The need for accurate selection of the best oocytes for in vitro fertilization protocols and thus, production of embryos has driven the search for oocyte quality markers from morphological criteria to biochemical parameters. Current studies are focused on the biochemical constituents of the follicular fluid and gene expression profiling of the cumulus cells. These parameters are, however, affected by factors that must be considered before making a judgment of the oocyte’s quality. These includes factors such as the type of hormonal stimulation protocol, age of oocyte donor and heat stress on the donor, all of which have been reported to influence the concentrations of many hormones, apolipoproteins, metabolites, fatty acids and growth factors in the follicular fluid and the expression of several genes in the cumulus cells. Another important point to note is species variation in the response to these extraneous influences, which thus calls for species targeted investigations. As reports are still scanty and investigations assumed to be very keen, we employed this review paper to bring attention of researchers and clinicians to those factors that may come to bear on the outcome of their investigations on oocyte and embryo quality.
Although circulating progesterone (P4) levels tend to change with the season, little is known about the seasonal changes of P4 synthesis-related proteins in the corpus luteum (CL) of mares. To examine these changes, seventy-four ovaries containing a CL were collected from Anglo-Norman mares at a local abattoir in Kumamoto, Japan (~N32°), five times during one year. The stages of the CLs were classified as early, mid and regressed by macroscopic observation of the CL and follicles. The mid CL, which had the highest P4 concentration, was used to evaluate the seasonal changes in P4 synthesis. The luteal P4 concentration and mRNA expression of luteinizing hormone receptor (LHCGR) were lowest during early winter and highest during late winter. The mRNA expressions of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) were lowest during early winter and increased during late winter. These results suggest that P4 synthesis in the CL is affected by the seasonal changes in the mRNA expressions of P4 synthesis-related proteins in mares.
The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term.
Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may have a wide range of applications in cell and gene therapy. However, the safety issues and the low efficiency associated with generating human iPSCs have limited their usage in clinical settings. The cell type used to create iPSCs can significantly influence the reprogramming efficiency and kinetics. Here, we show that amniotic fluid cells from the prenatal diagnosis of a β-thalassemia patient can be efficiently reprogrammed using a doxycycline (DOX)-inducible humanized version of the single lentiviral “stem cell cassette” vector flanked by loxP sites, which can be excised with Cre recombinase. We also demonstrated that the patient-derived iPSCs can be characterized based on the expression of pluripotency markers, and they can be differentiated into various somatic cell types in vitro and in vivo. Moreover, microarray analysis demonstrates a high correlation coefficient between human β-thalassemia iPS cells and human embryonic stem (hES) cells but a low correlation coefficient between human β-thalassemia amniotic fluid cells and human β-thalassemia iPS cells. Our data suggest that amniotic fluid cells may be an ideal human somatic cell resource for rapid and efficient generation of patient-specific iPS cells.
Gpr3, a member of the G protein-coupled receptor superfamily, was known as a critical factor for the maintenance of meiotic prophase arrest in oocytes via a Gs protein-mediated pathway. The present studies were conducted to examine the ovarian immunolocalization of Gpr3, its expression pattern in different stages of fetal, postnatal and developmental pigs and its effect on proliferation of ovarian granulosa cells in pigs. Immunohistochemical analysis indicated that Gpr3 was localized in egg nests, oocytes and granulosa cells (GCs) of the follicle ranging from the primordial to Graafian stages and the corpora lutea. Staining was faintly present in the corpora lutea and weak in GCs but was strong in oocytes. Real-time PCR and Western blotting indicated that Gpr3 mRNA and protein were both present in the different ages of ovaries, and there were wavy changes in the expression levels from postpartum 1 to 180 days. Moreover, both the mRNA and protein levels of Gpr3 were upregulated significantly during follicle growth, suggesting that Gpr3 might play potential roles in regulating ovarian follicle development in the pig. MTT and flow cytometry analyses indicated that Gpr3 knockdown significantly promoted proliferation of porcine GCs while increasing the proportion of cells in the S phase and the expression of Cyclin B1 and Cyclin D2, providing new insights into how Gpr3 signaling regulates the proliferation of porcine GCs. In conclusion, the stage- and cell-specific expression pattern of Gpr3 in the porcine ovary suggested that Gpr3 might play an important role during the entire process of follicular development and luteinization.
