Epigenetic systems play crucial roles in the differentiation of a mammalian fertilized egg into hundreds of cell types exhibiting distinct phenotypes, using a set of DNA molecules comprising about 3 billion nucleotides. Genome-wide analyses of epigenetic marks have revealed the remarkably well-established and well-maintained structure of the epigenome, consisting of DNA methylation and histone modifications that vary their state in a tissue type- and developmental stage-specific manner at numerous genomic loci. DNA methylation profiles comprising numerous tissue-dependent and differentially methylated regions (T-DMRs), found at such loci, are unique to every type of cell and tissue, and illuminate molecular networks that represent their phenotypes. T-DMRs are located in not only genic but also nongenic regions-including transposable genetic elements, such as short interspersed transposable element. Epigenetic studies indicate that the molecules that perform these modifications directly, such as DNA methyltransferases and eukaryotic histone methyltransferases, or indirectly, such as CpG-binding protein and noncoding RNAs-and combinations of these-contribute to the DNA methylation profile. It remains to be addressed how these molecules precisely find their target genomic loci.

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The peptidylarginine deiminases (PADs) are a family of calcium-dependent enzymes that post-translationally convert positively charged arginine residues to neutrally charged citrulline in a process called citrullination. There are five PAD family members (PAD1-4 and 6), each with unique tissue distribution patterns and functional roles including: cellular differentiation, nerve growth, apoptosis, inflammation, gene regulation, and early embryonic development. Previous review articles have focused on the expression and function of PADs and on their catalytic activity, citrullination, while other, more recent reviews have addressed the role of these enzymes in disease [1-3]. What has not been previously reviewed in any level of detail is the role that PAD proteins play in female reproduction. Given that: (1) PAD family members are highly represented in female reproductive tissues, (2) that some of the earlier PAD literature suggests that PADs play a critical role in female reproduction, and (3) that our studies have demonstrated that oocyte and early embryo restricted PAD6 is essential for female reproduction, we felt that a more comprehensive review of this topic was warranted.

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Hemochorial placentation is characterized by trophoblast-directed uterine spiral artery remodeling. The rat and human both possess hemochorial placentation and exhibit remarkable similarities regarding the depth of trophoblast invasion and the extent of uterine vascular modification. In vitro and in vivo research methodologies have been established using the rat as an animal model to investigate the extravillous/invasive trophoblast lineage. With these research approaches, two signaling pathways controlling the differentiation and invasion of the trophoblast cell lineage have been identified: i) hypoxia/hypoxia inducible factor and ii) phosphatidylinositol 3-kinase/AKT/Fos like antigen 1. Dissection of these pathways has facilitated identification of fundamental regulators of the invasive trophoblast cell lineage.

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The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P›0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P%lsaquo;0.05) on days 11-16 relative to other days of the cycle. The highest mRNA expression of PGRMC1, SERBP1 and PR was found during pregnancy. There were no changes (P›0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P‹0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

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The objective of the present study was to clarify the possible role of the zona pellucida (ZP) in early development of rat embryos and to determine the effect of glycosaminoglycans on the development of ZP-free 8-cell embryos before or after embryo transfer at the blastocyst stage. Eight-cell embryos were divided into three groups comprised of, 1) intact controls, 2) embryos with the ZP was removed with acidic solution and 3) pairs of ZP-free 8-cell embryos aggregated in a small hollow. These embryos were cultured in a chemically defined mR1ECM for 24 h. Developmental ability to the blastocyst stage and mean cell number in the blastocyst was lower in ZP-free embryos than in intact controls. When these blastocysts were transferred, the farrowing rate and efficiency of embryos developed to term were also lower in ZP-free embryos, but not in the aggregated ones. Supplementation with hyaluronan (HA; 63-250 μg/ml) or heparan sulfate proteoglycan (HS; 15 μg/ml) significantly improved blastocyst formation of ZP-free embryos and the cell number in the blastocyst by reducing the incidence of apoptosis. However, there were no beneficial effects of HA or HS on farrowing and newborn rates after transfer of the blastocysts. In conclusion, the ZP plays roles in maintaining successful development of early rat embryos at least from the 8-cell stage not only to the blastocyst stage but also to posttransfer stages. Glycosaminoglycans, such as HA or HS, appear to contribute to successful cleavage during early development to the blastocyst stage but may be insufficient to maintain the posttransfer survival of ZP-free embryos.

