Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 56, Issue 3
June
Displaying 1-11 of 11 articles from this issue
Original Article
  • Haolin ZHANG, Xia SHENG, Xiao HU, Xiuwen LI, Hui XU, Mengyuan ZHANG, B ...
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 297-302
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: March 03, 2010
    JOURNAL FREE ACCESS
    The purpose of this study was to investigate seasonal changes of spermatogenesis and the cellular localization of P450c17 and P450arom in wild male ground squirrels during the breeding and non-breeding seasons. The testicular weight, testicular size and score count of spermatogenesis from April to September were measured, and histological and immunohistochemical observations of testicular tissues were performed in wild male ground squirrels. In addition, total proteins were extracted from testicular tissue in the breeding and non-breeding seasons and were used for Western blotting analysis for P450c17 and P450arom. There were marked variations in testicular weight, testicular size and score count of spermatogenesis from the breeding season (April) to the non-breeding season (September). Histologically, spermatogonia, primary spermatocytes, secondary spermatocytes and spermatozoa were identified in the breeding season (April). Immunolocalization of P450c17 was detected in Leydig cells and spermatozoa during the breeding season and was only found in Leydig cells during the non-breeding season. The positive signals of P450c17 by Western blotting were both observed in the breeding and non-breeding seasons. Immunolocalization of P450arom was observed in Leydig cells, Sertoli cells and all types of spermatogenic cells including mature-phase spermatozoa in the breeding season, while immunoreactivity for P450arom was not present in the testis of the non-breeding season. With P450arom antibody, a band was also only detected in the breeding season by Western blotting. These results suggest that the seasonal changes in testicular weight and size are correlated with spermatogenesis and immunolocalization of P450c17 and P450arom, and androgen and estrogen may play an important role in the spermatogenesis and testicular recrudescence and regression process.
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  • Ying WANG, Lingling HOU, Chunying LI, Weijun GUAN, Lina CHEN, Xiangche ...
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 303-308
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: March 15, 2010
    JOURNAL FREE ACCESS
    The aim of this study was to explore the isolation and culture process of Beijing fatty chicken primordial germ cells (PGCs) and investigate their biological characteristics. The PGCs isolated from the genital ridges of Beijing fatty chicken (Gallus domesticus) embryos after 5.5 days of incubation were co-cultured with mice embryonic fibroblasts (MEF). The results showed that the PGCs of the Beijing fatty chicken were positive for periodic acid Schiff (PAS) and alkaline phosphatase (AKP) staining. These cells could proliferate for a prolonged time in vitro and maintain diploid karyotype. Immunocytochemical staining showed that they expressed SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81, and real-time PCR showed that they expressed Cvh, CDH and Dazl. They could form simple embryoid bodies and differentiate into osteoblasts in vitro. In addition, after transfected with pEGFP-N3, pEYFP-N1 and pDsRed-N1 vectors by liposomal transfection, enhanced green, yellow and red fluorescent protein-positive cells could be visualized using a laser confocal microscope. The above results suggested that PGCs from the Beijing fatty chicken not only had strong self-renewal ability, but also had the potential to differentiate towards mesoblast cells. These cells are suitable for genetic manipulation as nuclear donors.
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  • Tetsuma MURASE, Noriaki IMAEDA, Hiroto YAMADA, Masaki TAKASU, Kazuo TA ...
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 309-314
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: March 03, 2010
    JOURNAL FREE ACCESS
    The present study investigated whether substitution of HEPES for bicarbonate in BTS (BTS-H) used to dilute boar ejaculates immediately after ejaculation could reduce the increased inducibility of the acrosome reaction by calcium and calcium ionophore A23187. When an ejaculate was split, diluted 5-fold with regular BTS (BTS-B) and BTS-H and stored at 17 C for 12 h or 60 h, the extender or storage time had no significant influence on sperm motility or viability measured by the eosin-nigrosin method. When spermatozoa diluted serially with BTS-B and stored (36 h) were stimulated with Ca2+ (3 mM) and A23187 (0.3 μM), the proportion of spermatozoa that underwent the acrosome reaction (% acrosome reactions) significantly increased as the magnifications of dilution increased (bicarbonate content almost unchanged by dilution). By contrast, the % acrosome reactions in spermatozoa similarly diluted and stored with BTS-H decreased with the increasing magnifications of dilution (bicarbonate decreased). Sperm motility immediately after the end of incubation without A23178 tended to be lower for BTS-H than BTS-B, and the ejaculates for BTS-H had a tendency to have a lower total protein in seminal plasma than those for BTS-B. These results implied that the samples for BTS-H could be used as a model for ejaculates possibly collected during summer and showing subfertility. When an ejaculate was split, diluted serially with BTS-B and BTS-H and stored, viability measured by staining with propidium iodide was extremely similar between the 2 extenders and among the different dilution magnifications, regardless of whether spermatozoa were washed (stored for 36-66 h) or not (stored for 66-72 h). These results suggest that boar ejaculate can be stored with BTS-H at least according to the results for sperm motility and viability and that hypersensitivity of spermatozoa to Ca2+ and A23187 potentially associated with boar subfertility could be lessened by diluting ejaculates with BTS-H.
