, an intermediate cleaving time, had higher potential to develop into blastocysts. The results of the present study suggest that the type of donor nucleus used for nuclear transfer affects the timing of first cleavage.

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In the corpus luteum of rats and mice, 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to a biologically inactive metabolite, 20α-dihydroprogesterone (20α-OHP). The reduction of progesterone by 20α-HSD is believed to be important for functional luteolysis in these rodent species. In addition to the corpus luteum, expression of 20α-HSD has been demonstrated in tissues such as the placenta, endometrial epithelia, and fetal skin, although the roles it plays in the latter tissues remain to be determined. To determine the contribution of 20α-HSD to functional luteolysis and to the rodent reproductive system more generally, we generated a strain of mice with targeted disruption of the 20α-HSD gene. In the 20α-HSD^-/- mice we obtained, which lacked the genomic region essential for catalytic reaction, neither 20α-HSD activity in the corpus luteum nor an increase in the serum concentrations of 20α-OHP during pseudopregnancy or pregnancy was detected. The durations of the estrous cycle, pseudopregnancy, and pregnancy were significantly prolonged in the 20α-HSD^-/- mice, although the serum progesterone levels decreased to levels low enough for delivery of pups at term of pregnancy. In addition, the number of pups, especially live pups, was markedly decreased in the 20α-HSD^-/- mice. These findings suggest that the role of 20α-HSD in functional luteolysis is relatively minor but that it is involved in the survival of newborn mice.

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The effects of bisphenol A (BPA) on placentation have not been fully determined. The aim of this study was to clarify the structural changes of the placenta, abortion rate, and survival of neonates after BPA administration in mice. BPA (10 mg/kg/day) was administered to pregnant mice (BPA mice) subcutaneously from the first day of pregnancy (Day 0) to Day 7 (8 days total). The number of embryos and weights of whole uteri were measured on Days 10 and 12. Morphological changes in the placentae were examined by light microscopy on the corresponding days of pregnancy. The number of neonates was also counted. Survival rates were periodically calculated for neonates from the first day after parturition (P-Day 0) to P-Day 56. The number of embryos and weight of the uterus on Days 10 and 12 were significantly decreased by BPA injection. No notable differences were recognized between the left and right uteri. The proportion of the labyrinthine zone per whole placenta in the BPA mice became lower than that in the controls, and that of the metrial gland was higher in the BPA mice. The intervillous spaces of the placenta were narrower in the BPA mice. Degenerative changes were found in the trophoblastic giant cells and spongiotrophoblast layers of the BPA mice. The number of BPA mouse neonates was drastically decreased within 3 days after birth, and no mice survived after P-Day 56. The results suggest that BPA not only disrupts placental functions and leads to abortion through chronic stimulation of gene expression by binding to DNA but that it also affects the mortality of neonates through indirect exposure of embryos.

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Several studies have indicated that prolactin (PRL) assumes oligomeric, proteolytically cleaved, phosphorylated and glycosylated forms. Phosphorylated PRL (PPRL) is considered to be the most important posttranslationally modified form in the rat. In the present study, we examined whether or not PRL is present in the mouse pituitary gland in the phosphorylated form. Mouse pituitary PRL was digested with acid phosphatase, resolved by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue, and then immunoblotted against the anti-PRL, anti-phosphoserine and anti-phosphothreonine antibodies. We also examined whether PRL is phosphorylated by protein kinases and semi-quantified the ratios of PPRL to PRL in the pituitary gland. The results indicated that three types of PRL are present in the pituitary glands of both male and female mice. One was non-phosphorylated (isoform 1), and the other two were immunoreactive to anti-phosphoserine (isoform 2) and/or anti-phosphothreonine (isoform 3) antibodies. The ratio between isoforms 2 and 1 of the 30-day-old female mice was higher than that of the 20-day-old female mice. However, the ratios among the three isoforms in the male pituitary glands did not differ with age. The ratio of PPRL to isoform 1 was obviously reduced after ovariectomy (OVX), and it recovered with estrogen replacement. These results suggest that estrogen influences PRL phosphorylation in female mice.

