Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 56, Issue 6
Displaying 1-14 of 14 articles from this issue
Review
  • Mohammad MONIRUZZAMAN, Takashi MIYANO
    Article type: Review
    2010 Volume 56 Issue 6 Pages 559-566
    Published: 2010
    Released on J-STAGE: January 15, 2011
    JOURNAL FREE ACCESS
    Mammalian ovaries are endowed with a huge number of small oocytes (primordial oocytes) in primordial follicles. A small number of primordial oocytes start to grow, while others remain quiescent. Little is known about the mechanism regulating the activation of primordial oocytes. Recently, we found that primordial follicles in mature cows and prepubertal pigs took longer to initiate growth in xenografts compared with those in neonatal animals. We think that primordial oocytes in adult mammals are different from those in neonatal mammals. In this review, we summarize the results regarding the activation of primordial oocytes in neonatal and adult ovaries of different species and propose a model in which ovaries of neonatal mammals contain a mixed population of both quiescent and activated primordial oocytes, while almost all primordial oocytes are quiescent in adult females. The dormancy of primordial oocytes may be required to reserve the non-growing oocyte pool for the long reproductive life in mammals. FOXO3 is considered one of the molecules responsible for the dormancy of primordial oocytes in adult ovaries. These quiescent primordial oocytes are activated, perhaps by certain mechanisms involving the interaction between stimulatory and inhibitory factors, to enter the growth phase.
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Original Article
  • Michiyo ISHIDA, Makoto YOSHIDA, Shinya FUKUTA, Kenji UEMURA, Mieko IIJ ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 567-574
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: July 20, 2010
    JOURNAL FREE ACCESS
    Prolactin (PRL) has long been known to be a hormone responsible for mammary gland development and lactation in females, whereas its role in males is still unclear. Thus, we investigated male mouse (m) PRL protein and mRNA expression in spermatozoa at various differentiation stages in the testes. Quantitative RT-PCR and in situ hybridization detected the expression of PRL not only in Leydig cells but also in germ cells, in particular in spermatogonia. The nucleotide sequence of testis PRL mRNA was the same as that in the pituitary. The mPRL was detected in Leydig cells and in round and elongated spermatids of the testes by immunohistochemistry. Immunoblotting detected 2 forms of mPRL in the testes, one form was 23-kDa PRL, and the other form was smaller than full-length PRL. Based on these results, we focused on N-terminal cleaved PRL to determine its involvement in spermatogenesis. Immunohistochemistry using two sets of antibodies, one that recognized full-length PRL and N-terminal cleaved PRL and another that recognized full-length PRL and C-terminal cleaved PRL, suggested that intact PRL was localized in the nucleus of round spermatids, while N-terminal cleaved PRL variants were localized in the Golgi apparatus of the sperrmatid nuclei of round spermatids, cytoplasms of elongated spermatids and in the spermatozoa tails. These findings suggest that PRL is ectopically expressed in the spermiogenesis and spermatogenesis and that cleaved PRL variants were localized in the Golgi apparatus of spermatids and in spermatozoa tails.
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  • Changyong CHOE, Yong-Won SHIN, Eun-Jin KIM, Sang-Rae CHO, Hyun-Jong KI ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 575-582
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: July 20, 2010
    JOURNAL FREE ACCESS
    Various methods have been used to remove reactive oxygen species (ROS) generated from in vitro culture (IVC) conditions that can cause cell injury or death, including the application of low oxygen (O2) tension and the addition of antioxidants. The beneficial effects of antioxidants and O2 tension on IVC of porcine embryos, however, are controversial among researchers. In this study, we sought to determine the effects and optimal concentrations of antioxidants for the development of porcine embryos in an IVC system. Specifically, we examined the synergistic effects of antioxidants on development to the blastocyst stage in a culture system supplemented with L-cysteine during IVM. Of the antioxidants tested (melatonin, glutathione (GSH), β-mercaptoethanol (β-ME), N-acetylcysteine (NAC) and dithiothreitol (DTT)), addition of GSH (1 mM) or β-ME (25 μM) significantly increased development to the blastocyst stage compared with the controls without antioxidant treatment (22.2 ± 4.2% for 1 mM GSH, 25.9 ± 2.2% for 25 μM β-ME and 12-13% for the control, P<0.05). In addition, the mean cell number per blastocyst was increased by approximately 1.7-fold in the presence of GSH or β -ME. These GSH- and β-ME-induced increases in development to the blastocyst stage and total cell number, however, were not mimicked by melatonin, NAC or DTT, all of which are ROS scavengers. The combination of GSH or β-ME with L-cysteine significantly reduced high O2 tension-induced ROS production (P<0.05). These results suggest that a combination of 1 mM GSH or 25 μM β-ME with 1 mM L-cysteine could be used for production of high quality porcine blastocysts in IVC systems.
