The only commercially viable method of sexing mammalian sperm is to use a flow cytometer to measure sperm DNA content via fluorescence of the DNA-bound fluorophore Hoechst 33342, and then sort sperm into three populations, probably X, probably Y, and undetermined. Millions of insemination doses of sexed sperm are produced annually by this procedure. Although accuracy of sexing usually exceeds 90%, this procedure of sexing one sperm at a time has serious limitations, including cost, sort rates, and damage to sperm resulting in lowered fertility, but not abnormalities in offspring. Suggested areas for research include determining how sperm are damaged and where in the process of fertilization and embryonic development the infertility is manifest. Pre and post sorting procedures are done in approximately hourly batches, and these might be changed to continuous procedures. Numerous genetic, physical, and immunological procedures for sexing millions of sperm in parallel have been proposed, but none appears to be suitable for commercialization at this time due to issues of accuracy, repeatability, damage to sperm, and other problems. However, increasing numbers of reports are appearing concerning improvements in these procedures, and it appears inevitable that one or more of them eventually will prove to be efficacious. In developing such procedures, it is critical to monitor sexing accuracy regularly by rapid and inexpensive procedures such as fluorescence in situ hybridization, quantitative PCR, or sort reanalysis by flow cytometry. Furthermore, monitoring fertility of sexed sperm such as in vitro fertilization should be integral to the development process. Intellectual property issues could be substantive.
In mammalian preovulatory follicles, LH stimulation induces the ovulation process, including follicular wall rupture, granulosa cell luteinization, cumulus cell expansion and meiotic maturation of the oocyte. The receptor for LH (LHCGR) is expressed mostly in granulosa cells of preovulatory follicles, and is rarely expressed in cumulus cells or oocytes. The expression level in granulosa cells dramatically decreases after ovulation stimuli. Thus, a potent factor(s) secreted by granulosa cells is required to stimulate not only granulosa cells via an autocrine manner but also cumulus cells and/or oocytes via a paracrine pathway. Recent reports showed that granulosa cells and cumulus cells express EGF-like factors that activate the EGF receptor (EGFR)-mitogen-activated protein kinase3/1 (MAPK3/1) (also known as extracellular signal-regulated kinase1/2 (ERK1/2)) pathway in both cell types. EGF-like factors are composed of a signal sequence, transmembrane domain and EGF domain, suggesting that release of the EGF domain by a specific enzyme is essential for interaction with the EGFR to induce the ovulation process. In our studies, TACE/ADAM17, which is known to be a proteolytic enzyme of EGF-like factors in many types of tissue, was found to be expressed in FSH/LH-stimulated granulosa cells and cumulus cells together with activation of the EGFR-MAPK3/1 pathway. When TACE/ADAM17 activity was decreased by a specific inhibitor or siRNA technique, granulosa cell luteinization, cumulus expansion and oocyte maturation were suppressed in an in vitro culture. Thus, TACE/ADAM17 is one of the key genes expressed in both granulosa cells and cumulus cells for induction of the ovulation process.
Induced pluripotent stem (iPS) cells derived from disease patients are an invaluable resource for biomedical research and may provide a source for replacement therapies. In this study, we have generated iPS cells from Asian patients with chronic degenerative diseases of the nervous system, including spinal muscular atrophy (SMA), Parkinson disease (PD) and amyotrophic lateral sclerosis (ALS) by transduction with four factors (KLF4, SOX2, OCT4 and c-MYC). All of the iPS cells showed pluripotency similar to that of human embryonic stem cells (hESCs) and were able to differentiate into various somatic cell types in vitro and in vivo. Furthermore, the iPS cells also can be committed to differentiate into neural cells, the cell type that is affected in chronic degenerative diseases. Therefore, the patient-specific iPS cells we generated offer a cellular model in which to investigate disease mechanisms, discover and test novel drugs and develop new therapies for chronic neurodegenerative diseases.
