Sperm induce proinflammatory responses in the uterus and peripheral blood immune cells of artificially inseminated cows.

This in vivo study aimed to investigate local and systemic immune responses induced by sperm in cows after artificial insemination (AI). Initially, 12 multiparous Japanese Black cows were subjected to intrauterine AI (AI group, n = 6) or saline infusion (control group, n = 6). The uterine body and horn ipsilateral to the ovulatory follicle were mini-flushed with 2 ml of RPMI-1640 medium at different time points (0, 1, 6, 10, 24, 48 h, and 7 days after AI), centrifuged, and the sediments were examined under a light microscope. Vaginal smears were prepared at 0, 1, 6, and 10 h after AI to investigate the sperm backflow. Subsequently, another experiment was conducted by assigning cows to three groups: intrauterine AI (AI group, n = 5), heat-inactivated AI (Heat-AI group, n = 5), or saline infusion (control group, n = 5). Blood samples were collected, and polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were separated and analyzed for gene expression using real-time PCR. The results showed that most sperm were rapidly transported either forward into the uterine horn or backward into the vagina within 1 h after AI. The PMNs migrated into the uterine lumen 6 hours after AI. Only active sperm-induced proinflammatory responses in PMNs and PBMCs via upregulation of TNFa, IL8, IL1B, and PGES and downregulation of IL10 at 6 h after AI. These data provide evidence that sperm generate transient proinflammatory responses locally in the uterus and systemically in the peripheral immune cells, which may be prerequisites for uterine clearance, embryo receptivity, and implantation in cows.

Potential of preimplantation genomic selection using the blastomere separation technique in bovine in vitro fertilized embryos.

Preimplantation genomic selection combined with an in vitro embryo production system is expected as a means of accelerating genetic improvement in cattle. While micromanipulation-based biopsy approaches are often used to collect embryonic cells for genetic testing, they require expensive equipment and sophisticated skills, hindering the adoption of this system. In the present study, to develop a simple method for preimplantation genomic selection using the blastomere separation (BS) technique in bovine in vitro fertilized embryos, we examined the accuracy of single nucleotide polymorphism (SNP) genotyping and optimal cryopreservation method in demi-blastocysts produced by the BS technique. We demonstrated reliable SNP genotyping using DNA derived from demi-blastocysts. We indicated a suitable equilibrium time in vitrification solution for demi-blastocysts and succeeded obtaining pregnancies by the transfer of vitrified demi-blastocysts. In conclusion, our findings suggest that the BS technique provides a simple method for preimplantation genomic selection in bovine in vitro fertilized embryos.

Expression of C-C motif chemokines and their receptors in bovine placentomes at spontaneous and induced parturition.

In bovine placentomes, the inflammatory response is considered important for the detachment of the fetal membrane from the caruncle after parturition. Glucocorticoids, a trigger of the onset of parturition, facilitate functional maturation of placentomes via prostaglandin (PG) and estrogen production in cattle. This study investigated how exogeneous glucocorticoids, which exert immunosuppressive effects, affect placental inflammation at parturition. Placentomes were collected immediately after spontaneous or induced parturition. Parturition was conventionally induced using PGF2α or dexamethasone or with a combination of triamcinolone acetonide and high-dose betamethasone (TABET treatment). Polymerase chain reaction (PCR) array analysis indicated that 9/13 C-C motif chemokine ligands (CCLs) were upregulated > two-fold in spontaneous parturition, with CCL2 and CCL8 being highly expressed. The expressions of CCL2, CCL8, C-C motif chemokine receptor 1 (CCR1), and CCR5 in caruncles were significantly higher in spontaneous parturition than in induced parturition. Although the clinical dose of dexamethasone did not influence the expression of these CCLs and CCRs, TABET treatment increased CCR1 expression. CCL8, CCR1, CCR2, and CCR5 were localized in the caruncular epithelial cells. CCR2 was also localized in the epithelial cells of the cotyledonary villi. This study is the first report to reveal the disruption in CCL and CCR expression in bovine placentomes at induced parturition. Enhanced glucocorticoid exposure for the induction of parturition may upregulate CCR1 expression in placentomes, but the treatment does not adequately promote CCL expression. Additionally, immunohistochemistry suggested that the CCL-CCR system is involved in the functional regulation of maternal and fetal epithelial cells in placentomes at parturition.

