Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
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Original Article
  • Nan ZHANG, Wei MAO, Ying ZHANG, Na HUANG, Bo LIU, Long GAO, Shuangyi Z ...
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 101-108
    Released: April 13, 2018
    [Advance publication] Released: December 23, 2017
    JOURNALS FREE ACCESS

    Oviductal glycoprotein 1 (OVGP1), an oviductin, is involved in the maintenance of sperm viability and motility and contributes to sperm capacitation in the oviduct. In this study, the regulatory effects exerted by prostaglandin E2 (PGE2) and F (PGF) on OVGP1 expression via their corresponding receptors in bovine oviductal epithelial cells (BOECs) were investigated. BOECs were cultured in vitro, and their expression of receptors of PGE2 (PTGER1, PTGER2, PTGER3, and PTGER4) and PGF (PTGFR) was measured using RT-qPCR. Ca2+ concentration was determined with a fluorescence-based method and cAMP was quantified by enzyme-linked immunosorbent assays to verify activation of PTGER2 and PTGFR by their corresponding agonists in these cells. OVGP1 mRNA and protein expression was measured using RT-qPCR and western blotting, respectively, following PTGER2 and PTGFR agonist-induced activation. PTGER1, PTGER2, PTGER4, and PTGFR were found to be present in BOECs; however, PTGER3 expression was not detected. OVGP1 expression was significantly promoted by 10–6 M butaprost (a PTGER2 agonist) and decreased by 10–6 M fluprostenol (a PTGFR agonist). In addition, 3 μM H-89 (a PKA inhibitor) and 3 μM U0126 (an ERK inhibitor) effectively inhibited PGE2-induced upregulation of OVGP1, and 5 μM chelerythrine chloride (a PKC inhibitor) and 3 μM U0126 negated OVGP1 downregulation by PGF. In conclusion, this study demonstrates that OVGP1 expression in BOECs is enhanced by PGE2 via PTGER2-cAMP-PKA signaling, and reduced by PGF through the PTGFR-Ca2+-PKC pathway.

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  • Hisashi FUNAKURA, Ayumi SHIKI, Yuji TSUBAKISHITA, Shogo MIDO, Hiromu K ...
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 109-115
    Released: April 13, 2018
    [Advance publication] Released: January 05, 2018
    JOURNALS FREE ACCESS

    This study aimed to clarify the feasibility of a novel timed artificial insemination (TAI) protocol using ultrasonography, and to determine the associations between the ovarian component and fertility. In Experiment 1, 272 Japanese Black cows with a corpus luteum (CL) ≥ 18 mm in diameter were divided randomly into either the TRT group (134 cows that were administered gonadotropin-releasing hormone [GnRH] 56 h [day 2] after prostaglandin F [PGF] administration [day 0], followed by TAI 16–20 h later) or the CN-1 group (138 cows that were administered PGF followed by AI after estrus detection). In addition, the CN-2 group was designated for 306 cows given PGF and inseminated after estrus detection in the past two years at the same farms. In Experiment 2, 38 cows had the same treatment as the TRT group, and the sizes of follicles and CL were video-recorded on days 0 and 2. In Experiment 1, the AI and ovulation synchronization rates were higher in the TRT group than those in the CN-1 group (100 vs. 87.0% and 89.2 vs. 33.3%, respectively) (P < 0.01). The pregnancy rate in the TRT group (60.4%) was higher than that in the CN-2 group (45.1%) (P < 0.05). In Experiment 2, cows with a larger CL diameter and greater CL volume on day 0 had a higher pregnancy outcome (P < 0.05). In conclusion, this protocol was effective for improving pregnancy rates in beef herds, and fertility was associated with the CL size at the time of PGF administration.

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  • Jinsha LIU, Keiji MOCHIDA, Ayumi HASEGAWA, Kimiko INOUE, Atsuo OGURA
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 117-127
    Released: April 13, 2018
    [Advance publication] Released: December 21, 2017
    JOURNALS FREE ACCESS

    Although it is known that the susceptibility of mouse spermatozoa to freezing-thawing varies greatly with genetic background, the underlying mechanisms remain to be elucidated. In this study, to map genetic regions responsible for the susceptibility of spermatozoa to freezing-thawing, we performed in vitro fertilization using spermatozoa from recombinant inbred mice derived from the C57BL/6J and DBA/2J strains, whose spermatozoa showed distinct fertilization abilities after freezing. Genome-wide interval mapping identified two suggestive quantitative trait loci (QTL) associated with fertilization on chromosomes 1 and 11. The strongest QTL on chromosome 11 included 70 genes at 59.237260–61.324742 Mb and another QTL on chromosome 1 included 43 genes at 153.969506–158.217850 Mb. These regions included at least 15 genes involved with testicular expression and possibly with capacitation or sperm motility. Specifically, the Abl2 gene on chromosome 1, which may affect subcellular actin distribution, had polymorphisms between C57BL/6J and DBA/2J that caused at least three amino acid substitutions. A correlation analysis using recombinant inbred strains revealed that the fertilization rate was strongly correlated with the capacitation rate of frozen-thawed spermatozoa after preincubation. This result is consistent with the fact that C57BL/6J frozen-thawed spermatozoa recover their fertilization capacity following treatment with methyl-β-cyclodextrin to enhance sperm capacitation. Thus, our data provide important clues to the molecular mechanisms underlying cryodamage to mouse spermatozoa.

