Expression and localization of anti-Müllerian hormone and its receptors in bovine corpus luteum.

Although anti-Müllerian hormone (AMH) is involved in the regulation of granulosa cell function in female animals, its role in tissues other than ovarian follicles remains poorly understood. It has also been suggested that cows with high circulating AMH concentrations have increased fertility; however, the mechanism has not been elucidated. This study was conducted to identify the presence of the AMH-signaling system and its target cells in the bovine corpus luteum formed from an ovulated follicle. Immunoblotting revealed that the proteolytically cleaved C-terminal region in AMH (AMHC), a biologically active peptide, was present in trace amounts in the early corpus luteum and significantly increased during the mid to regressed stages. AMHC and cleaved N-terminal region (AMHN) in AMH generate a noncovalent isoform that improves the activity of AMH signaling. An immunohistochemical analysis revealed that AMHC, AMHN, and type II AMH receptor (AMHR2) were localized to luteal cells during the entire estrous cycle. AMH in the corpus luteum seemed to be newly synthesized since AMH expression was detected. These findings suggest that AMH signaling is involved in the regulation of luteal cell function through an autocrine and post-translational processing mechanism. The level of AMHR2 and mRNA expression of AMHR2 and type I AMH receptors (activin-like kinase 2, 3, and 6) were highest in the mid stage. Thus, AMH signaling in the corpus luteum may also be regulated by changes in the receptor levels. Since the transforming growth factor-beta superfamily, to which AMH belongs, is a multifunctional polypeptide growth factor, further studies are needed to evaluate whether AMH signaling has a role in facilitating or inhibiting luteal cell functions.

Calving prediction with continuous measurement of subcutaneous tissue glucose concentration in pregnant cows.

To reduce losses of dams and calves due to unfortunate events, such as dystocia and freezing to death, identifying the onset of calving and providing necessary assistance are crucial. Prepartum increase in blood glucose concentration is a known indicator to detect labor in pregnant cows. However, some issues, including the need for frequent blood sampling and stress on cows, must be resolved before establishing a method for anticipating calving using changes in blood glucose concentrations. Herein, instead of measuring the blood glucose concentrations, subcutaneous tissue glucose concentration (tGLU) was measured in peripartum primiparous (n = 6) and multiparous (n = 8) cows at 15 min intervals using a wearable sensor. A transient increase in tGLU was observed in the peripartum period, with peak individual concentrations occurring between 2.8 h before and 3.5 h after calving. tGLU in primiparous cows was significantly higher than that in multiparous cows. To account for individual variations in basal tGLU, the maximum relative increase in the 3-h moving average of tGLU (Max MA) was used to predict calving. Cutoff points for Max MA were established by parity, with receiver operating characteristic analysis predicting calving within 24, 18, 12, and 6 h. Except for one multiparous cow that showed an increase in tGLU just before calving, all cows reached at least two cutoff points and calving was predicted successfully. The time interval between reaching the tGLU cutoff points that predicted calving within 12 h and actual calving was 12.3 ± 5.6 h. In conclusion, this study demonstrated the potential role of tGLU as a predictive indicator of calving in cows. Advancements in machine learning-based prediction algorithms and bovine-optimized sensors will help in increasing the accuracy of calving prediction using tGLU.

Genetic relationships among emu populations in Japanese farms based on mitochondrial and microsatellite DNA polymorphisms.

Emus (Dromaius novaehollandiae) are expected to become a novel poultry species for producing eggs, meat, and oil. In our previous studies, Japanese emu populations were predicted to have reduced genetic diversity through inbreeding. For a sustainable emu industry in Japan, it is necessary to understand the current genetic structure and relationships in dispersed farms. In this study, we investigated the genetic structure and relationships of six Japanese emu farms based on mitochondrial DNA and microsatellite polymorphisms. We analyzed the DNA sequences of the mitochondrial D-loop region in 157 individuals and detected four haplotypes with four nucleotide substitution sites (Hap-a, Hap-b, Hap-c, and Hap-d). Analysis of molecular variance revealed that 43.6% of total variance was "among population," and the FST value was 0.436 with significant genetic differentiation (P < 0.001). In microsatellite analysis, the expected (HE ) and observed (HO ) heterozygosities were 0.53-0.64 and 0.44-0.59, respectively. Phylogenetic trees and STRUCTURE analysis revealed that the six Japanese farmed emu populations could be divided into four genetically differentiated groups. Therefore, we identified genetic resources that may be useful in extending the genetic diversity of Japanese farms and are predicted to contribute to the conservation and reconstruction of populations.