The present study was conducted to elucidate the profile of circulating gonadotropins and gonadal hormones from birth to puberty and relationship between gonadal seasonality and hormonal secretion in both sexes of Thoroughbred horses. Spring-born colts (n=6) and fillies (n=9) were blood sampled weekly from jugular vein from birth to 60 weeks of age. Circulating FSH, LH, prolactin, testosterone, progesterone, estradiol-17β, and immunoreactive (ir)-inhibin were measured by radioimmunoassay. In both sexes, the steroid hormones levels were remarkably high at birth, rapidly dropped within a week and remained at the lower levels until the start of second spring after birth. Ir-inhibin was also high during the birth, remaining lowest during winter and again increasing towards the second summer. There was an increase in FSH concentration in foals during the first summer months after birth and in the next summer, the FSH concentration along with that of LH increased significantly. The seasonal increase in circulating prolactin was remarkable even in the first year, and no differences were noted between the two summers. These results clearly demonstrated that the hypothalamo-pituitary axis is already responsive to changes in photoperiod and secrete prolactin similar to adult horses, but pituitary gonadotrophs for FSH and LH secretion is less sensitive. When the values of these hormones in the second breeding season after birth were compared with adult values of the respective sex in the breeding season, no significant differences were observed, indicating that spring-born fillies and colts have already attained the stage of puberty at the second breeding season after birth.
The intraovarian function of gonadally produced inhibin and activin has been extensively studied in experimental models for decades, yet their presence and function have been rarely reported in wild rodents. With our seasonal breeding model, the wild ground squirrel, we aimed to investigate the possible roles of these peptides in the seasonal folliculogenesis. Immunohistochemical staining and Western blotting have been used to detect the cellular localization and expression patterns of inhibin/activin subunits (α, βA and βB). In the breeding season ovary, all three subunits were present in granulosa cells, theca cells of antral follicles and interstitial cells, with the strongest immunostaining in granulosa cells. Following ovulation, the corpora lutea become a major site of inhibin/activin synthesis. In the nonbreeding season ovary, inhibin/activin α and βA subunits were weakly immunopositive in granulosa cells of early stage follicles, while βB subunit was undetectable. The expression level of inhibin/activin subunit proteins were generally higher in the ovaries of the breeding season, and then decreased to a relatively low level during the nonbreeding season. The dynamic expression of inhibin/activin subunits indicated that they might play important paracrine and/or autocrine roles during the seasonal folliculogenesis of the wild ground squirrel.
In this study, we investigated the immunoreactivity of vascular endothelial growth factor (VEGF) and its receptors flt-1 (VEGFR1) and the kinase domain receptor (KDR/Flk-1, VEGFR2) in the uteri of the wild ground squirrels during the estrous period, early pregnancy and nonbreeding period. Cellular localizations of VEGF, VEGFR1 and VEGFR2 were detected by immunohistochemistry, and total proteins were extracted from uterine tissue in the estrous period, early pregnancy and nonbreeding period for Western blotting analysis. In addition, plasma estradiol-17β and progesterone concentrations were measured by radioimmunoassay. Stronger positive staining of VEGF was found in luminal epithelial cells and glandular cells, and its receptors (VEGFR1 and VEGFR2) were observed in stromal cells in the estrous period and early pregnancy compared with the nonbreeding period. The protein levels of VEGF, VEGFR1 and VEGFR2 were significantly higher in the estrous period and early pregnancy as compared with the nonbreeding period. Besides, plasma estradiol-17β and progesterone concentrations were higher in the estrous period and early pregnancy than in the nonbreeding period, suggesting that the immunoreactivities of VEGF, VEGFR1 and VEGFR2 were correlated with changes in plasma estradiol-17β and progesterone concentrations. These results suggested that VEGF and its receptors may be involved in the regulation of seasonal changes in the uterine functions of wild female ground squirrels.
Transgenic rats show spermatid-specific ectopic expression of the reporter gene, herpes simplex virus type1 thymidine kinase (HSV1-TK), in the testes and have demonstrated male infertility. However, the disruption of spermatogenesis and the underlying molecular mechanisms in these transgenic animals have not been well clarified. In this study, light and electron microscopic observations were performed to characterize the morphological changes in the testes. To explore the molecular mechanisms of male infertility in the HSV1-TK transgenic rat, cDNA microarray and quantitative real-time PCR analyses were performed. The seminiferous tubules of 3-month-old transgenic rats showed morphological alterations including seminiferous epithelial sloughing, vacuolization, and degeneration of spermatogenic cells, suggesting a failure of Sertoli-germ cell interaction. Components of the epididymal lumen from transgenic rats included abnormal spermatozoa, degenerating round spermatids and abnormal elongated spermatids indicating an appearance of direct impairment of spermiogenesis. cDNA microarray and real-time PCRanalyses revealed significant changes (P<0.05) in the gene expression level in six genes, testin, versican, mamdc1, fgf7, ostf1 and cnot7. Among them, testin drew most of our attention, since the testin gene is a sensitive marker for disruption of Sertoli-germ cell adhesion. Thus, our results suggest that the accumulation of HSV1-TK in the spermatids not only directly interferes with spermiogenesis but also disrupts spermatogenesis through a disruption of Sertoli-germ cell adhesions. It is important to explore the testicular actions of the HSV1-TK protein in transgenic experimental models and thereby gain clues to find an appropriate treatment for HSV-infected patients exhibiting human male infertility, as has been recently observed.