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Uterine endometrial stromal cells differentiate into decidual cells during the late secretory phase of the menstrual cycle and pregnancy. However, the biochemical mechanisms of decidualization have yet to be definitively elucidated. In the present study, we transfected primary human endometrial stromal cell with a temperature-sensitive mutant of simian virus 40 large T antigen and thereby established an immortalized stromal cell line (EtsT) in order to examine the role of stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics, in stromal cell differentiation. When treated with the decidual stimulus dibutyryl-cAMP (db-cAMP) or forskolin, the fibroblastic cell-shaped EtsT cells transformed into large- and round-shaped cells and secreted large amounts of the decidual markers prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1). Analysis of the stathmin protein levels in the db-cAMP- and forskolin-treated EtsT cells revealed that the total and phosphorylated protein levels dropped as decidualization progressed. Suppression of stathmin expression by transfection with small interfering RNA (siRNA) suppressed EtsT cell proliferation. It also abolished db-cAMP-induced PRL and IGFBP-1 mRNA expression and protein secretion. Thus, stathmin expression can be considered an integral factor regulating the initial stage of the process of human endometrial stromal cell differentiation.

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Antiandrogens can cause reproductive disorders in humans and wild animals. In the present study, we tested whether the fungicide N-(3, 5-dichlorophenyl) succinimide (NDPS) acts as an antiandrogen using in vitro and in vivo assays. A transient transfection system based on luciferase activity was utilized for in vitro analysis of the antiandrogeic activity of NDPS. Hershberger assay was used to analyze the antiandrogenic activity of NDPS in rats. The expressions of the androgen-responsive genes testosterone-repressed prostatic message-2 (TRPM-2) and prostate specific binding protein polypeptide C3 (PBP C3) in the rat ventral prostate were measured using real-time PCR. Our results indicated that NDPS can block 5-dehydrotestosterone (DHT)-induced androgen receptor (AR) activity in transiently transfected HepG2 cells (-5 log M). In the Hershberger assay, the weights of the seminal vesicles and levator ani/bulbocavernosus muscles were significantly decreased (P<0.05) in all NDPS groups, and the weights of the ventral prostate, dorsolateral prostate, and Cowper's glands were significantly decreased (P<0.05) in the 100 and 200 mg/kg NDPS groups. NDPS only decreased (P<0.05) the expression of PBP C3 and had no effect on the level of TRPM-2 (P>0.05). In conclusion, NDPS is a moderate antiandrogen that elicits antiandrogenic effects at least partly by antagonizing AR and increasing the expression of PBP C3.

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Glutamate is the dominant excitatory neurotransmitter in a large number of physiological processes including neuroendocrine regulation. Some pharmacological studies have shown that different subtypes of glutamate receptor, such as the N-methyl-D-aspartic acid (NMDA) and α-amino-3-hydroxy-5-methy-4-isoxazolepropionic acid (AMPA) receptors, are involved in stress-induced adrenocorticotropin (ACTH) and prolactin secretion. However, the roles of the respective glutamate receptors and the mechanism of ACTH and prolactin secretion during stress via these receptors have not been investigated in detail. In the present study, we evaluated the role of AMPA-type glutamate receptor in ACTH and prolactin regulation under restraint stress in adult male rats. Male rats pretreated with a selective AMPA receptor antagonist, 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX; 50 μg), through a lateral ventricle cannula were stressed by immobilization. Administration of NBQX inhibited ACTH and prolactin secretion in response to restraint stress. However, NBQX had no significant effects on the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, as measured by the accumulation of 3, 4-dihydroxyphenylalanine (DOPA). In addition, administration of NBQX suppressed stress-induced prolactin secretion in the male rats pretreated with α-MT, an inhibitor of dopamine synthesis, and infused with dopamine solution (2.5 μg/200 μl/10 min). These results indicated that the effects of NBQX on prolactin secretion might be mediated by non-dopamine mechanisms. The contents of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the median eminence (ME) of the male rats decreased during restraint stress; however, the fluctuations in CRH and AVP were eliminated by NBQX administration. These results suggest that stress-induced ACTH and prolactin release mediated by neurotransmission via AMPA receptors might be partly attributable to hypophysiotropic regulatory factors in the hypothalamus.