Effect of autophagy induction and cathepsin B inhibition on developmental competence of poor quality bovine oocytes.

The present study investigated the effect of autophagy induction and cathepsin B (CTSB) inhibition on developmental competence of poor quality oocytes. Bovine cumulus oocyte complexes (COCs) were classified as good or poor according to their morphology. Autophagy activity was detected in good and poor germinal vesicle (GV) oocytes. Then E-64, a CTSB inhibitor, rapamycin (Rapa), an autophagy inducer, and combined administration was achieved during invitro maturation (IVM) of poor quality COCs followed by detection of autophagy activity. In the next experiment, E-64, Rapa, and E64 + Rapa, were added during IVM to good and poor quality COCs followed by invitro fertilization and culture for 8 days to investigate whether inhibition of CTSB and/or induction of autophagy improve embryonic development and quality. Autophagy activity was significantly lower in poor quality GV oocytes than in good quality ones. E-64, Rapa and E-64 + Rapa treatment during IVM significantly increased autophagy activity in poor quality oocytes. Addition of Rapa in good quality COCs did not increase the blastocyst rate, whereas E-64 increased the blastocyst rate and total cell number (TCN) with decreasing TUNEL-positive cells. In contrast, Rapa treatment in poor quality COCs significantly increased the blastocyst rate and TCN with decreasing TUNEL-positive cells. These results indicate oocyte quality has different responses to intracellular autophagy induction and CTSB activity control by potential autophagy and catabolic status, however, synergetic effect of autophagy induction and CTSB inhibition can increase developmental competence of both good and poor quality COCs, especially rescue effect in poor quality COCs.

Dynamic status of lysosomal cathepsin in bovine oocytes and preimplantation embryos.

Lysosomal cathepsin, in particular cathepsin B (CTSB), plays an important role in implantation, pregnancy, and embryonic development. However, little is known about the mechanism related to the dynamic status of lysosomal cathepsins in bovine oocytes and preimplantation embryos. In the present study, we investigated the dynamics of gene expression, activity, and immunolocalization of CTSB, as well as the activities of lysosome, in bovine oocytes and preimplantation embryos. After gene expression analysis of several cathepsin-related genes, transcript levels of CTSB, CTSD and CTSZ were highest in Metaphase II (MII) oocytes followed by a significant decrease from the 8-cell embryo stage. Activity of CTSB showed a significant increase in 1-cell and morula stage embryos. Lysosomal activity was also significant higher in 1-cell and morula stages, which was consistent with CTSB activities. However, immunolocalization of CTSB did not show the similar pattern of CTSB and lysosomal activities. We also found significantly higher expression levels of CTSB transcript in the trophectoderm (TE) compared to inner cell mass (ICM), whereas activity and immunolocalization of CTSB showed an opposite pattern, i.e. significantly higher in ICM than TE. These patterns were confirmed by the same analysis using separated ICM and TE. Our results suggest that lysosomal CTSB has a pivotal role during embryonic development and differentiation, especially fertilization and the differentiation period.

Potential of preimplantation genomic selection for carcass traits in Japanese Black cattle.

Preimplantation genomic selection using genomic estimated breeding values (GEBVs) based on single nucleotide polymorphism (SNP) genotypes is expected to accelerate genetic improvement in cattle. To develop a preimplantation genomic selection system for carcass traits in Japanese Black cattle, we investigated the accuracy of genomic evaluation of carcass traits using biopsied embryonic cells (Experiment 1); we also performed an empirical evaluation for embryo transfer (ET) of vitrified GEBV-evaluated blastocysts to assess the efficiency of the preimplantation genomic selection system (Experiment 2). In Experiment 1, the mean call rate for SNP genotyping using approximately 15 biopsied cells was 98.1 ± 0.3%, whereas that for approximately 5 biopsied cells was 91.5 ± 2.4%. The mean concordance rate for called genotypes between ~15-cell biopsies and the corresponding biopsied embryos was 99.9 ± 0.02%. The GEBVs for carcass weight, ribeye area, and marbling score calculated from ~15-cell biopsies closely matched those from the corresponding calves produced by ET. In Experiment 2, a total of 208 in vivo blastocysts were biopsied (~15-cell) and the biopsied cells were processed for SNP genotyping, where 88.5% of the samples were found to be suitable for GEBV calculation. Large variations in GEBVs for carcass traits were observed among full-sib embryos and, among the embryos, some presented higher GEBVs for ribeye area and marbling score than their parents. The conception rate following ET of vitrified GEBV-evaluated blastocysts was 41.9% (13/31). These findings suggest the possible application of preimplantation genomic selection for carcass traits in Japanese Black cattle.