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We examined the comprehensive epigenetic status, including histone H3 and H4 acetylation, DNA methylation and level of mRNA transcripts of bovine somatic cell nuclear transfer (SCNT) embryos treated with trichostatin A (TSA), along with their full-term developmental efficacy. Treatment with 50 nM TSA enhanced early developmental competence; increased acetylation of two histones, H3K9K14 and H4K8, at the blastocyst stage; and maintained the DNA methylation status of the satelliteI sequence in bovine SCNT embryos. The difference in IGFBP-3 transcript levels between in vivo and SCNT embryos disappeared in SCNT embryos after treatment with 50 nM TSA. Pregnancy, full-term developmental competence and body weight at birth of offspring did not differ between SCNT embryos treated with 50 nM TSA and untreated embryos. These results suggest that treatment with TSA improves preimplantation development and changes the epigenetic status but does not promote the full-term development competence in bovine SCNT embryos.

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Anti-Müllerian hormone (AMH) and testosterone (T) profiles in blood were investigated before and after an hCG stimulation test to assess their sensitivity and specificity for the existence of a functional cryptorchid testis in Japanese Black calves. The hCG (3,000 IU) was administered on Day 0, and peripheral blood was collected on Days 0 (just before hCG injection), 5 and 7 in intact male calves (Intact; n=19), bilateral castrated calves (Castrated; n=17), unilateral cryptorchid calves, which abdominal testis could been extracted (Uni-crypto; n=9). Castration of a descended testis was carried in the Castrated and Uni-Crypto groups on Day -14. The AMH detectability and the optimum cut-off point for T levels using the receiver operating characteristic curve were verified to characterize the cryptorchid testis. AMH values on Day 0 were 21.1 ± 5.1 and 29.0 ± 7.5 ng/ml in the Intact and Uni-crypto groups, respectively (Mean ± SEM). AMH levels were under the detection limit in the Castrated group (i.e., < 0.006 ng/ml). T showed its peak levels on Day 5 in the Intact group (26.8 ± 4.2 ng/ml), while it remained low in the Castrated group (< 0.9 ng/ml) and did not show a significant difference in the Uni-crypto group. The detectable levels for AMH was 0.006 ng/ml, and the optimum cut-off point for T was 0.9 ng/ml; the sensitivity and specificity for evaluation of testicular descent into the scrotum were 1.0 for both the AMH and T levels. The detection rates in the Uni-crypto group using them were 1.0 and 0.57 for AMH on Day 0 and T on Days 5 or 7, respectively. In conclusion, plasma AMH profiles could be used as a novel biomarker to evaluate the existence of a functional cryptorchid testis in Japanese Black calves.

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Several types of cells, including blastoderm cells, primordial germ cells, and embryonic germ cells were injected into early-stage recipient embryos to produce chimera avians and to gain insights into cell development. However, a limited number of studies of avian adult stem cells have also been conducted. This study is, to the best of our knowledge, the first to evaluate chicken bone marrow cells’ (chBMC) ability to differentiate into multiple cell lineages and capability to generate chimera chicks. We induced random differentiation of chBMCs in vitro and injected immunologically selected pluripotent cells in chBMCs into the blastoderms of recipient eggs. The multipotency of BMCs from the barred Plymouth rock (BPR) was confirmed via AP staining, RT-PCR, immunocytochemistry, and FACS using specific markers, such as Oct-4 and SSEA-1, 3 and 4. Isolated chBMCs were found to be able to induce in vitro differentiation to multiple cell lineages. Approximately 5,000 chBMCs were injected into the blastoderms of white leghorn (WL) recipients and proved able to contribute to the generation of somatic chimera chicks with a frequency of 2.7% (2 of 73). Confirmation of chimerism in hatched chicks was achieved via PCR analysis using D-loop-specific primers of BPR and WL. Our study demonstrated the successful production of chimera chicks using chBMC. Therefore, we propose that the use of adult chBMCs may constitute a new possible approach to the production of chimera poultry, and may provide helpful studies in avian developmental biology.