To avoid the problems associated with twinning in dairy cattle, one of the embryos may be eliminated. This study compares the effect on pregnancy maintenance of two embryo reduction techniques, manual rupture (MR) and transvaginal ultrasound-guided aspiration (TUGA) of allanto-amniotic fluid, in Holstein-Friesian cows with multiple pregnancies. In the first experiment, 61 lactating cows bearing unilateral twins (n=27), bilateral twins (n=30) or triplets/quadruplets (n=4) were subjected to MR (n=45) or TUGA using a 17-G neddle (n=16) on day 28–34 of gestation. In 21 and 10 cows undergoing MR and TUGA embryo reduction, respectively, pregnancy loss occurred before day 90 (46.7 vs. 62.5%, P= 0.28). Through binary logistic regression, the type of pregnancy was identified as the only variable significantly affecting pregnancy maintenance (P=0.03). Based on the odds ratio, the risk of pregnancy loss was 4.1 times higher for unilateral twins than for bilateral twins (70.4 vs. 36.7%, respectively, P=0.01). No effect was detected on pregnancy maintenance of the technique used (P=0.17) or of the interaction technique by type of pregnancy (P=0.22). In the second experiment, a 22-G needle was used to perform TUGA on 22 lactating cows. The pregnancy loss rates were 44.4% (4/9), 18.2% (2/11) and 50% (1/2) for cows bearing unilateral twins, bilateral twins and triplets, respectively. The total pregnancy loss rate following TUGA using the 22-G needle tended to be lower than that using the 17-G needle (31.8 vs. 62.5%; P=0.06). Our results suggest that TUGA using a 22-G needle could be the method of choice to perform embryo reduction in cows carrying multiple pregnancies.
The temporal progressive increase of interferon tau (IFNτ) secretion from the bovine trophoblast is a major embryonic signal of establishing pregnancy. Here, we cultured and isolated bovine trophoblast cells (BTs) from IVM/IVF oocytes and in vitro produced blastocysts, used them, for the first time, as donor cells for nuclear transfer and compared them with adult fibroblasts (AFs) as donor cells. BTs were reprogrammed in enucleated oocytes to blastocysts with similar efficiency to AFs (14.5% and 15.6% respectively, P≤0.05). The levels of IFNτ, CDX2 and OCT4 expression in IVF-, BT- and AF-derived blastocysts were analyzed using reverse transcription polymerase chain reaction and reverse transcription quantitative polymerase chain reaction (RT-PCR and RT-qPCR). IVF-produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid, expanded and hatching blastocysts. RT-PCR and RT-qPCR studies showed that IFNτ expression was higher in BT-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and BT-derived blastocysts showed a progressive increase in IFNτ expression as blastocyst development advanced when it compared with AF-derived blastocysts. OCT4 was inversely related with IFNτ expression, while CDX2 was found to be directly related with IFNτ temporal expression. Persistence of high expression of IFNτ and CDX2 was found to be higher in BT-derived embryos than in IVF- or AF-derived embryos. In conclusion, using BTs expressing IFNτ as donor cells for bovine NT could be a useful tool for understanding the IFNτ genetics and epigenetics.
Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P<0.05). No embryos irradiated with 9 Gy of X-rays survived to hatching. X-irradiation significantly reduced the number of endogenous PGCs in the embryonic gonads at stage 27 in a dose-dependent manner compared with nonirradiated controls. The numbers of endogenous PGCs in the 3-, 6- and 9-Gy-irradiated groups were 21.0, 9.6 and 4.6% of the nonirradiated control numbers, respectively. Sets of 100 donor PGCs were subsequently transferred intravascularly into embryos irradiated with 3 Gy X-rays and nonirradiated control embryos. Genetic cross-test analysis revealed that the germline transmission rate in the 3-Gy-irradiated group was significantly higher than in the control group (27.5 vs. 5.6%; P<0.05). In conclusion, X-irradiation reduced the number of endogenous PGCs and increased the germline transmission of transferred PGCs in chimeric chickens.