Evidence that interferon-tau secreted from Day-7 embryo in vivo generates anti-inflammatory immune response in the bovine uterus.

Recent studies suggest that Day-7 bovine embryo starts to communicate with the uterine epithelium through interferon-tau (IFNT) signaling. However, immune modulatory role of IFNT in the uterus just after the embryo moves from the oviduct is unclear. We aimed to examine the hypothesis that Day-7 bovine embryo secretes IFNT in the uterus, which induces anti-inflammatory response in immune cells. The uterine flush (UF) with multiple embryos was collected from Day-7 donor pregnant cows and peripheral blood mononuclear cells (PBMCs) were then cultured in UF. Transcripts detected in PBMCs revealed that UF from pregnant cows down-regulated pro-inflammatory cytokines (TNFA, IL1B) and up-regulated anti-inflammatory cytokine (IL10) expression, with activation of interferon-stimulated genes (ISGs; ISG15, OAS1) as compared with UF from non-pregnant cows. An addition of specific anti-IFNT antibody to the UF inhibited the effect on PBMCs, indicating that IFNT is a major factor for such immune modulation. The observation that conditioned media from bovine uterine epithelial cells both stimulated with IFNT in vitro and supplemented with fresh IFNT induced similar PBMCs gene expression, confirming that IFNT directly acts on this immune crosstalk. This study shows that IFNT secreted from Day-7 embryo in vivo generates anti-inflammatory response in immune cells, which may provide immunological tolerance to accept the embryo.

Expression and localization of aquaporins 3 and 7 in bull spermatozoa and their relevance to sperm motility after cryopreservation.

Artificial insemination with cryopreserved semen is a well-developed technique commonly used for controlled reproduction in cattle. However, despite current technical advances, cryopreservation continues to damage bull spermatozoa, resulting in a loss of approximately 30 to 50% of viable spermatozoa post thawing. To further improve the efficiency of cryopreservation of bull spermatozoa, understanding the molecular mechanisms underlying the cryobiological properties that affect cryoinjuries during cryopreservation process of bull spermatozoa is required. In this study, we examined the expression and localization of aquaporin (AQP) 3 and AQP7 in fresh, cooled, and frozen-thawed bull spermatozoa. Furthermore, we investigated the relevance of AQP3 and AQP7 to motility and to membrane integrity in frozen-thawed bull spermatozoa. Western blotting against AQP3 and AQP7 in bull spermatozoa revealed bands with molecular weights of approximately 42 kDa and 53 kDa, respectively. In immunocytochemistry analyses, immunostaining of AQP3 was clearly observed in the principal piece of the sperm tail. Two immunostaining patterns were observed for AQP7 -pattern 1: diffuse staining in head and entire tail, and pattern 2: diffuse staining in head and clear staining in mid-piece. Cooling and freeze-thawing did not affect the localization pattern of AQP7 and the relative abundances of AQP3 and AQP7 evaluated by Western blotting. Furthermore, we demonstrated that the relative abundances of AQP3 and AQP7 varied among ejaculates, and they were positively related to sperm motility, particularly sperm velocity, post freeze-thawing. Our findings suggest that AQP3 and AQP7 are possibly involved in the tolerance to freeze-thawing in bull spermatozoa, particularly in the sperm's tail.

Production of calves by the transfer of cryopreserved bovine elongating conceptuses and possible application for preimplantation genomic selection.