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  • Doungrut TUNGMAHASUK, Numfa FUNGBUN, Titaree LAOHARATCHATATHANIN, Ryot ...
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 129-134
    Released: April 13, 2018
    [Advance publication] Released: December 15, 2017
    JOURNALS FREE ACCESS

    Although the expression of gonadotropin-releasing hormone (GnRH) in the ovaries is well established, its physiological role remains unknown. The aim of this study was to determine whether ovarian GnRH mediates the actions of human chorionic gonadotropin (hCG) in the granulosa cells of immature female rats. Follicular growth was induced by administration of pregnant mare serum gonadotropin (PMSG, 15 IU/0.15 ml) on day 25 after birth, and hCG (20 IU/0.2 ml) was administered on day 27 revealing the increase of plasma progesterone level. Primary cultures of granulosa cells were established from large follicles 2 days after PMSG treatment. Progesterone synthesis was augmented by hCG in a dose-dependent manner. Annexin A5 (ANXA5), a biomarker of GnRH, was expressed in the granulosa-luteal cells after hCG treatment, as shown by immunohistochemistry, suggesting that hCG treatment induced GnRH action. The GnRH mRNA level was increased by hCG, and treatment with GnRH agonist (GnRHa) increased ANXA5 mRNA levels in the primary cultures of granulosa cells. Concomitant incubation of GnRH (10–7 M) or GnRHa (fertirelin acetate, 10–8 M) with hCG suppressed progesterone synthesis during a 3 h incubation period. The mRNA expression of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) was synergistically stimulated and suppressed by hCG and GnRHa, respectively. GnRHa stimulated p21 expression, and GnRHa and hCG synergistically reduced the mRNA expression levels of p27 and FOXO1. These data suggest that GnRH induced by LH may have a role for the LH-mediated luteinization of granulosa cells. In addition, ANXA5 may be involved in GnRH action. GnRH-ANXA5 would be an important mechanism in cell differentiation.

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  • Tomochika SUGIURA, Shun AKIYOSHI, Fumihiro INOUE, Yojiro YANAGAWA, Mas ...
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 135-143
    Released: April 13, 2018
    [Advance publication] Released: February 03, 2018
    JOURNALS FREE ACCESS

    The objective of this study was to investigate cyclical changes in endometrial thickness in relation to progesterone (P4) and estradiol-17β (E2) concentrations during natural and induced estrus in 15 cows. In the prostaglandin (PG) F-induced estrus group, ultrasonography (USG) at 6-h intervals was used to determine endometrial thickness 48–24 h before the PGF treatment until 24 h after ovulation (ovulation = Day 0). In the natural estrus group, USG was performed every 48 h from Day 3 to Days 15–18 after the first ovulation, and then every 6 h until 24 h after ovulation. Endometrial thickness was standardized using Day 13 as a reference day. Blood was collected during every USG examination and plasma P4 and E2 concentrations were determined. Endometrial thickness of the induced estrus group (n = 11) was greater than that of the natural estrus group (n = 9) between 60 and 12 h before ovulation (P < 0.05). In the natural estrus group, prior to an increase in endometrial thickness, a decrease in P4 and an increase in E2 were detected. In the induced estrus group, based on the time of ovulation, an increase in endometrial thickness was detected at the same time of a decrease in P4 before an increase in E2. These results suggest that decreases in P4 concentrations may be a cue to changes in endometrial thickness, while increases in E2 concentrations appear to sustain and/or enhance these changes.

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  • Noriko FUNASAKA, Motoi YOSHIOKA, Toshiaki ISHIBASHI, Toshiyuki TATSUKA ...
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 145-152
    Released: April 13, 2018
    [Advance publication] Released: January 19, 2018
    JOURNALS FREE ACCESS

    We monitored annual fluctuations of gonadal steroid levels in three sexually mature captive finless porpoises (Neophocaena asiaeorientalis; two males and one female) from two different facilities over 56–91 months. Two animals (one male and one female) were held in an indoor tank with a sunroof (facility A) and the other male was held in an indoor tank without a sunroof (facility B). Water temperatures in both facilities reflected seasonal changes during the study period with a minor difference in the fluctuation pattern. Testosterone levels of the male in facility A were higher from spring to summer every year and exhibited a 12-month cycle. The female showed estrus cycles in 1-month intervals from summer to winter, excluding 2 anestrus years. In contrast, the period of higher testosterone levels of the male in facility B gradually initiated earlier over the years under a constant photoperiod (11.5L:12.5D) and exhibited a 9-month cycle during the first 52 months. After changing the light conditions to a natural photoperiod, its testosterone levels were high from early spring to summer for 3 consecutive years and exhibited a 12-month cycle. Our results showed that under a constant artificial photoperiod, the male in facility B failed to recognize the seasonal changes of a natural external environment, resulting in a 9-month, free-running hormone cycle.