This study analyzed the effect of corpus luteum (CL) formation during weeks 3–5 postpartum on the subsequent reproductive performance of dairy cows. Factors contributing to CL formation during the postpartum period were also determined. Data were collected from 1524 Holstein dairy cows on 18 farms using a single ultrasonographic examination to determine the presence or absence of a CL during weeks 3–5 postpartum. The dates of calving, AI, conception and cow parity were also collected. Data were acquired for a subset of 475 cows on five farms related to peripartum reproductive events and the body condition score (BCS) during weeks 3–5 postpartum. The hazard of first postpartum insemination by 150 days in milk (DIM) was higher for cows with a CL compared with herd mates without a CL during week 3 (hazard ratio [HR]: 1.40, P=0.007), week 4 (HR: 1.28, P=0.004) and week 5 postpartum (HR: 1.43, P<0.0001). Furthermore, the pregnancy hazard was also higher by 210 DIM for cows with a CL compared with cows without a CL during week 3 (HR: 1.56, P=0.0009), week 4 (HR: 1.28, P=0.006) and week 5 postpartum (HR: 1.20, P=0.04). Cows calved during autumn were more likely to have a CL than cows calved during spring (odds ratio [OR] =2.32, P=0.003). Primiparous cows were less likely to have a CL than multiparous cows (OR=0.63, P=0.03). Cows with a BCS < 3.00 were less likely to have a CL than cows with a BCS ≥ 3.00 (OR=0.51, P=0.0013). In conclusion, CL formation during weeks 3–5 postpartum was related to subsequent improved reproductive performance when compared with herd mates without a CL.
Differentiated oocytes acquire totipotency through fertilization. During this transition, genome-wide chromatin remodeling occurs, which leads to change in gene expression. However, the mechanism that underlies this global change in chromatin structure has not been fully elucidated. Histone variants play a key role in defining chromatin structure and are implicated in inheritance of epigenetic information. In this study, we analyzed the nuclear localization and expression of H3.1 to elucidate the role of this histone variant in chromatin remodeling during oogenesis and preimplantation development. Analysis using Flag-tagged H3.1 transgenic mice revealed that Flag-H3.1 was not present in differentiated oocytes or early preimplantation embryos before the morula stage, although Flag-H3.1 mRNA was expressed at all stages examined. In addition, the expression levels of endogenous H3.1 genes were low at the stages where H3.1 was not present in chromatin. These results suggest that H3.1 is not incorporated into chromatin due to the inactivity of the histone chaperone and low mRNA expression level. The significance of the dynamics of H3.1 is evaluated in terms of chromatin remodeling that takes place during development.
To investigate the effect of dietary supplementation of sulfamethazine (SMZ) on growth performance, gonadal development and hormonal changes, male and female Japanese quails (Coturnix japonica) were fed a control diet with or without SMZ (0.2%) from one day post hatching until 6 weeks of age. In male quail, the deviation in growth performance between SMZ and control chicks started at the 3rd week, and the disparity was significant at the 5th and 6th weeks. Hormonal analysis revealed a substantial increase in the pituitary and circulating LH (at the 5th and 6th weeks), testicular and circulating testosterone (at the 6th week) and plasma ir-inhibin (at 5th week) levels following feeding of the diet containing SMZ. The testicular size and weights were significantly larger at the 5th week, and histological analysis demonstrated an enlargement of seminiferous tubules, filling of the luminal fluid with spermatozoa and a number of interstitial cells. In female quail, the body and ovarian weights were considerably increased at the 6th week. The SMZ supplemented group showed a significant elevation in pituitary LH content (from the 4th week), plasma LH (at the 5th and 6th weeks), ir-inhibin (at the 3rd and 6th week) and progesterone (at the 2nd, 5th and 6th weeks) as compared with control chicks. These results indicated that SMZ was able to stimulate the secretion of gonadotropins and accordingly the gonadal hormones and that was associated with an early gonadal function in male (at the 5th week) and female (at the 6th week) Japanese quail.