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Interspecies/intergeneric mitochondrial heteroplasmy can occur in interspecies/intergeneric hybrid embryos or following nuclear transfer. In the present study, intergeneric buffalo (Bubalus bubalis) mitochondria (WB-mt) or interspecies murine (Mus spretus) mitochondria (M-mt) were injected into bovine (Bos taurus) oocytes, and the subsequent embryonic development was characterized. Fibroblast mitochondria (WB-mt or M-mt) were microinjected into in vitro matured bovine oocytes followed by oocyte activation by a combination of electrical stimulation and 6-dimethylaminopurine treatment. After seven days of culture, embryo development was evaluated. The copy number of specific mtDNA populations (introduced and native mtDNA) from heteroplasmic oocytes was estimated using real-time PCR. The results illustrated that oocytes injected with either WB-mt or M-mt can develop to the blastocyst stage (20.6% and 19.6%). Cleavage division rates and development to the morula stage in oocytes injected with WB-mt were lower (76.2% and 45.9%, respectively) in comparison with uninjected oocytes (89.2% and 59.1%, respectively) (P<0.05). However, no differences were found in comparing M-mt injected oocytes and controls (P>0.05). An increase in bovine mtDNA copy number was observed at the expanded blastocyst stage of injected embryos (P<0.01), while the number of injected mtDNA was stable throughout development. This study demonstrates that interspecies/intergeneric mitochondrial injected bovine oocytes have the ability to develop to the blastocyst stage after parthenogenetic activation and that injected mtDNA was neither selectively destroyed nor enhanced through development. Moreover, injected intergeneric mitochondria had a demonstrated influence on bovine parthenogenetic development and mtDNA replication.

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Mammalian fertilization requires sperm to penetrate the cumulus to reach the oocyte. Although sperm hyaluronidase has long been believed to participate in the penetration process, our previous works revealed that neither of two sperm hyaluronidases, SPAM1 and HYAL5, are essential for fertilization. In this study, we have produced double-knockout mice lacking SPAM1 and either one of two sperm serine proteases, ACR and PRSS21, and characterized the mutant sperm. The SPAM1/ACR- and SPAM1/PRSS21-deficient males were fertile, whereas epididymal sperm of the mutant mice exhibited a reduced capacity to fertilize the oocytes in vitro. Despite normal motility, the ability of sperm to traverse the cumulus matrix was more severely impaired by the loss of SPAM1 and ACR or SPAM1 and PRSS21 than by the loss of only SPAM1. Moreover, SPAM1/ACR- and SPAM1/PRSS21-deficient sperm accumulated on the surface (outer edge) of the cumulus more abundantly than SPAM1-deficient sperm. These results suggest that ACR or PRSS21 or both may function cooperatively with SPAM1 in sperm/cumulus penetration.

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Alpha-Zearalenol (α-ZEA) is one of derivatives from Zearalenone (ZEA) which impacts mammalian reproduction and development. Previous studies have shown that pigs are sensitive to the estradiol-like effects of α-ZEA. However, the effect of α-ZEA for the early embryonic development has not been fully studied. The objective of this study was to identify the direct toxicity of α-ZEA on porcine preimplantation embryonic development, embryo quality and expression of developmentally important genes. Presumptive zygotes were cultured in porcine zygote medium 3 (PZM-3) in the presence of α-ZEA (n=2,957) or 17β-estradiol (E2) (n=1,333) dissolved in 0.1% Dimethyl Sulfoxide (DMSO) from 24 to 84 h post insemination followed by determination of apoptotic cell numbers and transcript levels of BAX, BCL2L1 and POU5F1 in blastocysts. Cleavage rates on day 2 were significantly decreased in 10, 30 and 60 μM α-ZEA groups; whereas blastocyst rates on day 6 were significantly decreased in the 30 and 60 μM of α-ZEA groups. Only the 100 μM E2 group significantly decreased cleavage and blastocyst rates. Total cell numbers (TCN) in blastocysts were significantly lower in the 10 μM α-ZEA group, but no differences in apoptotic cell rates were found. The expression levels of POU5F1 and BCL2L1 transcripts were similar; however, levels of BAX transcripts and the BAX/BCL2L1 ratio were increased in both α-ZEA groups. Since α-ZEA and E2 did not elicit similar effects, results suggest that α-ZEA might impact porcine preimplantation embryonic development through pathways other than estrogen receptor binding.

