Cytokinesis and cell division during pre-implantation embryonic development occur as an orchestrated spatiotemporal program. Cleavage, compaction, and blastulation in pre-implantation embryos are essential for successful implantation and pregnancy. Their alteration is associated with chromosomal imbalance and loss of developmental competence. In this study, we evaluated the time of cleavage and compaction as predictors for in vitro pre- and peri-implantation development and in utero implantation potential by time-lapse monitoring. Mouse 2-cell embryos were collected on 1.5 days post coitum (dpc) and were individually cultured to the outgrowth (OG) stage (7.5 dpc). Developmental stages were classified as 3-cell, 4-cell, 8-cell, morula, blastocyst, and OG. Cut-off times for successful blastocyst development were determined by receiver operating characteristic curve analysis. When cut-off times were set as 9 h for the third cleavage from the 2- to 4-cell stage, and 40 h for compaction from the 2-cell to morula stage, blastocyst and OG development rates, respectively, were significantly higher (P < 0.0001). Embryos were grouped according to the above cut-off time and transferred to the contralateral uterine horn on 3.5 dpc. Implantation rates in utero on 5.5 dpc were significantly higher in early third cleaved (≤ 9 h from 2- to 4-cell) and early compacted embryos (≤ 40 h from 2-cell to morula) than those in delayed embryos (P < 0.05). Therefore, the time of the third cleavage from 2- to the 4-cell stage and compaction from 2-cell to morula stage may be a useful morphokinetic parameter for predicting developmental potential, including successful implantation and pregnancy in human in vitro fertilization-embryo transfer programs.
From previous cDNA subtraction studies analyzing gene expression in equine endometrium, high lipocalin 2 (LCN2) mRNA expression was found in the gravid endometrium. In the uterus, LCN2 may transport hydrophobic molecules and siderophores with iron, or may form a complex with another protein, however, the expression of uterine LCN2 beyond day 20 of equine pregnancy and its receptor has not been characterized. To study the expression and potential roles of uterine LCN2 from pre-implantation to mid-gestation period, stage-specific endometrial samples were obtained from day 13 (day 0 = ovulation) cyclic and days 13, 19, 25, and 60 to 131 pregnant mares. Expression of LCN2 mRNA increased in day 19 gravid endometrium and was abundant from day 60 onward. The expression of LCN2 mRNA was localized to the glandular epithelium. LCN2 protein was detected in day 25 gravid endometrium and luminal fluid, and the protein was localized to the glandular epithelium and luminal cavity, whereas LCN2 receptor expression was found in luminal and glandular epithelium and trophectoderm throughout the experimental period. The presence of matrix metalloproteinase-9 (MMP9) was also examined because MMP9 is known to form a complex with LCN2. Although MMP9 and LCN2 were both found in luminal fluid from day 25 pregnant uterus, the complex of these proteins was not detected. Localization of the receptor in the trophectoderm suggests that endometrial LCN2 could play a role in carrying small substances from the mother to fetus in the equine species.
Transcription factor TEA domain family transcription factor 4 (Tead4) is one of the key factors involved in the differentiation of the trophectoderm (TE) in murine embryos. However, knowledge on the roles of TEAD4 in preimplantation development during bovine embryos is currently limited. This study examined the transcript and protein expression patterns of TEAD4 and attempted to elucidate the functions of TEAD4 during bovine preimplantation development using RNA interference. TEAD4 mRNA was found to be upregulated between the 16-cell and morula stages, and nuclear localization of the TEAD4 protein was detected at the morula stage, as well as in subsequent developmental stages. TEAD4 downregulation did not affect embryonic development until the blastocyst stage, and TEAD4-downregulated embryos were capable of forming the TE under both 5% and 21% O2 conditions. Results of gene expression analysis showed that TEAD4 downregulation did not affect the expression levels of POU class 5 transcription factor 1 (OCT-4), NANOG, caudal-type homeobox 2 (CDX2), GATA binding protein 3 (GATA3), and interferon-tau (IFNT). In conclusion, TEAD4 might be dispensable for development until the blastocyst stage and TE differentiation in bovine embryos.
Thermotaxis that sperm migrate to higher temperature area has been confirmed in rabbit and human. In this study, we examined the migration ability of bull sperm in a temperature gradient to confirm thermotaxis and elucidate the involvement of calcium in such thermotaxis, as well as the relation between sperm capacitation and bull fertility. Thermotaxis was evaluated in a temperature gradient of 34–42ºC using a cross-type column 22-mm long, 40-mm wide, and 100-μm deep. Significantly more sperm migrated to the high-temperature area of 39ºC in a 2ºC temperature gradient, and to 40ºC in a 1ºC temperature gradient. In calcium-free, BAPTA containing medium, and EGTA containing medium, the migrated sperm ratio in the two temperature areas was almost the same. In media containing lanthanum, ruthenium red, and 2APB, we could not confirm thermotaxis. Pre- and post-capacitated sperm migrated to the high-temperature area, expressing thermotaxis. The sperm from high-fertility bulls showed clear thermotaxis. Based on these results, thermotaxis of bull sperm was confirmed and the involvement of both calcium channels and intracellular stored calcium in thermotaxis was suggested. Although the sample size of bulls was quite small, the difference in thermotaxis may have been associated with bull fertility. Sperm thermotaxis evaluation has potential as a predictor of bull fertility.