The aim of this study was to evaluate the effect of classical and non-classical major histocompatibility complex (MHC) on the reproduction in the dairy cow. Nine pairs of MHC-I genes were chosen according to their homology and possible function, and their transcription levels in maternal peripheral blood mononuclear cells (PBMCs) from all three trimesters and transcription levels in fetal tissues were compared to evaluate their contributions to cattle reproduction. The results showed that three non-classical genes were variably expressed in PBMCs of pregnant cows. MICB was downregulated in the first and second trimesters (P<0.05), but recovered back to the level in replacement heifers in the last trimester (P>0.05). BoLA-NC1* was upregulated in the first and last trimesters (P<0.001) but no different in the second trimester (P>0.05). BoLA-NC3* was upregulated in all trimesters (P<0.001). On the other hand, MICB was upregulated in fetal ear tissues (P<0.001), and BoLA-NC1* was almost silent in both fetal placenta and ear tissues (P<0.001); however, BoLA-NC3* was upregulated in both the fetal placenta and ear tissues (P<0.001). These results suggested that non-classical gene BoLA-NC1* increased maternal immunity against the fetus, which was inhibited by BoLA-NC3*. BoLA-NC3* also inhibited fetal autoimmunity. Apoptosis of the fetal placenta could reduce itself expressing MICB, and upregulated expression of MICB in ear tissues was favorable for the fetus to escape autoimmunity. On the other hand, downregulated expression of MICB in the fetal placenta allows for placental decoherence from the maternal placentome, which was beneficial to fetus delivery. Although classical genes were expressed differentially, their effects were restricted because of heavy chain deficiency.
Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig.
We conducted this study to analyze apoptotic changes in the bovine placentome at spontaneous and induced parturition. Cows delivered i) after the administration of dexamethasone followed by prostaglandin F2α and estriol, ii) after the administration of prostaglandin F2α and estriol or iii) spontaneously. Prepartum changes in plasma progesterone and estradiol-17β concentrations were similar between spontaneous and induced parturition. Messenger RNA of BCL2-related protein A1 (BCL2A1), an antiapoptotic gene, was expressed by trophoblast binucleate cells and caruncular epithelial cells. Quantitative RT-PCR showed that the expression of BCL2A1 mRNA in cotyledonary and caruncular portions was significantly lower in spontaneous parturition than induced parturition. The expression of BCL2-associated X protein (BAX) mRNA, a proapoptotic gene, was significantly higher in cotyledons at spontaneous parturition than parturition induced without dexamethasone. Caspase-3 (CASP3) mRNA and pre-activated CASP3 protein were predominantly detected in caruncular epithelial cells regardless of how parturition proceeded. Activated CASP3 protein was found in trophoblast uninucleate cells and binucleate cells rather than caruncular epithelial cells. In spontaneous parturition, intense staining of activated CASP3 was detected in caruncular epithelial cells. Spontaneous and dexamethasone-induced parturition increased apoptotic cells in the placentome compared with parturition induced without dexamethasone. The number of binucleate cells was significantly decreased in spontaneous parturition. The present results suggest that although the clinical dose of dexamethasone induces apoptosis in the placentome at term, neither dexamethasone nor prostaglandin F2α evoke normal physiological changes in the placentome during delivery such as a change in the balance of apoptosis-related genes and disappearance of binucleate cells.
Runx3 is a transcription factor that belongs to the Runx family. We studied the localization of Runx3 mRNA in the mouse uterus, and its function in the mouse endometrium using Runx3 knockout (Runx3–/–) mice. Runx3 mRNA was detected in the endometrial luminal epithelial cells, glandular epithelial cells and stromal cells below the epithelial cell layer on the luminal side. The uteri of Runx3–/– mice were smaller than those of wt mice. The endometrial layer and uterine glands of Runx3–/– mice were less developed than those of wild-type mice, and the endometrial stromal layer was thinner. Transforming growth factor β1 and β3 (TGFβ1 and β3) mRNA levels in endometrial stromal cells of Runx3–/– mice were low compared with those of wild-type mice. Estradiol-17β (E2) increased Tgfb2 mRNA levels in endometrial stromal cells of Runx3–/– mice, but not in those of wild-type mice. E2 increased epidermal growth factor (EGF) mRNA levels in endometrial stromal cells of wild-type mice, but did not increase those of Runx3–/– mice. The diminished Tgfb1 and Tgfb3 mRNA expressions may lead to the reduced proliferation of endometrial stromal cells. Alterations of E2-associated expressions of Tgfb2 and Egf mRNA in endometrial stromal cells of Runx3–/– mice may be associated with suppression of E2-dependent endometrial epithelial cell proliferation in Runx3–/– mice. Thus, Runx3 is likely to be a regulatory factor responsible for endometrial growth.