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  • Shizuka MIZUMACHI, Taiki ARITOMI, Kuniaki SASAKI, Kazuei MATSUBARA, Yu ...
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 153-160
    Released: April 13, 2018
    [Advance publication] Released: February 16, 2018
    JOURNALS FREE ACCESS

    Macromolecular crowded culture medium formed by addition of polyvinylpyrrolidone (PVP; molecular weight = 360 000), positively influences the viability, growth, and development of bovine oocytes. Owing to its apparently various effects, uncovering the specific mechanisms of crowding responsible for these outcomes is important. The present study was conducted to determine the effects of crowding on oocytes with a particular focus on the intimacy of contacts between oocyte and cumulus/granulosa cells. Growing mouse oocyte-granulosa cell complexes were cultured for 10 days in a modified α-minimum essential medium, supplemented with PVP at a concentration of 0%, 1%, 2%, or 3% (w/v). Although the complexes developed in all groups, 2% and 3% PVP medium induced a substantial morphological modification, and a larger proportion of oocytes associated with cumulus cells survived in 3% PVP medium than in the 0% or 1% PVP medium. No significant difference was found in the frequencies of polar body extrusion (78–88%) and blastocyst formation (approximately 40%) after in vitro fertilization among the experimental groups. Confocal laser scanning microscopy indicated a higher number of transzonal processes reaching the oocyte from cumulus cells in 2% PVP medium than in 0% PVP medium. Transmission electron microscopy depicted close adhesion of the oocyte with cumulus cells in 2% PVP medium —bearing a resemblance to their in vivo counterparts— and loose adhesion in 0% PVP medium. In conclusion, we found that a mechanism for the action of crowded conditions involves the strengthening of contacts and communication between oocytes and companion cumulus/granulosa cells.

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    Editor’s picks

    Cover Story :
    Recently, the effects of macromolecular crowding on cultured cells have gathered increasing interest. Crowded culture medium prepared by the addition of polyvinylpyrrolidone (PVP; Mw 360,000) positively influences the viability of oocytes during in vitro growth. Mizumachi et al. found that crowding affects a wide range of factors, including oocyte viability, complex morphology, and intimate conjunction of oocytes with cumulus/granulosa cells across the zona pellucida (Mizumachi et al. Macromolecular crowded conditions strengthen contacts between mouse oocytes and companion granulosa cells during in vitro growth, pp. 153-160). Confocal laser scanning microscopy indicated a higher number of transzonal processes (TZPs) reaching the oocyte from cumulus cells in 2% PVP medium than in 0% PVP medium.

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  • Kohtaro MORITA, Mikiko TOKORO, Yuki HATANAKA, Chika HIGUCHI, Haruka IK ...
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 161-171
    Released: April 13, 2018
    [Advance publication] Released: March 02, 2018
    JOURNALS FREE ACCESS

    Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.

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  • Hui PENG, Jianchao HUO, Yuyun GAO, Jing CHEN, Xiang YU, Tianfang XIAO
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 173-177
    Released: April 13, 2018
    [Advance publication] Released: February 09, 2018
    JOURNALS FREE ACCESS

    Fas-associated protein factor 1 (FAF1) is a Fas-associated protein that functions in multiple cellular processes. Previous research showed that mutations in Faf1 led to the lethality of cleavage stage embryos in a mouse model. The aim of the present study was to analyze the expression pattern, localization, and function of FAF1 in meiotic resumption of mouse oocytes. FAF1 was exclusively expressed in oocytes at various follicular stages within the ovary and was predominantly localized in the cytoplasm of growing oocytes. Furthermore, Faf1 mRNA and protein were persistently present during oocyte maturation and Faf1 mRNA levels were similar in the germinal vesicle (GV), GV breakdown (GVBD), and metaphase II (MII) stages of oocytes. Moreover, knockdown of Faf1 in GV-stage oocytes led to a significantly decreased rate of GVBD. To our knowledge, these results provide the first evidence regarding a novel function of FAF1 in meiotic resumption in mouse oocytes.