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In this study, it was demonstrated that tetraploid-derived blastocyst embryos had very few Oct4-positive cells at the mid-blastocyst stage and that the inner cell mass at biomarkers Oct4, Sox2 and Klf4 was expressed at less than 10% of the level observed in diploid blastocysts. In contrast, trophectoderm-related gene transcripts showed an approximately 10 to 40% increase. Of 32,996 individual mouse genes evaluated by microarray, 50 genes were differentially expressed between tetraploid or diploid and parthenote embryos at the blastocyst stage (P<0.05). Of these 50 genes, 28 were more highly expressed in tetraploid-derived blastocysts, whereas 22 were more highly downregulated. However, some genes involved in receptor activity, cell adhesion molecule, calcium ion binding, protein biosynthesis, redox processes, transport, and transcription showed a significant decrease or increase in gene expression in the tetraploid-derived blastocyst embryos. Thus, microarray analysis can be used as a tool to screen for underlying defects responsible for the development of tetraploid-derived embryos.

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The platelet-derived growth factor (PDGF) system is expressed and can exert its biological role in the male reproductive system including the maintenance of morphological structure and function of the epididymis. The aim of this study was to clarify the relationship between the PDGF system and seasonal changes in morphology of the wild ground squirrel epididymis during the breeding and nonbreeding seasons. Hematoxylin-eosin (HE) staining was used to observe the epididymal morphology and histology. Immunohistochemistry and Western blotting were performed to detect the immunoreactivities of PDGF-A and B and PDGFR-α. Significant seasonal changes in epididymal morphology were observed in the breeding and nonbreeding seasons. The proportions of the three compartments (interstitial tissue, epithelium and lumen of the duct) revealed distinct variances. Strong immunostaining of PDGF-A was present in the myoid cell and on the sperm in the breeding season, whereas there was a faint signal in the myoid cell in the nonbreeding season. PDGFR-α was expressed in all cell types of the epithelium throughout the whole seasonal cycle, and immunostaining of PDGFR-α in the breeding season was significantly stronger compared with that of the nonbreeding season. PDGF-B was not detected in the epididymis of wild ground squirrels. These results suggested that seasonal morphological changes in epididymis were correlated with immunoreactivities of PDGF-A and its receptor PDGFR-α and that PDGF-A and PDGFR-α might function as paracrine, autocrine or apocrine factors in wild ground squirrels.

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We have previously suggested that TEX19, a mammalian-specific protein of which two paralogs exist in rodents, could be implicated in stem cell self-renewal and pluripotency. We have established here the expression profiles of Tex19.1 and Tex19.2 during mouse development and adulthood. We show that both genes are coexpressed in the ectoderm and then in primordial germ cells (PGCs). They are also coexpressed in the testis from embryonic day 13.5 to adulthood, whereas only Tex19.1 transcripts are detected in the developing and adult ovary as well as in the placenta and its precursor tissue, the ectoplacental cone. The presence of both Tex19.1 and Tex19.2 in PGCs, gonocytes and spermatocytes opens the possibility that these two genes could play redundant functions in male germ cells. Furthermore, the placental expression of Tex19.1 can explain why Tex19.1 knockout mice show embryonic lethality, in addition to testis defects.