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  • Rebecca R. PAYTON, Louisa A. RISPOLI, J. Lannett EDWARDS
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 583-592
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: July 20, 2010
    JOURNAL FREE ACCESS
    Results described herein provide insight regarding certain features of gamete RNA and how they compare to cumulus cell RNA. In particular, 28S/18S rRNA ratio and size distribution of RNA molecules differed in total RNA from oocytes versus surrounding cumulus cells. Specifically, oocyte total RNA had a lower rRNA ratio and an increased abundance of smaller RNA sizes compared to RNA from surrounding cumulus. Extensive efforts demonstrated that observed differences were repeatable whether oocyte maturation occurred in vitro or in vivo, and were similar between the nuclear stages examined. Features of oocyte RNA were conserved across six mammalian species, yet differed from surrounding cumulus. Profiles of sperm RNA were also examined but had no discernible ribosomal RNA peaks and were conserved across four mammalian species. Because the oocyte and spermatozoon are highly specialized cells representing unique molecular entities required for proper embryo development, dissimilarities described herein likely represent real gamete versus cumulus RNA differences.
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  • Thanh Quang DANG-NGUYEN, Kazuhiro KIKUCHI, Tamas SOMFAI, Manabu OZAWA, ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 593-600
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: July 20, 2010
    JOURNAL FREE ACCESS
    The following selection markers for in vitro-produced porcine embryos were investigated: the timing, pattern and evenness of the first cleavage and the timing of the second cleavage. The embryos that cleaved by 30 h post-insemination (hpi) developed to blastocysts at a significantly higher rate (60.9%) and with a significantly higher cell number (33.6 cells) than those of embryos cleaved by 36 hpi (26.4% and 23.6 cells, respectively, P<0.05). Blastocyst proportions derived from 2- and 3-cell embryos cleaved by 30 hpi (68.2 and 65.3%, respectively) were significantly higher than those of 4- and >4-cell embryos (46.3 and 42.6%, respectively, P<0.05). The cell number per blastocyst generated from 2-cell embryos was significantly greater (37.3 cells) than those from 3-, 4- and >4-cell embryos (23.6-27.8 cells, P<0.05). Among embryos cleaved by 30 hpi, the blastocysts derived from evenly cleaved embryos (40.6 cells) were of significantly better quality than those derived from unevenly cleaved embryos (33.2 cells, P<0.05), although their blastocyst rates did not differ. The evenly cleaved embryos that underwent subsequent cleavage within 18 h had significantly higher blastocyst rates (72.7-81.0%) and quality (36.2-40.9 cells) than those without subsequent cleavage (48.3% and 22.5 cells, respectively, P<0.05) during the same period. In conclusion, the timing, pattern and evenness of the first cleavage and the timing of the second cleavage affected the developmental competence and quality of in vitro-produced porcine embryos.
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  • Irena BARNETOVA, Helena FULKA, Josef FULKA, JR
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 601-606
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: July 28, 2010
    JOURNAL FREE ACCESS
    Both the sperm and oocyte are terminally differentiated cells, but within a very short post-fertilization period, their genomes are converted into a totipotent zygote. The process of this transformation has been studied in a number of mammals as well as in the pig, for which very inconsistent results have been published. To clarify these inconsistencies, we have used the interspecies intracytoplasmic sperm injection technique for embryo production and subsequent paternal genome remodeling evaluation. First, we injected boar sperm heads into ovulated and in vitro matured mouse oocytes. The boar spermatozoa consistently decondense in ovulated oocytes and form fully developed pronuclei with demethylated DNA (5-methylcytosine; 5-MeC). Additional labeling against other histone modifications (H3/K9 dimethylation, H3/K4 trimethylation) and HP1 (Heterochromatin Protein 1) revealed similarity to those changes that are typical for natural mouse zygotes. On the other hand, no decondensation and formation of male pronuclei were observed, in spite of obvious oocyte activation, in in vitro matured oocytes. For this reason, we have evaluated the reprogramming parameters of in vitro matured mouse oocytes in more detail. In mouse zygotes (intraspecific), both pronuclei were consistently formed, but no sperm head chromatin demethylation was detected after 5-MeC labeling. Our observations suggest that porcine sperm heads are capable of undergoing active demethylation in in vivo matured mouse oocytes. On the other hand, in vitro matured oocytes possess much lower sperm remodeling capabilities.