Preimplantation genomic selection based on single nucleotide polymorphism (SNP) genotypes is expected to accelerate genetic improvement in cattle. However, genome-wide genotyping at the early embryonic stage has several limitations, such as the technical difficulty of embryonic biopsy and low accuracy of genotyping resulting from a limited number of biopsied cells. After hatching from the zona pellucida, the morphology of the bovine embryo changes from spherical to filamentous, in a process known as elongation. The bovine nonsurgical elongating conceptus transfer technique was recently developed and applied for sexing without requiring specialized skills for biopsy. In order to develop a bovine preimplantation genomic selection system combined with the elongating conceptus transfer technique, we examined the accuracy of genotyping by SNP chip analysis using the DNA from elongating conceptuses (Experiment 1) and optimal cryopreservation methods for elongating conceptuses (Experiment 2). In Experiment 1, the call rates of SNP chip analysis following whole genome amplification in biopsied cells from two elongating conceptuses were 95.14% and 99.32%, which were sufficient for estimating genomic breeding value. In Experiment 2, the rates of dead cells in elongating conceptuses cryopreserved by slow freezing were comparable to those in fresh elongating conceptuses. In addition, we obtained healthy calves by the transfer of elongating conceptuses cryopreserved by slow freezing. Our findings indicate that the elongating conceptus transfer technology enables preimplantation genomic selection in cattle based on SNP chip analysis. Further studies on the optimization of cryopreservation methods for elongating conceptuses are required for practical application of the selection system.

Long-term changes in plasma anti-Müllerian hormone concentration and the relationship with superovulatory response in Japanese Black cattle.

The concentration of circulating anti-Müllerian hormone (AMH) in cattle is a useful endocrine marker for ovarian response to superovulation. Although the AMH concentration undergoes little variation throughout the estrous cycle, its long-term changes remain incompletely understood. Here, we investigated the relationship between superovulation response and plasma AMH concentration in Japanese Black cattle and the long-term changes in plasma AMH concentration of embryo donor cows and heifers. The median, 25th percentile, and 75th percentile of AMH concentrations in 222 mature animals were 0.265, 0.118, and 0.488 ng/ml, respectively. The numbers of ova/embryos, fertilized embryos, and transferable embryos in a total of 295 superovulations were significantly different among the H (AMH ≥ 0.488 ng/ml), M (AMH 0.487-0.119 ng/ml), and L (AMH ≤ 0.118 ng/ml) groups. AMH concentrations during repeated superovulation in ten donor cows were significantly decreased after the third treatment. In heifers, the highest AMH concentration was observed in individuals during 2-13 months of age, with considerable individual variability. AMH concentrations of heifers at 10 or 11 months correlated with the number of ova/embryos during superovulation at 13-18 months (r = 0.641, P < 0.05). These results suggest that the 25th and 75th percentile values of AMH concentration would give a useful rough estimate of ovarian response; however, repeated superovulation may reduce the predictive accuracy of single measurements of AMH concentration. It would be possible to evaluate AMH concentration in heifers after approximately 11 months of age.

Genetic diagnosis of band 3 deficiency using a quenching probe (QProbe)-PCR assay in bovine embryos.

The present study was conducted to develop a simple and rapid procedure to determine the genotype of band 3 deficiency in bovine embryos by a novel real-time PCR method using a fluorescent quenching-based probe (QProbe-PCR). QProbe-PCR successfully distinguished wild type and R664X mutant alleles by melting curve analysis. Minimal amounts of DNA template were required for the detection of wild type/wild type alleles, mutant/mutant alleles, and wild type/mutant alleles; their amounts were 10 pg, 25 pg, and 50 pg, respectively. When 10 blastomeres were used as a DNA sample, accuracies of genotyping by QProbe-PCR were 100% and 89% in embryos homozygous for the wild type allele and heterozygous for the wild type and mutant alleles, respectively. QProbe-PCR takes approximately 2 h for genotyping and requires lesser time than the conventional method using PCR-RFLP, which requires digestion with a restriction enzyme and electrophoresis. Our data showed that QProbe-PCR is a useful method for rapid analysis of the genetic deficiency in preimplantation embryos. Reduction in the time required for genotyping enabled the transfer of genetically selected embryos to recipient cows on the day of embryo collection. These results suggest that determination of the genotype for the genetic deficiency in embryos is useful to select animals free from the genetic disease, and it also makes it possible to produce an animal model homozygous for the mutation.