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The changes in histone acetylation are not always consistent in various cell types and at different developmental stages. We immunostained specific antibodies against acetylated lysine 9 of histone H3 and acetylated lysines 5 and 12 of histone H4 in an effort to understand the detailed changes in histone acetylation during sheep oocyte meiosis. We found that the acetylation fluorescence signals of H3/K9 and H4/K12 on chromatin appeared intensively in the germinal vesicle (GV), late-GV (L-GV), and germinal vesicle breakdown (GVBD) stages and became weak in metaphase I (MI); however staining reappeared in anaphase I-telophase-I (AI-TI) and metaphase II (MII). Furthermore, staining was detected in the first polar bodies. The fluorescence signals of H4/K5 first appeared in the MI stage and became intensive in the AI-TI stage; however they were barely detectable in MII stage chromosomes and first polar bodies. We conclude that the acetylation patterns of H3/K9 and H4/K12 during oocyte meiotic maturation are similar and that the pattern of H4/K5 is unique.

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The present study was conducted to investigate whether goat fetal myoblasts with no inherent adipogenic potential can be induced to transdifferentiate into adipocytes. Goat fetal myoblasts were transiently transfected by the adipogenic transcription factors, peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα). Both PPARγ and C/EBPα were capable of inducing adipogenic transdifferentiation as indicated by the appearance of mature adipocytes when the transfected cells were cultured in adipogenic differentiation medium (ADM). Ectopic expression of PPARγ induced endogenous C/EBPα expression and vice versa only when the cells were cultured in ADM. Removal of troglitazone, a PPARγ agonist, from the ADM resulted in a dramatic decline in the number of adipocytes, indicating that PPARγ stimulation is necessary to induce adipogenic transdifferentiation of goat fetal myoblasts. These results demonstrate for the first time that primary cultured myoblasts can be transdifferentiated into adipocytes.

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Sanguinarine (SA), a phytobiotic from Sanguinaria Canadensis, has been demonstrated to inhibit vessel growth. Current restrictions on the use of antibiotic growth promoters have motivated addition of this alkaloid as a naturally appetizing feed additive for farm animals. However, concern may araise since angiogenesis is a fundamental event in ovarian follicle growth. Therefore, the aim of this study was to evaluate the potential negative role of SA in follicular angiogenesis. For this purpose, we studied the effect of 300 nM SA on the production of vascular endothelial growth factor (VEGF) by swine granulosa cells from follicles >5 mm and on the activation of Akt, the main effector of the VEGF signalling pathway. In addition, the potential interference of SA in vessel development was tested in an in vitro angiogenesis bioassay. SA inhibited both VEGF production and VEGF-induced Akt activation in swine granulosa cells. Moreover, it was able to block vessel growth induced by VEGF. Taken together, our results suggest that SA could be detrimental to follicular angiogenesis, and therefore supplementation of feed with this alkaloid should be carefully considered.

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Sterility in male hypogonadic (hgn/hgn) rats results from congenital testicular dysplasia caused by a single recessive gene hgn on rat chromosome 10. We recently identified an insertion mutation in the Spag5/astrin gene of hgn/hgn rats that may cause defective proliferation of immature Sertoli cells in the postnatal hgn/hgn testis. Since the pathological alterations were present in the testes at birth, we examined the involvement of defective mitosis and apoptotic cell death in embryonic development of hgn/hgn testes. Testicular hypoplasia was apparent at embryonic day (ED) 18.5. Immunostaining of hgn/hgn testes at ED 21.5 with antibody to GATA-4, which is specific for fetal Sertoli cells in the seminiferous cords, showed that the significant decrease in the number of fetal Sertoli cells was accompanied by a two fold increase in their mitotic index and abnormal mitosis and apoptosis. Prior to this, we observed a decrease in the number of BrdU-labeled cells, an increase in the number of TUNEL-positive apoptotic cells, and presence of MIS-positive apoptotic cells in hgn/hgn testes on ED 17.5 and 18.5. These results suggest that the Spag5 mutation may cause a reduction in mitotic activity and an increase in apoptosis of fetal Sertoli cells in hgn/hgn testes.