Carcass traits and fat quality of breeding emu (Dromaius novaehollandiae) in Northern Japan.

Characterization of carcass traits and fat quality is important to effectively produce and genetically improve emus. We investigated carcass traits in 309 emus. The meat production of female emus showed a significantly higher value than that of males (P < 0.01). The fat weight of male (9.232 ± 3.156 kg) was larger than that of the female (7.772 ± 2.697 kg). The fat yield (fat weight per kg of body weight) was strongly correlated to body weight (r = 0.79 and r = 0.75 in male and female, respectively). The fat melting points of females and males were 19.19 ± 3.39°C and 19.39 ± 3.39°C, respectively, without significant difference. Since the fat melting point did not correlate to body and fat weights, we predicted that it was an independent trait from body growth and was highly influenced by genetic elements. Percentages of palmitic, stearic, oleic, linoleic, and α-linolenic acids were 22.27 ± 3.50%, 9.37 ± 1.90%, 54.11 ± 5.17%, 13.54 ± 7.80% and 0.71 ± 0.59%, respectively. Among them, linoleic acid contents showed a wide individual difference (range 0.3-19.9%). The oleic/stearic acid ratio showed a negative correlation to the fat melting point. These results suggest that the fat melting point is a good indicator of C18:1/C18:0 ratio in emu fat.

Impaired placentomal interferon signaling as the possible cause of retained fetal membrane in parturition-induced cows.

Although hormonal induction of parturition in cattle results in the successful delivery of healthy calves, the risk of retained fetal membrane is significantly increased. In a previous study, a combination of the long-acting glucocorticoid, triamcinolone acetonide, with a high dose of betamethasone partially normalized the placentomal gene expression during parturition; however, the incidence of retained fetal membrane remained high. This study further explored placentomal dysfunction and aimed to elucidate the mechanism of retained fetal membrane in parturition-induced cows. In this study, transcriptome analysis revealed that enhanced glucocorticoid exposure normalized the expression of a substantial fraction of genes in the cotyledons. In contrast, a significant reduction in the multiple signaling pathway activities, including interferon signaling, was found in the caruncles during induced parturition. Real-time PCR showed that the expression of interferon-tau in the caruncles, but not interferon-alpha or interferon-gamma, was significantly lower in induced parturition than spontaneous parturition. Interferon-stimulated gene expression was also significantly decreased in the caruncles during induced parturition. These results indicate that interferon signaling could be important for immunological control in placentomes during parturition. Additionally, this suggests that interferon-tau might be a pivotal ligand for interferon receptors in the caruncles. This study revealed that peripheral blood leukocytes in prepartum cows transcribed interferon-tau. Macrophage infiltration in the placentome is known to participate in the detachment of the fetal membrane from the caruncle. Thus, this study raised the possibility that immune cells migrating into the caruncles at parturition may act as a source of ligands that activate interferon signaling.

Synthesis of mixed chitin esters with long fatty and bulky acyl substituents in ionic liquid.

This study revealed that mixed chitin esters with long fatty and bulky acyl substituents were efficiently synthesized by acylation using acyl chlorides in the presence of pyridine and N,N-dimethyl-4-aminopyridine in an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), at 100 °C for 24 h. A stearoyl group was selected as the first substituent, which was combined with different long fatty and bulky acyl groups as the second substituents. In addition to IR analysis of the products, which suggested progress of the acylation, 1H NMR measurement was allowed for structural confirmation for high degrees of substitution (DSs) of the desired derivatives in CDCl3/CF3CO2H solvents. Crystalline structures and thermal property of the products were evaluated by powder X-ray diffraction and differential scanning calorimetry measurements, respectively. All the products showed film formability by casting from solutions in chloroform or chloroform/trifluoroacetic acid solvents. The occurrence of halogen exchange between acyl chlorides and AMIMBr in the present system was speculated to produce highly reactive acyl bromides in situ, which efficiently reacted with hydroxy groups in chitin to obtain high DS products.

Development of 49 novel microsatellite markers from Next-generation sequencing data and a robust method for parentage tests in the emu (Dromaius novaehollandiae).