In mammalian ovaries, the majority of follicles are lost before ovulation by atresia. This degenerative process is initiated or caused by granulosa cell apoptosis. To reveal the androgen-dependent mechanism of selective follicular atresia, the culture model system for agonism and antagonism of the androgen receptor has been established. We examined the influence of an androgen receptor antagonist, 2-hydroxyflutamide (2-Hf), on the incidence of apoptosis in cultured porcine granulosa cells. They were incubated (6 and 12-h) in the presence of testosterone (T, 10–7M), 2-Hf (1.7×10–4 M) or both T and 2-Hf (T+2-Hf), and then analyzed by flow cytometry with fluorescein labelled annexin V. To better imitate in vivo conditions, the intact porcine follicles (6–8 mm in diameter) have been incubated in an organ culture system with the addition of the same factors. Sections obtained from follicles fixed after culture were stained with hematoxylin and eosin, and the presence of apoptosis-related DNA strand breaks was evaluated by the TUNEL method. Estradiol and progesterone concentrations in the culture media were measured by radioimmunoassays. The addition of T or 2-Hf to the culture media caused an increase in the number of apoptotic granulosa cells, while treatment with T+2-Hf decreased it in both in vitro and organotypic models. Follicles cultured with the addition of T or 2-Hf exhibited morphological changes indicating follicular atresia. Granulosal estradiol secretion was considerably stimulated by T+2-Hf. The highest increase in follicular estradiol secretion was observed after the anti-androgen addition. In both granulosal and follicular cultures, the production of progesterone declined in the presence of T or 2-Hf but increased after their simultaneous addition. In conclusion, androgen receptor antagonist 2–Hf attenuates induction of granulosa cell apoptosis in the presence of a high T level. The nature of this protective mechanism as yet is unknown and requires further research.
Persistence of the corpus luteum (CL) in cattle usually occurs during the puerperium and is associated with interference of prostaglandin (PG) F2α release from the uterus. The objective of the present study was to determine for the first time the gene expressions in the persistent CL compared with the CL of pregnancy and cyclic CL. Three types of CL biopsy samples were collected from 32 lactating Holstein cows: (1) CL persisting for 29 to 33 days after the first ovulation postpartum (persistent CL, n=9), (2) CL between days 29 and 33 of early pregnancy (CL of pregnancy, n=8) and (3) CL between days 10 and 13 of the estrous cycle (cyclic CL, n=27). mRNA expression of 2',5'-oligoadenylate synthetase-1 was upregulated only in the CL of pregnancy, confirming exposure to interferon-τ (IFNT) produced by trophoblasts in pregnant cows. mRNA expressions of immune tolerance-related factors (PGES and forkhead/winged helix transcription factor 3) were upregulated in the CL of pregnancy but not in the persistent CL, suggesting that IFNT controls upregulation of these genes. mRNA expression relating to some of the major systems such as lymphangiogenesis, inflammation and apoptosis were similarly upregulated in the persistent CL and the CL of pregnancy but not in the cyclic CL. The results suggest that the persistent CL may survive for a long period without changes in local immune tolerance but develops several major systems required for CL maintenance similar to the CL of pregnancy.
Signal transducer and activator of transcription-3 (Stat3) plays a central role in interleukin-6 (IL-6)-mediated cell proliferation by inhibiting apoptosis in a variety of cell types. The Stat3 pathway is essential for embryonic development. The aim of this study was to determine the effects of recombinant IL-6 on the viability and development of porcine diploid parthenotes cultured in vitro. Four-cell parthenotes, derived in vitro, were cultured to the blastocyst stage, with or without recombinant IL-6. The addition of 10 or 100 ng/ml of recombinant swine IL-6 into PZM3 medium increased the development rate of parthenotes to the blastocyst stage (P<0.05). When supplemented with 10 ng/ml of recombinant swine IL-6, the number of parthenotes at the blastocyst stage increased (P<0.05) and apoptosis decreased (P<0.05). Real-time RT-PCR experiments revealed that the addition of recombinant swine IL-6 decreased the mRNA expression of the pro-apoptotic gene Caspase3 (P<0.01) but increased the expression levels of the anti-apoptotic genes Bcl2l1 and Survivin. IL-6 receptors and Stat3 mRNA expression were upregulated after treatment with 10 ng/ml recombinant swine IL-6. Immunoblots and fluorescence labeling experiments showed that the levels of phosphorylated Stat3 were upregulated. These results suggest that recombinant swine IL-6 prevents apoptosis of porcine parthenotes and enhances porcine embryo viability through the IL-6/Stat3 signaling pathway in vitro.