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We studied the effects of administering estradiol benzoate (EB) plus progesterone (P4) as part of a CIDR-based protocol during the growth or static phases of dominant follicle development on follicular wave emergence, follicular growth, synchrony of ovulation and pregnancy rate following CIDR withdrawal, treatment with PGF_2α and GnRH, and fixed-time artificial insemination (TAI). Forty-one previously synchronized lactating Holstein dairy cows were randomly allocated to three treatment groups. The control group (n=14) received a CIDR on the third day after ovulation only (Day 0). The two treatment groups were administered CIDRs comprising 2 mg EB and 50 mg P4 either on the third (T1, n=14) or eighth day (T2, n=13) after ovulation (Day 0). All cows received PGF_2α after CIDR removal on Day 7, GnRH on Day 9, and TAI 16 h after GnRH treatment. The proportion of cows with follicular wave emergence within 8 days of treatment differed (P<0.01) among the control (14.3%), T1 (85.7%), and T2 groups (92.9%). However, the mean intervals between treatment and wave emergence were not significantly different. There were significant differences in the diameters of the dominant follicles on Day 7 (P<0.01) and in preovulatory follicles on Day 9 (P<0.01), with the largest follicles observed in the control group and the smallest follicles observed in the T2 group. In contrast, the numbers of cows showing synchronous ovulation after GnRH treatment (92.9 to 100.0%) and pregnancy following TAI (46.2 to 50.0%) were similar between the treatment groups. The results showed that, irrespective of the phase (growth or static) of the dominant follicle, administration of 2 mg EB plus 50 mg P4 to CIDR-treated lactating dairy cows induced consistent follicular wave emergence and development, synchronous ovulation after GnRH administration, and similar pregnancy rates following TAI.

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Movement of water and cryoprotectants through the plasma membrane needs to be accelerated for successful cryopreservation of zebrafish oocytes/embryos, which are much larger than their mammalian counterparts. Aquaporin-3 is a water/solute channel that can transport not only water but also various cryoprotectants. In this study, we attempted to increase the permeability of immature zebrafish oocytes at stage III to water and cryoprotectants by exogenous expression of rat aquaporin-3. Immature zebrafish oocytes were injected with rat aquaporin-3 cRNA and cultured for 5-12 h. Permeability to water and cryoprotectants was then determined based on changes in the volumes of the oocytes in a hypertonic sucrose solution and various cryoprotectant solutions at 25 C. The permeability to water of the aquaporin-3 cRNA-injected oocytes was three times higher than that of intact and water-injected oocytes. The permeability of the aquaporin-3 cRNA-injected oocytes to ethylene glycol, glycerol, propylene glycol, and DMSO was also 2-4 times higher than that of intact oocytes. Thus, the permeability of immature zebrafish oocytes to water and cryoprotectants was enhanced by exogenous expression of aquaporin-3. Cryopreservation of teleost oocytes may be realized through a further increase in permeability.

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The development of cleavage stage preimplantation embryos is disrupted by exposure to heat shock, such as high temperatures in the summer season. In this study, we investigated whether addition of anthocyanins, which are strong scavengers of reactive oxygen species (ROS), improves development and intracellular redox status of heat-exposed bovine preimplantation embryos by reduction of heat shock-derived oxidative stress. After in vitro fertilization (IVF), embryos were cultured at 38.5 C through Day 8 (Day 0=day of IVF) with 0, 0.1, 1 and 10 μg/ml anthocyanins (non-heat-shocked group). On Day 2, embryos were cultured at 41.5 C for 6 h with 0, 0.1, 1 and 10 μg/ml anthocyanins followed by culture at 38.5 C until Day 8 (HS group). After exposure to heat shock, the intracellular ROS and glutathione (GSH) contents of individual embryos were measured in the non-heat-shocked and HS groups using fluorescent probes. On Day 8, the blastocysts formation rates of the embryos and total cell numbers of blastocysts were evaluated. Embryos exposed to heat shock without anthocyanins showed a significant decrease in blastocyst formation rate and GSH content (P<0.05) and an increase in intracellular ROS (P<0.05) compared with non-heat-shocked embryos. In contrast, addition of 0.1 μg/ml anthocyanins significantly (P<0.05) improved the blastocyst formation rate of the heat-shocked embryos. Addition of any dose of anthocyanins produced a significant decrease in the ROS levels (P<0.05) and tended to increase the GSH levels under heat-shock conditions. However, addition of higher concentrations (1 and 10 μg/ml) of anthocyanins to the culture media under heat shock did not improve the development of embryos. These results indicate that anthocyanins maintain the intracellular redox balance of heat-shocked bovine embryos by reducing intracellular oxidative stress and increasing the GSH levels. Thus, alterations of the redox state using natural antioxidative polyphenols is a useful approach for reducing heat shock-derived oxidative stress.