Embryo sexing and sex chromosomal chimerism analysis by loop-mediated isothermal amplification in cattle and water buffaloes.

In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes.

Differences in apoptotic status in the bovine placentome between spontaneous and induced parturition.

We conducted this study to analyze apoptotic changes in the bovine placentome at spontaneous and induced parturition. Cows delivered i) after the administration of dexamethasone followed by prostaglandin F(2α) and estriol, ii) after the administration of prostaglandin F(2α) and estriol or iii) spontaneously. Prepartum changes in plasma progesterone and estradiol-17ß concentrations were similar between spontaneous and induced parturition. Messenger RNA of BCL2-related protein A1 (BCL2A1), an antiapoptotic gene, was expressed by trophoblast binucleate cells and caruncular epithelial cells. Quantitative RT-PCR showed that the expression of BCL2A1 mRNA in cotyledonary and caruncular portions was significantly lower in spontaneous parturition than induced parturition. The expression of BCL2-associated X protein (BAX) mRNA, a proapoptotic gene, was significantly higher in cotyledons at spontaneous parturition than parturition induced without dexamethasone. Caspase-3 (CASP3) mRNA and pre-activated CASP3 protein were predominantly detected in caruncular epithelial cells regardless of how parturition proceeded. Activated CASP3 protein was found in trophoblast uninucleate cells and binucleate cells rather than caruncular epithelial cells. In spontaneous parturition, intense staining of activated CASP3 was detected in caruncular epithelial cells. Spontaneous and dexamethasone-induced parturition increased apoptotic cells in the placentome compared with parturition induced without dexamethasone. The number of binucleate cells was significantly decreased in spontaneous parturition. The present results suggest that although the clinical dose of dexamethasone induces apoptosis in the placentome at term, neither dexamethasone nor prostaglandin F(2α) evoke normal physiological changes in the placentome during delivery such as a change in the balance of apoptosis-related genes and disappearance of binucleate cells.

Prepartum maternal plasma glucose concentrations and placental glucose transporter mRNA expression in cows carrying somatic cell clone fetuses.

In this study, the plasma glucose concentrations of cows carrying a somatic cell clone fetus during late pregnancy and placental glucose transporter (GLUT) mRNA levels at parturition were examined. Parturition was induced using dexamethasone, prostaglandin F(2α) and estriol in cows bearing a clone (Clone) or a fetus fertilized in vivo as a control (DEX). Plasma glucose concentrations were measured in the cows (days 257 and 271 of pregnancy and at parturition) and newborn calves. Cotyledon and caruncle tissues removed just after parturition were used for mRNA extraction. Expression of mRNA was also analyzed in control cows that were induced to undergo parturition without dexamethasone (PG) or that spontaneously delivered (SP). The glucose concentrations of the Clone group were significantly low at all points examined, but those of the calves were normal. The increase in the maternal glucose concentration from day 257 to parturition was significantly lower in the Clone group. Glucose concentrations were negatively correlated with birth weight for clones (day 257; r=-0.584, day 271; r=-0.286, parturition; r=-0.549). There was no difference in mRNA levels in the cotyledons among the animals examined. In the caruncles, the Clone and PG groups showed significantly higher GLUT1 and GLUT3 mRNA levels than the SP group, and the GLUT3 mRNA level was significantly higher in the Clone group than in the DEX group. The glucocorticoid receptor α mRNA level was significantly lower in the SP group than in the DEX group. Although spontaneous parturition and administration of dexamethasone suppressed the placental GLUT mRNA levels, the action was not observed in clone pregnancy. These results raise the possibility of facilitation of glucose transportation through the placenta to meet increased nutritional requirements of overgrown clone fetuses.

DNA methylation status of bovine blastocyst embryos obtained from various procedures.