The current study was performed to investigate the effect of oocyte donor status, including age and body weight, on metaphase II (MII) oocyte recovery using two superovulation methods in cynomolgus monkeys. The use of Method A [recombinant gonadotrophin (75 IU/kg, 3 ×, 3-day intervals) and human chorionic gonadotropin (hCG)] led to great increases in ovary size and the mean number of MII oocytes retrieved in age- and body-weight-dependent manner; in contrast, both the parameters were similar in Method B [recombinant gonadotrophin (60 IU, twice daily, 6 days), recombinant gonadotropin and recombinant human luteinizing hormone (rhLH) (60 IU, twice daily, 3 days), and hCG]. Importantly, Method A showed maximal MII oocyte recovery rate in > 60-month-old or 4.5–5.0-kg female monkeys, whereas Method B was equally effective regardless of the donor age and body weight. These results indicate that superovulatory responses depend on the interaction between oocyte donor status and the superovulation method used in cynomolgus monkeys.
DNA methylation in transcriptional regulatory regions is crucial for gene expression. The DNA methylation status of the edges of CpG islands, called CpG island shore, is involved in tissue/cell-type-specific gene expression. Haploinsufficiency diseases are caused by inheritance of one mutated null allele and are classified as autosomal dominant. However, in the same pedigree, phenotypic variances are observed despite the inheritance of the identical mutated null allele, including Fibrillin1 (FBN1), which is responsible for development of the haploinsufficient Marfan disease. In this study, we examined the relationship between gene expression and DNA methylation patterns of the FBN1 CpG island shore focusing on transcriptionally active hypomethylated alleles (Hypo-alleles). No difference in the DNA methylation level of FBN1 CpG island shore was observed in porcine fetal fibroblast (PFF) and the liver, whereas FBN1 expression was higher in PFF than in the liver. However, Hypo-allele ratio of the FBN1 CpG island shore in PFF was higher than that in the liver, indicating that Hypo-allele ratio of the FBN1 CpG island shore likely correlated with FBN1 expression level. In addition, oocyte-derived DNA hypermethylation in preimplantation embryos was erased until the blastocyst stage, and re-methylation of the FBN1 CpG island shore was observed with prolonged in vitro culture of blastocysts. These results suggest that the establishment of the DNA methylation pattern within the FBN1 CpG island shore occurs after the blastocyst stage, likely during organogenesis. In conclusion, Hypo-allele ratios of the FBN1 CpG island shore correlated with FBN1 expression levels in porcine tissues.
The fertilized oocyte begins cleavage, leading to zygotic gene activation (ZGA), which re-activates the resting genome to acquire totipotency. In this process, genomic function is regulated by the dynamic structural conversion in the nucleus. Indeed, a considerable number of genes that are essential for embryonic development are located near the pericentromeric regions, wherein the heterochromatin is formed. These genes are repressed transcriptionally in somatic cells. Three-dimensional fluorescence in situ hybridization (3D-FISH) enables the visualization of the intranuclear spatial arrangement, such as gene loci, chromosomal domains, and chromosome territories (CTs). However, the 3D-FISH approach in mammalian embryos has been limited to certain repeated sequences because of its unfavorable properties. In this study, we developed an easy-to-use chamber device (EASI-FISH chamber) for 3D-FISH in early embryos, and visualized, for the first time, the spatial arrangements of pericentromeric regions, the ZGA-activated gene (Zscan4) loci, and CTs (chromosome 7), simultaneously during the early cleavage stage of mouse embryos by 3D-FISH. As a result, it was revealed that morphological changes of the pericentromeric regions and CTs, and relocation of the Zscan4 loci in CTs, occurred in the 1- to 4-cell stage embryos, which was different from those in somatic cells. This convenient and reproducible 3D-FISH technique for mammalian embryos represents a valuable tool that will provide insights into the nuclear dynamics of development.
The aim of this study was to investigate the effect of different heparin concentrations in the course of sexed in vitro fertilization (IVF), on bovine embryonic development and development to term following embryo transfer (ET). With a total of 9156 oocytes for IVF, sorted as well as unsorted sperm from four bulls had different heparin requirements for achieving the highest rate of development in vitro. However, when optimal heparin concentrations were used (40 to 80 µg/ml), the performance of X-sorted sperm (0.3 × 106/ml/IVF droplet) from all four bulls, as judged by blastocyst development (Bulls A, B, C, and D: 25.2, 19.7, 25.1, and 9.8%, respectively), was significantly increased, and the blastocyst rate was comparable to that observed with unsorted sperm at certain heparin concentrations within the four bulls. We determined that near-optimal blastocyst development was possible with sorted sperm from all four bulls, when a heparin concentration of 40 µg/ml was used. Pregnancy rates at d 70 post ET ranged from 39.1 to 40.3% (P > 0.05), and the calving rates ranged from 34.4 to 35.1% (P > 0.05), when heparin was used at a concentration of 10 μg/ml (n = 236), 20 μg/ml (n = 189), and 40 μg/ml (n = 305), respectively. Our study demonstrates that, although the sorted sperm of different bulls performed optimally over a range of heparin concentrations, a generally accepted heparin concentration of 40 µg/ml can be set for sexed IVF. This improvement is beneficial when sexed embryo production by ovum pickup and IVF is an essential component of genetic breeding programs.