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The majority of somatic cell nuclear transferred (SCNT) embryos die before or after implantation. Many studies have focused on morphological remodeling of the donor nucleus and its associated cytoskeletal structures in the early events of nuclear transfer. However, little is known about the 2-cell stage of SCNT embryos after the first division. In this study, we compared the morphological status of chromosomal division during the 1-cell stage to the 2-cell stage in SCNT embryos with that in intracytoplasmic sperm injection (ICSI) embryos. The microtubules and cytoplasmic asters, which are related to chromatin segregation, disappeared at the pronuclear stage, although formation of the first mitotic spindle was normal in both the SCNT and ICSI embryos. However, nuclear fragmentation was observed in 30% of the 2-cell SCNT embryos and 12% of the 2-cell ICSI embryos. Nuclear fragmentation was present in both blastomeres of these embryos. No apoptotic DNA fragmentation was observed in TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays for either the SCNT or ICSI embryos. In both the SCNT and ICSI embryos, the distribution of chromosomes in the first mitotic spindle was disturbed during the process of division from the 1-cell stage to the 2-cell stage. These results suggest that loss of SCNT embryos just before or after implantation may be due to an abnormal chromosome distribution at the 2-cell stage.

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In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken β-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4- to 8-cell stage at 60 hpf. Only 4- to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P<0.05) than embryos with weak or absent luminescence. However, embryos with mosaic luminescence developed at a much lower rate (P<0.05) than those with whole-embryo luminescence, even if the embryos exhibited strong luminescence. Our results indicate that precise and uniform embryonic gene expression at the 4- to 8-cell stage at 60 hpf may be closely related to development of bovine NT embryos to the blastocyst stage.

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To clarify the mechanism that impairs development of in vitro grown (IVG) oocytes, we assessed whether the developmental disability of IVG oocytes is caused by cytoplasmic dysfunction. First, we assessed the cleavage of nuclear-substituted oocytes cultured in vitro. The nuclei, but not the cytoplasm, of the IVG oocytes were able to support subsequent cleavage after artificial activation. The mitochondrial activity of the oocytes increased as the follicles grew. However, the mitochondrial activity of the IVG oocytes was significantly lower than that of ovulated oocytes and oocytes recovered from follicles with diameters of more than 300 μm. Furthermore, the expression levels of mitochondrial transcriptional factor A (TFAM) in the oocytes increased in a similar manner. However, the expression levels of TFAM in the IVG oocytes was significantly lower than that of ovulated oocytes and oocytes recovered from follicles with diameters of more than 300 μm. Taken together, these results indicate that the low developmental competence of IVG oocytes is caused by a cytoplasm deficiency due to low mitochondrial activity.