DNA methylation is an important factor for the regulation of gene expression in early embryos. It is well known that the satellite I sequence is more heavily methylated in bovine somatic cell nuclear transfer (NT-SC) embryos than in embryos derived from in vitro fertilization (IVF). However, the methylation status of bovine embryos obtained by other procedures is not well known. To clarify DNA methylation levels of bovine embryos obtained from various procedures, we examined satellite I sequences in bovine blastocyst (BC) embryos derived from NT-SC, NT using embryonic blastomeres (NT-EM), in vivo (Vivo), IVF and parthenogenetic treatment (PA). Furthermore, in order to evaluate the efficacy of DNA demethylation by the NT procedure, we determined the DNA methylation levels in bovine embryos in which NT was recapitulated (Re-NT). Although the DNA methylation levels in the NT-SC embryos were higher than those in the other embryos, the NT-EM embryos exhibited lower DNA methylation levels. The satellite I sequence in the NT-SC embryos was more demethylated than that in the donor cells. Although the DNA methylation level in the individual NT-SC embryos showed variation, the full-term developmental efficacy of these embryos were not different. These findings suggest that the methylation level of the satellite I sequence at the BC stage is not related to the abnormalities of bovine embryos produced by NT-SC. There was no difference in methylation levels between Re-NT and NT-SC embryos. Our results indicated that the DNA methylation status differed among embryos produced by various methods and that at least some of the demethylation of the donor cell genome occurred in the recipient cytoplast after NT-SC, but the demethylation ability of the NT procedure was noted in the first NT but not in the second NT.

Day-7 embryos generate an anti-inflammatory immune response in peripheral blood immune cells in superovulated cows.

PROBLEM: Recently, we demonstrated both in vitro and in vivo that an immunological crosstalk between Day-7 embryo and immune cells exists locally in the uterus in cows. The peripheral immune response to early embryos at Day-7 of pregnancy in cows remains largely unknown. Thus, we aimed to investigate the response of peripheral blood immune cells in the presence of multiple Day-7 embryos in the uterus in donor cows of embryo transfer program. METHOD OF STUDY: Superovulated cows were either inseminated (n = 13) at 12-18 hours post-estrus or remained without inseminations (n = 6). Blood was collected following insemination (Day-1) and immediately after uterine flushing (Day-7). Polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood for analysis of gene expression. RESULTS: Interferon-stimulated genes (ISGs: ISG15 and OAS1) were increased in both PMNs and PBMCs, with up-regulation of PTGES and anti-inflammatory cytokines (TGFB1 and IL10) expressions at Day-7 of post-inseminations in cows, when compared to those at Day-7 in non-inseminated cows. CONCLUSION: The findings indicate that the presence of multiple embryos in the uterus generates an anti-inflammatory immune response in peripheral blood immune cells at Day-7 of pregnancy in cows, which might help to accept semi-allogenic embryo in the uterus.

Enhanced glucocorticoid exposure facilitates the expression of genes involved in prostaglandin and estrogen syntheses in bovine placentomes at induced parturition.

The mechanism by which the fetal membrane detaches after parturition in cattle is poorly understood, but the upregulation of placentomal prostaglandin and estrogen synthesis are considered to be important. This study investigated whether enhanced glucocorticoid exposure affected the functional maturation of placentomes at induced parturition. Placentomes were collected immediately after spontaneous (beef; n = 5, dairy; n = 5) or induced parturition in beef and dairy cattle. Parturition was induced conventionally using prostaglandin F2α (beef; n = 7, dairy; n = 6) or dexamethasone (beef; n = 6) or with a combination of triamcinolone acetonide (a long-acting glucocorticoid) and a high dose of betamethasone (TABET treatment, beef; n = 6, dairy; n = 9). Gene expression levels and protein localization in placentomes were analyzed by RT-qPCR and immunohistochemistry, respectively. Compared with the conventional methods, TABET treatment resulted in upregulated PTGS2 expression in cotyledons. The expression levels of PTGS2 and PGES were positively correlated in both cotyledons and caruncles. TABET treatment also upregulated the expression of CYP17A1, but not of CYP19A1, in cotyledons. The results revealed, for the first time, that PLA2G4A was localized in microvascular endothelial cells in the cotyledonary villi and the maternal septum. PTGS2 and PGES were colocalized in mononucleated cells of the cotyledonary villi and caruncle epithelial cells adjacent to the chorionic plate. TABET treatment upregulated the expression of placentomal genes involved in PGE2 synthesis and the conversion of pregnenolone to androstenedione. Thus, enhanced glucocorticoid exposure might partially facilitate the functional maturation of placentomes at induced parturition in cattle.

Effects of genetic background on responses to superovulation in Japanese Black cattle.