To predict the fertility of frozen-thawed bull spermatozoa, a sperm penetration assay (SPA) using zona-free hamster oocytes was optimized, and the assay results were compared with data from field fertility expressed as the non-return rate (NRR). To increase sperm penetration, the spermatozoa were pre-incubated and coincubated with oocytes in media containing various concentrations of heparin (0 to 50 μg/ml). Coincubation with 10 μg/ml heparin showed the highest sperm penetration (P<0.05); it is considered to be the optimized SPA method. Sperm fertility index values obtained from WSPA were significantly correlated with the historic average NRR of 46 bulls (P<0.01). To determine the normal range for SPA, we established the lower limits of the sperm fertility index and set the cut-off value at 2.55, at which point the NRR was more than 70%, using the receiver operating characteristic curve. The overall accuracy for the 46 bulls was 95.7% (44/46) for both the low and high NRR, with a sensitivity of 95.5% (21/22) and a specificity of 95.8%. This protocol would make it easier to discriminate bulls according to their sperm fertilizing ability.
Phosphorylation of histone H3 at Ser10 (H3S10P) has been linked to a variety of cellular processes, such as chromosome condensation and gene activation/silencing. Remarkably, in mammalian somatic cells, H3S10P initiates in the pericentromeric heterochromatin during the late G2 phase, and phosphorylation spreads throughout the chromosomes arms in prophase, being maintained until the onset of anaphase when it gets dephosphorylated. Considerable studies have been carried out about H3S10P in different organisms; however, there is little information about this histone modification in mammalian embryos. We hypothesized that this epigenetic modification could also be a marker of pericentromeric heterochromatin in preimplantation embryos. We therefore followed the H3S10P distribution pattern in the G1/S and G2 phases through the entire preimplantation development in in vivo mouse embryos. We paid special attention to its localization relative to another pericentromeric heterochromatin marker, HP1β and performed immunoFISH using specific pericentromeric heterochromatin probes. Our results indicate that H3S10P presents a remarkable distribution pattern in preimplantation mouse embryos until the 4-cell stage and is a better marker of pericentromeric heterochromatin than HP1β. After the 8-cell stage, H3S10P kinetic is more similar to the somatic one, initiating during G2 in chromocenters and disappearing upon telophase. Based on these findings, we believe that H3S10P is a good marker of pericentromeric heterochromatin, especially in the late 1- and 2-cell stages as it labels both parental genomes and that it can be used to further investigate epigenetic regulation and heterochromatin mechanisms in early preimplantation embryos.
In the production of cattle nuclear transfer embryos, the production efficiency is affected by the oocyte developmental competence and successful enucleation rate. This study investigated the effect of treating oocytes with milrinone, a phosphodiesterase inhibitor, on these two characteristics. When cumulus–oocyte complexes (COCs) were cultured for 19 h with 0, 50 or 100 μM of milrinone, the enucleation rate was significantly improved by 100 μM milrinone. However, milrinone treatment during in vitro maturation (IVM) also delayed meiotic progression by at least 2 h, which would affect the examination of enucleation rate and developmental competence of oocytes. Thus, in the second experiment, meiotic resumption was temporarily inhibited with butyrolactone I (BL-I; 100 μM, 18 h) to decrease the delayed maturation caused by milrinone; this enabled a more accurate comparison of the effects of milrinone after oocyte maturation. In nuclear transfer embryo production, oocytes treated with milrinone (100 μM, 20 h) showed a significantly higher rate of enucleation compared with that of control oocytes. This improved enucleation rate was associated with a closer location of the metaphase plate to the first polar body in the treated oocytes compared with that in control oocytes. Furthermore, milrinone improved the frequency of development to the blastocyst stage in the resulting embryos. In conclusion, milrinone supplementation during IVM improved enucleation rates by rendering the metaphase plate in close proximity to the first polar body, and this treatment also improved oocyte developmental competence. These benefits additively improved the yield of cloned embryos that developed to the blastocyst stage.