The emu is a useful and new breed of poultry, but their genetic improvement has not advanced yet due to their very recent domestication. Pedigree information is difficult to record because of their complex reproduction system (polyandry). To identify parent-offspring relationships in the emu, parentage test based on polymorphic DNA markers have to be developed. In this study, we isolated more than 25,000 microsatellite (simple sequence repeat, SSR) regions from Next-generation sequencing data via the QDD pipeline and developed 49 SSR markers with polymorphism in the Japanese farmed emu. The dinucleotide motifs, (AC)n, (AT)n and (AG)n, were the most frequently detected and were found on 10,167 (38.55%), 8,114 (30.76%) and 4,796 (18.18%) contigs, respectively. Forty-nine novel SSR markers were characterized in 20 individuals and showed NA ranged from 2 to 12, with an average of 4.2. HE/HO ranged from 0.389/0.071 to 0.702/1.000 with an average of 0.601/0.515. PIC value ranged from 0.059 to 0.886 with an average of 0.528, and 17 of 49 markers showed a higher polymorphism than 0.500. Thirty-four individuals were genotyped using 12 markers, and CERVUS simulations based on genotype showed that parents of all offspring were identified with 0.9995-1.0 probability. Thus, 49 novel SSR markers and a robust method for parentage test for the Japanese emu were developed.

Mitochondrial DNA variations in Japanese farmed emu populations.

The emu (Dromaius novaehollandiae) is a new poultry. In this study, we investigated the haplotype composition of mitochondrial DNA among emu populations farmed in Japan. We sequenced the D-loop region in 109 individuals, and detected four substitution sites and three haplotypes (Hap-a, -b, and -c). Hap-a was the most frequently observed haplotype in the Japanese populations. Although Hap-c was a rare haplotype in not only Japanese but also Australian populations, it was detected with high frequency in the Japanese farmed population. The AMOVA indicated that 9% of total variance was "among population". The FST value was 0.087 and genetic differentiation was significant (P<0.01). These results may contribute to conserving the genetic resources available for the Japanese emu industry.

Development and characterization of ten novel microsatellite loci for the emu (Dromaius novaehollandiae) and genetic diversity of Japanese farm populations.

The emu (Dromaius novaehollandiae) is a useful poultry animal farmed for fat, meat, and eggs. Genetic structure and relationships among farmed emu populations in Japan are unknown and the number of microsatellite markers for genetic analysis of the emu is insufficient. In this study, we isolated 16 microsatellites from the emu genome and developed ten new microsatellite markers. These microsatellite markers were used to characterize three farm emu populations in Japan. The number of alleles ranged from 3 to 13 and the expected (HE) and observed heterozygosity (HO) of these microsatellite loci was 0.187-0.802 and 0.179-0.647, respectively. The polymorphic information content ranged from 0.176 to 0.786. Positive inbreeding coefficient (FIS) values were detected in all tested populations, and they ranged from 0.027 to 0.540. These results suggest that farm populations of the emu in Japan resulted from inbreeding. The fixation index (FST) values ranged from 0.026 to 0.061, and phylogenetic trees and population structure analysis confirmed no definitive genetic differentiation among the three populations. Therefore, these populations are at a relatively low level of genetic differentiation at present. The microsatellite markers developed in our study can be utilized for genetic analysis and preservation of genetic resources in the emu.

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.

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.

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.

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.

Facile acylation of α-chitin in ionic liquid.

Based on the fact that an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), efficiently dissolved α-chitin, this study investigated the development of facile acylation reactions of α-chitin in AMIMBr media. Under optimal conditions in the presence of pyridine and N,N-dimethyl-4-aminopyridine as base and catalyst, respectively, lauroylation of α-chitin smoothly took place using lauroyl chloride in AMIMBr at 100 °C for 24 h to produce a chitin laurate with a high degree of substitution (DS). Chitin acylates having different substituents were also synthesized by acylation of α-chitin using various acyl chlorides under the same conditions. In addition to IR analysis of the products, 1H NMR measurement was allowed for structure confirmation owing to the dissolution of the high DS derivatives in CDCl3/CF3CO2H mixed solvents.

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.

The Necessity of OCT-4 and CDX2 for Early Development and Gene Expression Involved in Differentiation of Inner Cell Mass and Trophectoderm Lineages in Bovine Embryos.