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  • Mikiko TOKORO, Seung-Wook SHIN, Satoshi NISHIKAWA, Hyang-Heun LEE, Yuk ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 607-615
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: August 11, 2010
    JOURNAL FREE ACCESS
    We investigated the contribution of phosphorylated RNA polymerase II (RNAP II) and dynamic epigenetic changes to the onset of minor zygotic gene activation (ZGA). Using immunofluorescence staining, we observed that the nuclear localization of RNAP II was initiated by 6 hours post insemination (hpi), whereas RNAP II phosphorylated at serine residue 5 of the carboxyl-terminal domain (CTD) was localized by 9 hpi, and then RNAP II phosphorylated at serine residue 2 of the CTD was localized in the nucleus of embryos by 12 hpi. In a transient gene expression assay using a plasmid reporter gene (pβ-actin/luciferase+/SV40) injected during 6-9 hpi into the male pronucleus, the luciferase+ gene was actively transcribed and translated by 13 and 15 hpi, respectively, indicating that a transcriptionally silent state persisted for at least 4 hours after injection. We found that the methylation status in the chicken β-actin promoter region of the plasmid reporter gene may not be associated with the transcriptionally silent state before minor ZGA. Exposure to trichostatin A did not induce premature expression of the silent reporter gene injected into 1-cell embryos containing histone deacetylase activity and did not affect the amount of luciferase produced per embryo. Acetylated histone H3 lysine 9/14 and acetylated histone H4 lysine 12 and 16 were enriched preferentially in the injected reporter gene at least until 13 hpi, which coincided with the transcriptionally active state. Taken together, these results suggest that deposition of selectively acetylated histones onto the chromatin of 1-cell embryos functions together with transcriptional elongation by RNAP II and that this sequential chromatin remodeling is involved in the molecular mechanism associated with the onset of minor ZGA in the preimplantation mouse embryo.
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  • Masafumi TETSUKA, Hiromi NISHIMOTO, Akio MIYAMOTO, Kiyoshi OKUDA, Seiz ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 616-622
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: August 11, 2010
    JOURNAL FREE ACCESS
    Glucocorticoids modulate ovarian function in cattle. However, their regulatory mechanisms have not been fully elucidated. In the present study, we examined gene expression of two glucocorticoid-metabolizing enzymes, a bidirectional 11β-HSD type 1 (11HSD1) and a dehydrogenase 11β-HSD type 2 (11HSD2), and glucocorticoid receptor (GR) in bovine follicles during follicular maturation and atresia. Granulosa cells (GCs) and theca interna layers (TIs) were harvested from follicles classified as small growing, dominant, preovulatory, early atretic and late atretic follicles. The expression levels of 11HSD1, 11HSD2 and GR mRNA were quantified by real-time PCR. In the healthy follicles, expression of 11HSD1 mRNA increased as follicles matured, both in GCs and TIs. A significant negative correlation was found between the concentration of cortisol in follicular fluid and the level of 11HSD1 mRNA in GCs. The expression of 11HSD2 and GR was either very low or largely unchanged during follicular maturation. In the atretic follicles, a drastic increase in the expression of 11HSD2 was observed both in GCs and TIs. To assess the effect of FSH on the expression of 11HSDs and GR, GCs were cultured with FSH (0-100 ng/ml) for up to 6 days. FSH increased 11HSD1 mRNA in a dose-dependent manner, but not 11HSD2, nor GR. Taken together, these results suggest that developmentally-regulated 11HSD1 plays a pivotal role in modulating the local glucocorticoid environment in maturing bovine follicles.