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  • Shujuan GUO, Xingyu YAN, Feifei SHI, Ke MA, Zi-Jiang CHEN, Cong ZHANG
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 179-186
    Released: April 13, 2018
    [Advance publication] Released: February 15, 2018
    JOURNALS FREE ACCESS

    The Snail gene family includes Snai1, Snai2, and Snai3 that encode zinc finger-containing transcriptional repressors in mammals. The expression and localization of SNAI1 and SNAI2 have been studied extensively during folliculogenesis, ovulation, luteinization, and embryogenesis in mice. However, the role of SNAI3 is unknown. In this study, we investigated the expression of SNAI3 during these processes. Our immunohistochemistry data showed that SNAI3 first appeared in oocytes by postnatal day (PD) 9. Following this, SNAI3 was found to be expressed consistently in theca and interstitial cells, along with oocytes. In gonadotropin-treated immature mice, the expression of SNAI3 did not change significantly during follicular development. The expression of SNAI3 was reduced during ovulation, after which it increased gradually during luteinization. Similar results were obtained from western blot analyses. Furthermore, real-time polymerase chain reaction (RT-PCR) analyses revealed varying mRNA levels of different Snail factors at a given time in gonadotropin-induced ovaries. During early embryo cleavage, SNAI3 was localized to the nucleus, except the nucleolus at the germinal vesicle and one-cell stages. From two- to eight-cell stages, SNAI3 was localized only to the nucleolus. Thereafter, SNAI3 was detected only in the cytoplasm, except during the blastocyst stage when it was localized to the nucleus of the trophectoderm and the inner cell mass. RT-PCR results showed that the expression of Snail superfamily genes was decreased during the blastocyst stage. From the eight-cell to morula stage, when compaction occurs that is a prerequisite for blastocyst formation, Snai3 mRNA was expressed at very low levels and was opposite to the highest expression level of the compaction-related gene, E-cadherin, at the eight-cell stage. Taken together, our results suggest that SNAI3 likely plays some roles during folliculogenesis, luteinization, and early embryonic development.

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  • Pasqualino LOI, Cesare GALLI, Giovanna LAZZARI, Kazutsugu MATSUKAWA, J ...
    Type: Original Article
    Volume 64 (2018) Issue 2 Pages 187-191
    Released: April 13, 2018
    [Advance publication] Released: February 15, 2018
    JOURNALS FREE ACCESS

    Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

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Technology Report
  • Kohta KIKUCHI, Keisuke KOZAI, Takuo HOJO, Miki SAKATANI, Kiyoshi OKUDA ...
    Type: Technology Report
    Volume 64 (2018) Issue 2 Pages 193-197
    Released: April 13, 2018
    [Advance publication] Released: January 07, 2018
    JOURNALS FREE ACCESS

    We investigated the electrical impedance of the reproductive tracts (vagina and uterine endometrial tissues) and the expression of mucus-related genes to identify the stage of the estrous cycle in mares. We first examined vaginal impedance in native Hokkaido mares during their estrous cycle and found no significant differences. However, impedance levels tended to decrease towards ovulation. Furthermore, we investigated the estrous cycle by measuring the electrical impedance of the uterine endometrial tissues obtained from carcasses of mares. We found that impedance levels in the endometrial tissues decreased in the regressed phase of the corpus luteum (CL). Expression of mucus-related genes (ATP1A1, CFTR, AQP3, and AQP5) varied at different stages of the estrous cycle. Among them, AQP3 expression was consistent with previous reports. We concluded that electrical impedance in the uterine endometrial tissues of mares could be potentially used to verify the presence of active CL in horses for experimental purposes. However, further studies are needed to determine the reference value and to identify the day of the estrous cycle in mares.

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  • Shuntaro IKEDA, Miki SUGIMOTO, Shinichi KUME
    Type: Technology Report
    Volume 64 (2018) Issue 2 Pages 199-202
    Released: April 13, 2018
    [Advance publication] Released: January 21, 2018
    JOURNALS FREE ACCESS

    Bovine preimplantation embryos exhibit dramatic biological changes between before and after the 8-16-cell stage. Here we report a simple lipofection method to transfect siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well (WOW) culture system. Bovine one-cell embryos produced in vitro were freed from the zona pellucida and cultured up to the 8-16-cell stage in WOW dishes. The 8-16-cell embryos were lipofected with siRNA and the transfection efficiency was assessed at 48 h of transfection. Lipofection with a red fluorescent non-targeting siRNA revealed the importance of zona removal for transfection of siRNA into embryos. Using this method, we knocked down the methionine adenosyltransferase 2A (MAT2A) gene, achieving a significant reduction in MAT2A expression (P < 0.05) concomitant with the marked inhibition of blastocyst development. Our proposed method, tentatively named ‘Octo-lipofection’, may be useful to analyze gene functions in bovine preimplantation embryos without expensive equipment and skill-intensive techniques.

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