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This study compared two types of controlled internal drug release (CIDR)-based timed artificial insemination (TAI) protocol for treatment of repeat breeder dairy cows. In the first trial of the experiment, 55 repeat breeder cows were randomly assigned to the following two treatments. (1) In the EB group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 1 mg estradiol benzoate (EB) plus 50 mg progesterone (P4; Day 0). On Day 7, they were given an injection of PGF_2α and the CIDR device was removed. The cows were given an injection of 1 mg EB on Day 8 and were subjected to TAI 30 h later (n=27). (2) In the gonadotrophin releasing hormone (GnRH) group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 250 μg gonadorelin (GnRH; Day 0). On Day 7, they were given an injection of PGF_2α and the CIDR device was removed. The cows were given an injection of 250 μg GnRH on Day 9 and were subjected to TAI 17 h later (n=28). In the second trial, 41 repeat breeder cows that were confirmed as not pregnant in the first trial were randomly assigned to the same two treatments used in the first trial (an EB group of 20 cows and a GnRH group of 21 cows). The ovaries of 15 cows from each group were examined by transrectal ultrasonography in order to observe the changes in ovarian structures, and blood samples were collected for analysis of serum P4 concentrations. The pregnancy rates following TAI in the first (18.5 vs. 32.1%) and second (40.0 vs. 38.1%) trials and the combined rates (27.7 vs. 34.7%) did not differ between the EB and GnRH groups. The proportions of cows with follicular wave emergence within 7 days did not differ between the EB (12/15) and GnRH groups (13/15). The interval to wave emergence was shorter (P<0.01) in the GnRH group than in the EB group, but there was no difference in the mean diameters of dominant follicles on Day 7 between the groups. Moreover, the proportions of cows with synchronized ovulation following a second EB or GnRH treatment did not differ between the groups. In conclusion, treatment with either EB or GnRH in a CIDR-based TAI protocol results in synchronous follicular wave emergence, follicular development, synchronous ovulation, and similar pregnancy rates for TAI in repeat breeder cows.

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The objective of the present study was to investigate the effect of addition of ghrelin to in vitro culture medium on preimplantation development of porcine in vitro fertilized and parthenogenetic embryos. In Experiment 1, we sought to compare the in vitro developmental competence of IVF and parthenogenetic embryos. No significant (P<0.05) differences were detected for cleavage rate or blastocyst rate between the in vitro fertilization (IVF)- and parthenogenetic activation-derived embryos. In Experiment 2, parthenogenetic embryos were cultured in Porcine Zygote Medium-3 containing various concentrations of ghrelin. The blastocyst rate was remakably (P<0.05) increased when 5 ng/ml (PA-5) and 500 ng/ml (PA-500) of ghrelin was added to in vitro culture medium compared with the other groups. Total cell number per blastocyst was slightly promoted in the ghrelin treatment groups compared with the controls. However, the ratio of inner cell mass (ICM) cell number/total cell number was significantly reduced in the PA-50 group compared with the controls (P<0.05). In Experiment 3, we cultured in vitro fertilized embryos in Porcine Zygote Medium-3 supplemented with ghrelin at different dosages. The rate of blasotcyst formation was markedly (P<0.05) elevated when 500 ng/ml ghrelin was added to culture medium (IVF-500) compared with the controls. Increased total cell numbers (P<0.05) were observed when in vitro fertilized embryos were cultured in IVF-50 and IVF-500 compared with the controls. However, the ratio of ICM cell number/total cell number was decreased in the ghrelin treatment groups compared with the controls (P<0.05). Taken together, the results suggest that ghrelin can enhance blastocyst formation of porcine in vitro fertilized and parthenogenetic embryos while exerting a negative effect on the structural integrity of the blastocysts.

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The aim of the present study was to examine the messenger RNA expressions of the endothelin and angiotensin systems during the periovulatory phase in gonadotrophin releasing hormone (GnRH)-treated cows. Ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (control, before LH surge), 3-5 h after GnRH (during LH surge), 10 h after GnRH; 20 h after GnRH, 25 h after GnRH (peri-ovulation), and early corpus luteum (CL) (Days 2-3). Expression of mRNA was investigated using quantitative real-time PCR. The expression of angiotensin converting enzyme (ACE) mRNA significantly decreased immediately after onset of the LH surge and remained at low levels. The levels of angiotensin II receptor type 1 (AT1R) and type 2 (AT2R) expression during the periovulatory period significantly decreased compared with other periods. The concentration of angiotensin II in follicular fluid began to increase 10 h after GnRH treatment and further increased as ovulation approached. The level of ET-1 mRNA significantly decreased 10 h after GnRH treatment compared with the levels before GnRH treatment and those of the early CL period. The expression of ETR-A and ETR-B mRNA during the periovulatory period were lower than in other periods. The expression of ECE-1 mRNA began to decrease in the LH surge period and significantly decrease in the periovulatory period compared with other periods. These results suggest that the vasoactive peptides angiotensin and endothelin may be associated with final maturation of follicles.