Current embryo vitrification methods with proven efficacy are based on the minimum volume cooling (MVC) concept by which embryos are vitrified and rewarmed ultrarapidly in a very small amount of cryopreserving solution to ensure the high viability of the embryos. However, these methods are not suitable for simultaneously vitrifying a large number of embryos. Here, we describe a novel vitrification method based on use of a hollow fiber device, which can easily hold as many as 40 mouse or 20 porcine embryos in less than 0.1 μl of solution. Survival rates of up to 100% were obtained for mouse embryos vitrified in the presence of 15% DMSO, 15% ethylene glycol and 0.5 M sucrose using the hollow fiber vitrification (HFV) method, regardless of the developmental stage of the embryos (1-cell, 2-cell, morula or blastocyst; n = 50/group). The HFV method was also proven to be effective for vitrifying porcine in vitro- and in vivo-derived embryos that are known to be highly cryosensitive. For porcine embryos, the blastocyst formation rate of in vitro maturation (IVM)-derived parthenogenetic morulae after vitrification (48/65, 73.8%) did not decrease significantly compared with non-vitrified embryos (59/65, 90.8%). Transfer of 72 in vivo-derived embryos vitrified at the morula/early blastocyst stages to 3 recipients gave rise to 29 (40.3%) piglets. These data demonstrate that the HFV method enables simultaneous vitrification of multiple embryos while still adhering to the MVC concept, and this new method is very effective for cryopreserving embryos of mice and pigs.
Elevated concentrations of circulating progesterone (P4) in the immediate post-ovulation period are associated with advancement of conceptus elongation in cattle. Superovulated (SOV) cattle have not only elevated plasma P4 concentrations but also multiple embryos in the uterus because of the formation of multiple corpora lutea. We examined the relationship between plasma P4 concentration and uterine glucose level in the immediate post-ovulation period and the presence and growth of multiple conceptuses in SOV cattle. SOV cattle were artificially inseminated with frozen-thawed semen at standing estrus (day 0), and the conceptuses were recovered by nonsurgical flushing of the uterus on day 13. In the SOV cattle, there were quadratic relationships between plasma P4 concentration on days 4, 5 and 7 and conceptus length and between number of conceptuses in the uterus and conceptus length. These results suggest that conceptus growth in SOV cattle is regulated by both systemic P4 level and number of conceptuses and that there are ranges of plasma P4 concentrations and numbers of conceptuses in the uterus that are suitable for conceptus growth and development. Plasma P4 concentrations on days 5 and 7, but not the numbers of conceptuses, were quadratically correlated with uterine glucose levels on day 13 in SOV cattle. In addition, conceptus length was positively correlated with uterine glucose level in SOV cattle. Accordingly, regardless of the number of conceptuses in the uterus, the plasma P4 concentration was well correlated with the regulation of conceptus growth via changes in uterine glucose levels in SOV cattle.
We examined the effects of the timing of cumulus cell removal from in vitro-matured (IVM) bovine oocytes on enucleation efficiency and subsequent in vitro development after nuclear transfer (NT). Cumulus cells were removed from IVM oocytes by pipetting, by low-speed vortexing and pipetting or by high-speed vortexing at 12, 15 or 18 h of IVM, and then denuded oocytes were further cultured for 6, 3 or 0 h, respectively (18 h of IVM in total). There was no difference in the rate of extrusion of the first polar body (PB1) among the groups. The success rate of blind enucleation of oocytes denuded at 12 h (before PB1 extrusion) by high-speed vortexing (81.7%) was significantly higher than that of oocytes denuded at 18 h by high-speed vortexing (67.8%) but similar to those of oocytes denuded by pipetting after low-speed vortexing (78.6–84.1%) or pipetting alone (84.6–86.9%). After high-speed vortexing, the percentage of oocytes in which metaphase II (MII) chromosomes were located adjacent to the PB1 tended to be lower for the oocytes denuded at 18 h than that for the oocytes denuded at 12 h. In contrast, by pipetting or low-speed vortexing and pipetting, the timing of denuding did not affect the relative location of MII chromosomes. The timing and method of denuding did not affect the fusion and blastocyst formation rates of NT embryos. These results suggest that high-speed vortexing is applicable only to cumulus cell removal from oocytes prior to PB1 extrusion, while pipetting or low-speed vortexing followed by pipetting is useful regardless of the PB1 formation status and leads to successful blind enucleation of IVM oocytes.
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