We investigated the effects of genetic background on the responses to superovulation in Japanese Black cattle. The genotype frequencies of GRIA1 and FSHR relating to ovulation and follicular development in each of the major bloodlines-Tajiri, Fujiyoshi, and Kedaka-were analyzed. The Tajiri line had the lowest frequency of G allele homozygosity of c.710A>G in GRIA1 among the three bloodlines, and deviation from Hardy-Weinberg equilibrium was detected. Genotype frequencies of c.337C>G, c.871A>G, and c.1973C>G in FSHR were in Hardy-Weinberg equilibrium in all bloodlines. The results of generalized linear mixed-model analyses showed that farm, levels of plasma anti-Müllerian hormone (AMH) concentration, age in months, repeated superovulation, c.337C>G in FSHR, and bloodlines had significant effects on the responses to superovulation. The number of transferable embryos in the group heterozygous for c.337C>G in FSHR was significantly higher than that in the group homozygous for the C allele. The Kedaka line showed a significantly higher number of ova/embryos, fertilized embryos, and transferable embryos than the Tajiri and Fujiyoshi lines. The concentration of circulating AMH is a useful endocrine marker for antral follicle counts. This study revealed the effects of genetic background on the responses to superovulation using levels of plasma AMH concentration as a covariate. The prominent effect of genetic background on superovulation in the Kedaka line requires additional studies to confirm the genomic regions and polymorphisms that are involved in the trait.

Excess estrogen sulfoconjugation as the possible cause for a poor sign of parturition in pregnant cows carrying somatic cell clone fetuses.

We conducted this study to elucidate a factor causing a poor sign of parturition and prolonged gestation, which is frequently observed in cows carrying somatic clone fetuses. Pre-partum rises in concentrations of plasma estrone and estradiol-17beta in the recipient cows pregnant with clones were subtle. By contrast, the plasma concentration of estrone sulfate in clone pregnancies increased gradually from pre-initiation of parturition induction whereas control cows that received in vivo-derived embryos showed a significant increase at parturition. Therefore, in clone pregnancies, the ratio of estrone/estrone sulfate was low during the pre-partum period compared with control. Messenger RNA expression of estrogen sulfotransferase (SULT1E1) in the placenta at parturition was significantly higher in clone pregnancies than control pregnancies and was localized in binucleate cells (BNC). SULT1E1 mRNA abundance was negatively and positively correlated with concentrations of maternal estrone and estrone sulfate at parturition respectively. Messenger RNA expressions of estrogen sulfatase (STS) and aromatase (CYP19) were similar between clone and control pregnancies and were localized in BNC and caruncular epithelial cells. STS and CYP19 mRNA abundances showed positive correlations with maternal estradiol-17beta concentration. The population of BNC in the placenta did not differ between clone and control pregnancies. Plasma cortisol concentration of vaginally delivered newborn clone calves was comparable with those of control, although cesarean section delivered clone calves showed a low concentration. These results suggest that excess estrogen sulfoconjugation is the reason for the perturbed low ratio of active to inactive estrogens and the resulting hormonal imbalance contributes to the lack of overt signs of readiness for parturition in cows pregnant with clones.

Rapid sex chromosomal chimerism analysis in heterosexual twin female calves by Loop-mediated Isothermal Amplification.

We attempted to apply an embryo sexing kit with Loop-mediated Isothermal Amplification (LAMP) to sex chromosomal chimerism analysis in heterosexual twin female calves. Peripheral blood was used for the amplification of male-specific DNA, derived from XY leukocytes. When blood samples were diluted 1:1000 in LAMP reaction mixture, hemoglobin or blood coagulation did not influence the turbidity measurement of the reaction mixture for detection of amplified DNA. This procedure detected the existence of XY leukocytes of 0.01% in female blood. Furthermore, all heterosexual twin female calves, bearing sex chromosomal chimerism based on karyotyping and PCR, showed male-specific DNA from peripheral blood by LAMP. These results indicated that the embryo sexing kit with LAMP was available for sensitive detection of sex chromosomal chimerism. This procedure made it possible to detect easily Y-chromosome specific DNA in a short interval compared with PCR, and was convenient for field application of freemartin diagnosis.