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Cloned animals have been produced in several mammalian species so far, although success rates to term are very low. Aberrations in gene expression derived from abnormal epigenetic status have been thought to be a cause of developmental abnormalities in clones, and several abnormalities in gene expression have already been detected in cloned animals and embryos. In this study, we examined the hypothesis that the poor survival rates of nuclear transfer (NT) embryos are partly due to aberrations in the regulation of expression of genes transcribed by RNA polymerases I and III, in addition to polymerase II. We produced cloned and in vitro fertilized mouse embryos that developed to the blastocyst stage, and the amounts of several genes were analyzed using individual embryos. We found that the amounts of mature 18S ribosomal RNA (rRNA) transcribed by RNA polymerase I were lower in NT embryos than in IVF embryos, but that the amounts of 47S rRNA and intermediates of mature rRNAs were higher in NT embryos. In addition, the amount of 7SK RNA transcribed by RNA polymerase III was lower in NT embryos than in IVF embryos. The transcripts of all but one of the genes transcribed by RNA polymerase II were not noticeably different between NT and IVF embryos. These results suggest that some of the transcripts produced by RNA polymerases I, II and III are aberrantly regulated in NT embryos.

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In a previous study, we found that 3-methyl-4-nitrophenol (PNMC), a component of diesel exhaust particles and also a degradation product of the insecticide fenitrothion, exhibits reproductive toxicity in the adult male Japanese quail. The present study investigated the toxicity of PNMC in the female Japanese quail and its ability to influence reproduction in immature females. The quail (21-day-old) were injected intramuscularly (im) with PNMC at doses 0.1, 1 or 10 mg/kg body weight daily for 3 days. There was no significant difference in body growth between the PNMC-administered and control birds. However, the weights of the oviducts were significantly lower in the PNMC-treated birds at all doses. Furthermore, the plasma concentrations of luteinizing hormone (LH) and estradiol-17 β were significantly decreased with 1 and 10 mg/kg of PNMC. These findings suggest that PNMC might influence the hypothalamo-pituitary-gonadal axis with decreasing in secretion of GnRH, LH and ovarian steroid hormones and subsequently disturb growth of the reproductive organs of immature female quail. This study indicates that PNMC induces reproductive toxicity at the central level and disrupts reproductive function in the immature female quail.

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This study was carried out to investigate the effects of minimum essential medium (MEM) vitamins during in vitro maturation (IVM)/in vitro culture (IVC) of porcine nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. Porcine cumulus-oocyte complexes (COCs) were divided into five groups, matured for 44 h in maturation medium with various concentrations of MEM vitamins (0, 0.05, 0.1, 0.2 and 0.4%), and observed for maturation rate. Also, COCs were matured in NUSU-23 media without MEM vitamins for 44 h and cultured in PZM-3 media with various concentrations of MEM vitamins (0, 0.05, 0.4 and 1.0%) for 6 days following nuclear transfer. Factorial (IVM/IVC) experiments were also performed in NCSU-23 medium with or without 0.05% MEM vitamins and PZM-3 medium with or without 0.4% MEM vitamins. They were then tested by examining in vitro development of the porcine reconstructed embryos. The maturation rates of the COCs treated with the MEM vitamins did not differ significantly among the MEM vitamin-treated groups. Addition of vitamins to culture medium did not affect development of porcine reconstructed embryos in vitro. However, addition of low concentrations of MEM vitamins only to maturation medium increased (P<0.05) the proportion of NT embryos developing into blastocysts compared with the control group. Addition of MEM vitamins to IVC medium did not enhance the developmental rate compared with the control group. Thus, addition of MEM vitamins to IVM medium could improve subsequent blastocyst development of porcine NT embryos.