The relationship between the growth of preantral and antral follicles and that of their oocytes in ovaries of domestic cats (Felis catus) was analyzed. Eight hundred and five pairs of follicles and oocytes from the ovaries of 51 female cats were collected, and only healthy and fresh follicles and oocytes with or without zona pellucida were used in this study. Immediately after collection, the diameters of follicles and their oocytes were measured. The relationship of the follicle diameter to the oocyte diameter was applied to four regression models and statistically analyzed. The best fitting model was found to be a hyperbolic regression (the coefficient of determination was 0.976 between the follicles and their oocytes with a zona pellucida, y=184x/(x+0.0738); the coefficient of determination was 0.983 between the follicles and their oocytes without a zona pellucida, y=122x/(x+0.0301)). The differentiated equations for the hyperbolic curves in the oocytes with or without a zona pellucida and the follicles were found to be y’=13.6/(x+0.0738)2 and y’=3.67/(x+0.0301)2, where y and x were the diameters of the oocytes (μm) and follicles (mm), respectively. When follicles grew to a size larger than 0.4 mm in diameter, the growth rates of their oocytes calculated by the differentiation equations showed an asymptotic depression around zero. Thus, it was suggested that when the follicles grew to a size larger than 0.4 mm in diameter, their oocytes reached full size and ceased to grow and that the zona pellucida stopped growing when the diameter of the follicles reached 0.3 mm in domestic cats.
Genomic imprinting confers allele-specific expression in less than 1% of genes, in a parent-of-origin specific fashion. In humans and mice the Peg1/Mest gene (Mest) is maternally repressed, and paternally expressed. Mest is expressed in embryogenic mesoderm-derived tissues and in adult brain, and paternal mutations in Mest lead to growth retardation and defective maternal behaviour. Despite our current understanding of mechanisms associated with the establishment of imprinting of Mest and other imprinted genes, it is unclear to what extent Mest imprinting needs to be maintained in adult tissues. Aberrations of imprinting are known to occur in certain rare syndromes, and involve either inherited mutations, or constitutive epigenetic alterations occurring soon after fertilization. Imprinting abnormalities may also occur in the aging somatic tissues of adult individuals. Here we report an occurrence of post-embryonic somatic variability of Mest allelic expression in a colony of mice where heterozygotes at the imprinted Mest locus for a mutation inherited from the father spontaneously expressed the normally silenced allele from the mother. In addition, a newly acquired ability to overcome the deficit in maternal reproductive behaviour had occurred in the mutant mice, but this appeared not to be directly linked to the Mest mutation. Our results suggest that at least one allele of Mest expression is required in the somatic tissues of adult individuals and that under certain conditions (such as in the presence of a Mest insertional mutation or in an altered genetic background), somatically acquired alterations of allelic expression at the Mest locus may occur.
In rats, artificial insemination (AI) is surgically performed as a general tool to obtain offspring using cryopreserved spermatozoa. Nonsurgical AI is a more desirable technology because it does not require any surgical procedures. However, there has never been a successful nonsurgical AI since frozen-thawed rat spermatozoa show low motility. We show here for the first time successful nonsurgical AI in rats using oxytocin treatment. Intraperitoneal injection of oxytocin (1/800 IU) immediately before nonsurgical AI significantly increased the number of sperm collected from the oviducts compared with that without oxytocin treatment. Therefore, to obtain pups, oxytocin was intraperitoneally injected into females mated with vasectomized males, and the rats were then used for nonsurgical AI. Seven of the 12 oxytocin-treated rats became pregnant after nonsurgical AI, and 37 pups were obtained. Only one rat (1/13) without oxytocin treatment was pregnant after nonsurgical AI, and only 1 pup was delivered. These results show success for the first time in obtaining offspring using frozen-thawed rat spermatozoa via nonsurgical AI. Our results also suggest the possibility that oxytocin treatment is effective for improvement of nonsurgical AI even in other species.
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