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  • Brahmasani SAMBASIVA RAO, Yelisetti UMA MAHESH, Uthanda Raman LAKSHMIK ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 623-629
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: August 11, 2010
    JOURNAL FREE ACCESS
    The ability to rescue gametes from endangered or wildlife species and to subsequently produce viable embryos holds tremendous potential as a means to increase the population size of endangered or wildlife species. The objective of this study was to assess the meiotic and developmental competence of oocytes recovered from postmortem ovaries of the Indian blackbuck. Oocytes collected from the ovaries of dead blackbucks were allowed to mature in vitro and then tested for developmental potential by activation with ionomycin followed by treatment with 6-dimethylaminopurine. The average number of oocytes recovered per ovary was 10.9, and recovery of the oocytes did not depend on the presence or absence of the corpus luteum, on the side, size and weight of the ovaries or on the type of oocytes recovered. The proportion of good quality oocytes showing cumulus expansion and extrusion of the first polar body were 79.3% and 46.1% when cultured with gonadotropins. In vitro maturation studies indicated that the proportion of oocytes that reached MII stage was significantly higher when good quality oocytes (68%) were used compared with fair quality oocytes (48%) when cultured in the presence of gonadotropins. Furthermore, fifty eight percent of the in vitro matured oocytes cleaved, and thirteen percent of the cleaved oocytes developed into blastocysts. These findings suggest that the oocytes recovered from postmortem ovaries of the blackbuck can be utilized for production of embryos.
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  • Takehiro HIMAKI, Satoshi WATANABE, Haiei CHI, Mitsutoshi YOSHIDA, Kazu ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 630-638
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: August 28, 2010
    JOURNAL FREE ACCESS
    Galα1-3Gal (α-Gal epitope) is the major xenoantigenic epitope responsible for hyperacute rejection upon pig-to-human xenotransplantation. Endo-β-galactosidase C (EndoGalC) from Clostridium perfringens can digest the α-Gal epitope. In this study, gene-engineered primary cultured porcine embryonic fibroblasts (PEF) expressing EndoGalC were obtained and subjected to somatic cell nuclear transfer (SCNT) to test whether xenograft-competent pigs can be created. The EndoGalC-expressing PEF clones exhibited highly reduced expression of α-Gal epitope, as revealed by cytochemical staining with BS-I-B4 isolectin, a lectin that specifically binds to α-Gal epitope, and FACS analysis. The pattern of low level of α-Gal epitope expression continued for at least 6 months (more than 10 generations) after isolation. SCNT of nuclei from these cells resulted in the generation of blastocysts that displayed nearly complete loss of α-Gal epitope from their cell surface. This is the first study to demonstrate that SCNT using EndoGalC-expressing PEFs as donors would be useful for production of genetically modified cloned pigs suitable for xenotransplantation.
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  • Genlin WANG, Qunwei CUI, Kaining CHENG, Xiangying ZHANG, Guangdong XIN ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 639-642
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: August 28, 2010
    JOURNAL FREE ACCESS
    The aim of the present study was to predict fetal sex at different time points of gestation in cattle by detecting the fetal SRY gene in cow plasma. Plasma DNA was extracted from the blood samples of 110 pregnant cows during the gestational period of 30 to 242 days. Nested PCR was employed to detect the fetal SRY, which the male fetus carries exclusively, in cow plasma. The cows positive for SRY were predicted to carry male fetuses. The results showed that the fetal DNA from cow plasma was successfully amplified and that fetuses could be sexed with an overall accuracy rate of 100% (43/43) for males and 91.0% (61/67) for females and with accuracy rates of 100% (3/3) for males and 85.7% (12/14) for females at 30 EN 59 days of gestation and 100% (40/40) for males and 92.5% (49/53) for females at more than 2 months of gestation, respectively. This suggests that the molecular method developed here could be used in sex prediction for fetuses.