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This study was conducted to determine whether meiotic maturation could be induced in ovarian oocytes from the American brown bear (Ursus arctos), a model for gamete "rescue" techniques for endangered ursids. The bears were euthanized, and their ovaries were transported to the laboratory within 4 h. The mean ovarian size was 2.4 × 1.8 cm (range: 2.0-3.3 × 1.5-2.2 cm). The ovaries obtained from the 2 brown bears yielded 97 oocytes (48.5/female), and 88 (90.7%) of them were morphologically classified as normal quality. Oocytes were in vitro matured at 38.5 C in 5% CO₂ for 24 or 48 h in TCM-199 supplemented with 10% FBS, 1 μg/ml estradiol-17β, and 10 μg/ml FSH. In Exp. 1, morphologic evaluation of matured oocytes was conducted by measuring the diameters of oocytes with a zona pellucida (ZP) or cytoplasm without a ZP. In Exp. 2, activation was induced by applying two 20 μsec DC pulses of 2.0 kV/cm delivered by an Electro Cell Fusion Generator. The activated oocytes were cultured in TCM-199 containing 2 mM of 6-dimethylaminopurine for 4 h, in Charles Rosenkrans (CR) 1 for 3 days and the in CR2 for another 4 days. The diameters of the matured bear oocytes with a ZP and with cytoplasm without a ZP (161.8 ± 6.0 and 135.3 ± 7.5 μm, respectively) were significantly (P<0.05) larger than those of bovine oocytes (150.7 ± 4.9 and 118.7 ± 7.5 μm). The maturation rates of the bear oocytes were 17.6 and 59.4% at 24 and 48 h of in vitro maturation, the percentage of activated oocytes that developed to the 2 or 4-cell stage was 31.6%; however, no blastocysts were observed. These results indicate that bear oocytes can develop to metaphase II in an in vitro culture system and that activated oocytes can develop to the 2 or 4-cell stages.

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Transcription factor p63, a member of the tumor suppressor p53 family, plays an important role in epithelial development, and its expression is found in many epithelial tissues. Our previous reports have shown that p63 is expressed in primordial and testicular germ cells. In this study, we investigated the expression and distribution of p63 isoforms (TAp63α, TAp63β, TAp63γ, ΔNp63α, ΔNp63β and ΔNp63γ) in the mouse ovary. Reverse transcription-polymerase chain reaction experiments with primers specific for different p63 isoforms demonstrated that transcripts encoding the transactivation domain and α isoforms were preferentially expressed in the ovary. Immunolocalization of p63 proteins, presumably that of full-length p63α isoform, was observed in the oocytes of primordial, primary and two-layered secondary follicles. However, positive staining was not detected in the oocytes of multi-layered secondary follicles and antral follicles as they continued to develop. This data suggests that p63 may be implicated in the meiosis and cell cycle control of germ cells in the mouse ovary.

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With respect to the assessment of residue situation and as a part of preclinical trials to determine the biological activities of potential gonadotropin releasing hormone (GnRH) residues in porcine organisms the GnRH agonist Gonadorelin[6-D-Phe] (D-Phe⁶-LHRH) was administered either enterally or intramuscularly (i.m.) to female Goettinger miniature pigs in order to evaluate the GnRH-induced luteinizing hormone (LH) surge. Gilts received an (i) enteral application of 10 mg D-Phe⁶-LHRH via a probang (enteral group, n=7), (ii) i.m. injection of 0.1 mg D-Phe⁶-LHRH (parenteral group, n=5), or (iii) saline injection (control group, n=4). The GnRH and saline applications were repeated every second day with up to seven repetitions. Blood samples were collected via previously fitted jugular catheters immediately before injections, over an 8 h period in 1 h intervals beginning 2 h after injections, and at 24, 26, 28 and 30 h after applications. Enteral application of D-Phe⁶-LHRH induced an LH surge in 23 of 30 treatments. All gilts in the parenteral group exhibited LH release after each D-Phe⁶-LHRH application (P<0.05), whereas no LH surges were observed after saline injection in the control group. A significant (P<0.05) LH rise to mean maximum LH concentrations of 3.25 ± 0.43 and 3.05 ± 0.26 ng/ml occurred in both the enteral and parenteral groups, but there was no difference in the time interval after GnRH (2.6 ± 0.3 vs. 2.3 ± 0.3 h) and the mean duration of LH peak (6.5 ± 0.4 and 6.8 ± 0.3 h) between the treatment groups. In conclusion, (i) enteral application of 10 mg D-Phe⁶-LHRH induced LH release in a physiological range from the pituitary of female minipigs, and (ii) neither an accumulative effect nor a cumulative LH response were found after repeated GnRH application. Furthermore, (iii) in regard to consumer protection and gonadotropin secretion, D-Phe⁶-LHRH residues can be excluded from having long-term effects.

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