The functions of POU class 5 transcription factor 1 (Oct-4) and caudal-type homeobox 2 (Cdx2) in the differentiation of the murine inner cell mass (ICM) and trophectoderm (TE) have been described in detail. However, little is known about the roles of OCT-4 and CDX2 in preimplantation bovine embryos. To elucidate their functions during early development in bovine embryos, we performed OCT-4 and CDX2 downregulation using RNA interference. We injected OCT-4- or CDX2-specific short interfering RNAs (siRNAs) into bovine zygotes. The rate of blastocyst development of OCT-4-downregulated embryos was lower compared with uninjected or control siRNA-injected embryos. Gene expression analysis revealed decreased CDX2 and fibroblast growth factor 4 expression in OCT-4-downregulated embryos. CDX2-downregulated embryos developed to the blastocyst stage; however, in most cases, blastocoel formation was delayed. Gene expression analysis revealed decreased GATA3 expression and elevated NANOG expression in CDX2-downregulated embryos. In conclusion, OCT-4 and CDX2 are essential for early development and gene expression involved in differentiation of ICM and TE lineages in bovine embryos.

Regulation of bovine oviductal NO synthesis by follicular steroids and prostaglandins.

Nitric oxide (NO) is a regulator of sperm motility, oocyte/embryo survival, and waves of contraction/relaxation in mammalian oviducts. As follicles control oviductal functions by two routes at least, (1) a systemic way via blood vessels before ovulation, (2) a direct way by entering of follicular fluid through fimbria at ovulation, we hypothesized that NO synthesis in the bovine oviduct is regulated by follicular steroids and prostaglandins (PGs). Quantification of mRNA expressions in the ampullary tissues showed that inducible NO synthase (NOS2) mRNA expression was highest on the day of ovulation (day 0). By contrast, NOS2 mRNA expression in the isthmus was highest on days 5-6 and lowest on days 19-21. Endothelial NOS (NOS3) mRNA expressions in either the ampulla or the isthmus did not change during the estrous cycle. PGE2 and PGF2α increased NOS2 mRNA expressions in cultured ampullary oviductal epithelial cells after 1-h incubation. These increases were suppressed by an antagonist of E-prostanoid receptor type 2, one of the PGE2 receptor. Estradiol-17ß decreased the expression of NOS2 mRNA expression in cultured isthmic epithelial cells 24h after treatment. This effect was suppressed by an antagonist of estrogen receptorα(ESR1). Expression of ESR1 was highest on days 19-21 in the isthmic tissues. The overall findings indicate region-specific difference of NO synthesis in the oviduct. PGs flowed from ruptured follicle may up-regulate NO synthesis in the oviductal epithelium, whereas circulating E2 seems to inhibit NO synthesis via ESR1 in the isthmus at the follicular stage.

Localization of TGF-ß and TGF-ß receptor in bovine term placentome and expression differences between spontaneous and induced parturition.

INTRODUCTION: Mechanisms of detachment of fetal membrane after parturition in cattle are poorly understood. Glucocorticoids trigger the initiation of parturition and may facilitate the placental maturation. We compared the disappearance of trophoblast binucleate cells (BNCs) and expression of transforming growth factor-ß (TGFB) in term placentomes between spontaneous and induced parturition to investigate the influences of glucocorticoids on the placental maturity. METHODS: Cows were delivered spontaneously (SP group) or after the administration of prostaglandin (PG) F(2)α (PG group); dexamethasone, PGF(2)α, and estriol (DEX group); and triamcinolone acetonide, PGF(2)α, and betamethasone (BET group) and placentomes were collected immediately after parturition. The number of BNCs in hematoxylin and eosin stained section was examined. Protein localization and mRNA levels of TGFB and its receptor (TGFBR) were analyzed using immunohistochemistry and qRT-PCR, respectively. RESULTS: TGFB1 is characteristically localized in the maternal septum in caruncle in contrast to TGFB2 and TGFB3, which are mainly found in cotyledonary villi and maternal epithelial cells. TGFBR1 and TGFBR2 colocalized in BNCs. The number of BNCs was lower in the SP group than in PG and DEX groups. mRNA levels of TGFB1, TGFBR1 and TGFBR2 in the SP group differed from PG and DEX groups. There was no difference between SP and BET groups in all analyses. DISCUSSION: These results indicate that parturition inductions using PGF(2)α or dexamethasone were not able to induce disappearance of BNCs and change of TGFB signaling. Results in the BET group suggest that investigation into types, dose, and dosage schedule of glucocorticoids may facilitate placental maturation.