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The aim of this study was to evaluate the effect of body weight (BW) change during the early postpartum period and BW on day 50 post partum on the subsequent expression of estrus and fertility at first insemination before day 90 post partum. The data were derived from 1036 postpartum cows reaching 90 days in milk. The following data were recorded for each animal: lactation number, daily milk production at day 50 post partum, calving date, days in milk at estrus and at AI, insemination date, insemination number, inseminating bull, AI technician and body weight at calving, at day 50 post partum and at AI. Of the 1036 cows registered, 817 (78.9%) showed first estrus before day 90 post partum and were inseminated. Using logistic regression procedures and based on the odds ratio a one unit (kg) increase in the daily milk production at day 50 post partum and a one unit (kg) decrease in the BW between calving and day 50 post partum were related to a 1.03-fold increase and a 0.97-fold decrease in the estrus expression rate before day 90 post partum, respectively. Of the 817 cows inseminated before day 90 post partum, 437 became pregnant (53.5%). A one unit increase in the BW at day 50 post partum produced a 1.003-fold increase in the early fertility rate. Pregnancy rate before day 90 was also influenced by the season at calving, bull providing semen and technician. In conclusion, the register of BW during the postpartum period may be a useful tool for evaluating the nutritional status and its relationship with the subsequent reproductive efficiency in dairy cows.

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Compared with advanced developmental stage embryos and somatic cells, fully grown mammalian oocytes contain specific nucleolus-like structures (NPB - nucleolus precursor bodies). It is commonly accepted that they serve as a store of material(s) from which typical nucleoli are gradually formed. Whilst nucleoli from somatic cells can be collected relatively easily for further biochemical analyses, a sufficient number of oocyte nucleoli is very difficult to obtain. We have found that isolated oocytes nucleoli fuse very efficiently when contact is established between them. Thus, well visible giant nucleoli can be obtained, relatively easily handled and then used for further biochemical analyses. With the use of colloidal gold staining, we estimated that a single fully grown mouse oocyte nucleolus contains approximately 1.6 ng of protein. We do believe that this approach will accelerate further research aiming at analyzing the composition of oocyte nucleoli in more detail.

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The objective of this study was to investigate the relationship between the plasma concentrations of vitamin A (VA), vitamin E (VE) and β-carotene (BC) during embryo collection in Japanese Black cows that had undergone superovulation treatment and the embryo collection results. Following superovulation treatment in 116 Japanese Black cows, we collected 1317 embryos by nonsurgical means seven days after artificial insemination. The collected embryos were classified into transferable embryos, unfertilized oocytes and degenerated embryos. After embryo collection, we collected blood samples from the cows and measured the plasma concentrations of VA, VE and BC. The cows were then divided into 2 groups depending on the plasma concentration of VA (L and H: < 80 IU/dl and ≥ 80 IU/dl), VE (L and H: < 150 μg/dl and ≥ 150 μg/dl) and BC (L and H: < 150 μg/dl and ≥ 150 μg/dl). As a result, the number of collected embryos in the H group of VE was significantly (P<0.01) higher than that in L groups. Furthermore, the number of transferable embryos was higher (P<0.05) in all VA, VE and BC H groups than in the L groups. The H group for BC showed a high ratio of transferable embryos compared with the L group (P<0.05). Consequently, the present study suggests that the plasma VE and BC concentrations are positively correlated with the embryo collection results.

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The aim of this study was to improve the reliability of predicting the superovulatory response in Japanese Black cattle. Follicle counts and plasma anti-Müllerian hormone concentrations were analyzed within four days prior to the initiation of superovulation. The single nucleotide polymorphism (guanine or adenine) of the ionotropic glutamate receptor AMPA1 was determined. The plasma anti-Müllerian hormone concentration was positively correlated (P<0.001) with the numbers of all follicles and small (<5 mm) follicles and with the numbers of ova/embryos (P<0.001), fertilized embryos (P<0.001) and transferable embryos (P=0.005). There was no significant difference in follicle counts and superovulatory responses between donor cows bearing guanine/adenine or guanine/guanine alleles of AMPA1. Donor cows with a high plasma anti-Müllerian hormone concentration and homozygous for the guanine-containing allele of AMPA1 were most responsive to superovulation. The results suggest that physiological and genetic markers of superovulation have a synergistic effect on the accuracy of predictions of responsiveness.

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