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  • Agnieszka BLITEK, Jolanta KIEWISZ, Agnieszka WACLAWIK, Monika M. KACZM ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 643-648
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: August 28, 2010
    JOURNAL FREE ACCESS
    The homeobox A (HOXA) family of genes is responsible for segmental development of the female reproductive tract during embryogenesis. However, HOXA10 has been shown to be essential not only for uterus development, but also for implantation. Persistent expression and steroid-dependent regulation of this gene has been demonstrated in adult human, primate, murine and canine uteri. Moreover, HOXA10-dependent expression of prostaglandin H synthase-2 (PGHS-2), a key enzyme in prostaglandin production, has been previously detected. The role of the HOXA10 gene in the porcine uterus is not well established. Therefore, the present studies were undertaken to 1) examine the effect of E2 and P4 on HOXA10 mRNA and protein content in the endometrium collected on day 9 of the estrous cycle and 2) determine the PGHS-2 protein expression and PGE2 and PGF2α secretion from endometrial tissue in response to steroid treatment. Endometrial explants collected from mature gilts on day 9 of the estrous cycle were incubated with E2 (1-100 nM), P4 (10-1000 nM) or E2 (10 nM) and P4 (100 nM) for 24 h. E2 alone or E2 in the presence of P4 increased HOXA10 mRNA expression in the endometrium (P<0.05). The HOXA10 protein level was upregulated in response to E2, P4 and both steroids administered simultaneously (P<0.05). Moreover, E2 and P4 stimulated PGHS-2 protein expression in cultured endometrial explants. PGE2, but not PGF2α, secretion increased in the presence of E2 (P<0.05). However, the release of both prostaglandins was decreased after treatment of endometrial explants with the highest dose of P4 (P<0.01). These results demonstrate that E2 and P4 are important regulators of HOXA10 gene expression in the adult porcine endometrium during the mid-luteal phase of the estrous cycle. Additionally, the similar profiles of endometrial HOXA10 and PGHS-2 expression in the presence of E2 and P4 indicate that both genes are simultaneously regulated by steroids in the porcine uterus.
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  • Sueo NIIMURA, Tadahiro OGATA, Ayano OKIMURA, Taro SATO, Yasuhiko UCHIY ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 649-654
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: August 28, 2010
    JOURNAL FREE ACCESS
    Morphological changes of cultured bovine blastocysts during hatching were observed using time-lapse videomicrography in order to investigate the patterns of the hatching process that occurred in the blastocysts and to determine whether the hatching patterns differed between blastocysts developed from fresh and cryopreserved embryos. Compacted morulae (CMs) were collected from superovulation-treated Japanese Black and Holstein dairy cattle and cultured in a medium in a CO2 culture chamber equipped with an inverted microscope at 38.5 C. Images of resultant blastocysts during the period from blastocoel formation to completion of hatching were taken at 4-sec intervals by a CCD color camera connected to an inverted microscope and recorded by a time-lapse video cassette recorder. In blastocysts developed from fresh CMs, hatching was found to begin with protrusion of trophectoderm cells from zonae pellucidae at the expanded stage. Protrusion of the cells occurred in any site of the trophectoderm. After protrusion, a large or small slit was formed in the zona pellucida in all blastocysts as a result of blastocyst expansion or enlargement of the protrusion. Then, blastocysts completely escaped from the zona pellucida through the slit in the state of expansion. From these findings, the hatching patterns of cattle blastocysts could be classified into 5 types. In blastocysts developed from frozen-thawed CMs, the hatching pattern and length of time needed for hatching are similar to those in blastocysts developed from fresh CMs. In addition, the pregnancy rate of recipients following transfer of frozen-thawed CMs (52.4%) did not differ from that of recipients following transfer with fresh CMs (58.3%). These results suggested that the quality of frozen-thawed cattle embryos is comparable to that of fresh embryos and that there could be a relationship between the hatching pattern of blastocysts and the viability of embryos after transfer.
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  • Seung-Wook SHIN, Mikiko TOKORO, Satoshi NISHIKAWA, Hyang-Heun LEE, Yuk ...
    Article type: Original Article
    2010 Volume 56 Issue 6 Pages 655-663
    Published: 2010
    Released on J-STAGE: January 15, 2011
    Advance online publication: August 28, 2010
    JOURNAL FREE ACCESS
    In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation. However, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins. Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryos. First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used these oocytes throughout this study. Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the G1 phase of the first cell cycle. Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i.e., the hsp70.1, MuERV-L, eif-1a and zscan4d genes. As a result, we found that onset of expression of the four examined ZGA genes was delayed in both normally developed 2-cell embryos and arrested 1-cell embryos. Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos.
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