This study investigated the correlation between the body surface temperature (BST) and core body temperature of ewes and changes in BST during the prepartum stage in pregnant ewes. Four non-pregnant adult ewes were used in the first experiment. The BST of the upper neck, vaginal temperature (VT), and ambient temperature (AT) were measured every 10 min for seven days and analyzed for correlations. The mean (± SD) BST and VT of ewes during the study period were 35.4 ± 1.7°C and 39.1 ± 0.4°C, respectively, with a correlation of r = 0.62, P < 0.001. This finding suggested that the BST was associated with core body temperature in ewes. In the subsequent experiment, seven pregnant ewes in their third trimester were used to evaluate changes in BST measured at the upper neck 72 h before parturition. The mean BST at –24–0 h (0 h = time of parturition) was significantly lower than that at –72– –48 h and –48– –24 h (P < 0.05). The BST tended to decrease toward parturition; all BST measurements at –16– –3 h were significantly lower than those at –72 h (P < 0.05). A clear circadian rhythm in the BST was observed at two days and the day before parturition and an unclear circadian rhythm was observed on the day of parturition. Therefore, these findings indicate that the BST also decreases before parturition, as do vaginal and rectal temperatures.
The objective of this study was to compare the cAMP and cGMP levels in cumulus-oocyte complexes (COCs) derived from the middle follicles (MFs, 3−6 mm in diameter) and small follicles (SFs, 1–3 mm in diameter) of pre-pubertal gilts during the first 24-h period of maturation in vitro (IVM). Both cAMP and cGMP levels in MF- and SF-derived oocytes did not change during this period. Although the cAMP levels increased in the COCs at 10 and 20 h after the start of IVM, the levels of cAMP were significantly higher in MF-derived COCs than in SF-derived COCs at 20 h after the start of IVM. On the other hand, the cGMP levels in COCs decreased to basal levels between 10 and 20 h after the start of the IVM, whereas cGMP levels were lower in SF-derived COCs than in MF-derived COCs during the first 10 h. The number of cumulus cells was larger in the MF-derived COCs than in the SF-derived COCs during the first 20-h period of IVM. The estimated cAMP level per cumulus cell at 10 h after the start of the IVM was higher in SF-derived COCs than in MF-derived COCs, whereas the estimated cGMP level per cumulus cell was no different between MF- and SF-derived COCs. From these results, we conclude that cAMP and cGMP levels in COCs, but not in oocytes, drastically change during the first 20-h period of IVM, and that both cAMP and cGMP levels significantly differ between MF- and SF-derived COCs.
Reproduction is regulated by gonadotropins secreted from gonadotrophs. The production and secretion of gonadotropins are mainly regulated by gonadotropin-releasing hormone (GnRH). Agonists or antagonists that influence GnRH action on gonadotrophs are important to regulate reproduction; however, these factors have not been fully characterized due to the lack of simple and easy-to-use techniques to detect gonadotropin secretion from gonadotropin-producing cells. In the present study, we found that Gaussia luciferase (Gluc), which was expressed in LβT2 cells, can be secreted like a luteinizing-hormone (LH) upon stimulation with GnRH. The Gluc secreted into the medium was easily monitored as luminescence signals. The detection range of the GnRH-induced Gluc activity was comparable to that of the enzyme-linked immunosorbent assay for LH. In addition, when the Gluc was expressed in AtT20 cells, which produce adrenocorticotropic hormone (ACTH), the Gluc activity in the medium increased in parallel with the ACTH secretion upon stimulation with corticotropin-releasing hormone. Thus, the Gluc assay in the present study can be easily used for high-throughput screening of factors that influence LH or ACTH secretion from LβT2 or AtT20 cells, respectively.
Anti-Müllerian hormone (AMH) is secreted from the preantral and small antral follicles. It regulates follicle development and inhibits follicular atresia. This study examined how age, parity, and time after parturition affect plasma AMH concentrations in Japanese Black cows. We measured plasma AMH concentrations in primiparous, secundiparous, and multiparous (third parity or higher) cows at four time points: day 2 (day 0 = parturition), day 8, 2 days before first postpartum ovulation (pre-1stOv), and 12 days after first ovulation (post-1stOV). We observed a positive correlation between plasma AMH concentration and age (in months) and parity on day 2, day 8, and post-1stOV, but not on pre-1stOv. The multiparous cows had higher AMH concentrations than primiparous cows throughout the postpartum period (P < 0.05). Our results indicate that age and parity significantly influence plasma AMH concentrations in Japanese Black cows during the voluntary waiting period.