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  • Yasuhisa YAMASHITA, Ikko KAWASHIMA, Yosuke GUNJI, Mitsugu HISHINUMA, M ...
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 315-323
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: February 18, 2010
    JOURNAL FREE ACCESS
    During in vitro maturation of porcine cumulus-oocyte complexes (COCs), progesterone was secreted from cumulus cells and acted on the cumulus cells themselves, which required for cumulus expansion and oocyte maturation. EGF-like factor (amphiregulin, AREG; epiregulin, EREG) and its protease, TACE/ADAM17, are also expressed in cumulus cells, and thereby, soluble EGF domain was acted on the EGF receptor expressed on cumulus cells. In this study, we examined the relationship between progesterone function and EGF-like factor stimuli in cumulus cells of porcine COCs. When COCs were cultured with FSH and LH, Areg, Ereg and Tace/Adam17 were expressed in cumulus cells. Treatment with a progesterone receptor (PGR) antagonist, RU486, did not affect the Areg and Ereg mRNA expression levels at any culture time points. However, the Tace/Adam17 mRNA level, protein level and its activity were significantly suppressed by RU486 at the 30 or 40 h time point. At 20 h of culture, phosphorylation of ERK1/2 and the expressions of target genes (Has2, Tnfaip6 and Ptgs2) were not suppressed by RU486; however, at 40 h, ERK1/2 phosphorylation and the target gene expression levels were significantly downregulated by RU486 in cumulus cells. Furthermore, the negative effects of RU486 at 40 h were overcome by the addition of EGF. These results indicated that the level of TACE/ADAM17 in cumulus cells was regulated by the progesterone-PGR pathway during in vitro maturation of porcine COCs. Therefore, we concluded that the progesterone-induced TACE/ADAM17 leads to production of soluble EGF domain from cumulus cells, which enhances functional changes of cumulus cells and progresses meiotic maturation of oocytes during in vitro maturation of porcine COCs.
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  • Xinjie CHEN, Yong FAN, Xiaolin LONG, Xiaofang SUN
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 324-329
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: March 03, 2010
    JOURNAL FREE ACCESS
    After fertilization, male and female gametes undergo extensive reprogramming to restore totipotency. Both DNA methylation and histone modification are important epigenetic reprogramming events. Previous studies have reported that the paternal pronucleus of the human zygote is actively demethylated to some extent, while the maternal pronucleus remains methylated. However, to our knowledge, the relationship between DNA methylation and H3K9 dimethylation patterns in human embryos has not been reported. In this study, we examined the dynamic DNA methylation and H3K9 dimethylation patterns in triploid and bipronucleated zygotes and early developing embryos. We sought to gain further insight into the relationship between DNA methylation and H3K9 dimethylation and to investigate whether removing a pronucleus from triploid zygotes affects DNA methylation and H3K9 dimethylation patterns. We found that active DNA demethylation of the two male pronuclei occurred in tripronuclear human zygotes while the female pronucleus remained methylated at 20 h post-insemination. In tripronuclear human zygotes, H3K9 was hypomethylated in the two paternal pronuclei relative to the maternal pronucleus. Our data show that there are no differences in the DNA methylation and H3K9 dimethylation patterns between tripronuclear and corrected bipronuclear human zygotes. However, correction of 3PN human zygotes dispermic in origin could not improve subsequent embryo development. In conclusion, DNA methylation and H3K9 dimethylation patterns are well correlated in tripronuclear zygotes and embryos; early embryo development is not affected by removal of a male pronucleus. Our results imply that limited developmental potential of either 3PN or corrected 2PN embryos may not be caused by the abnormalities in DNA methylation or H3K9 dimethylation modification.
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  • Kiho LEE, Chunmin WANG, John M CHAILLE, Zoltan MACHATY
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 330-335
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: February 18, 2010
    JOURNAL FREE ACCESS
    The effects of resveratrol (a phytoalexin with a wide variety of pharmacological activities) on pig embryos produced by parthenogenesis and/or in vitro fertilization have been investigated. First, parthenogenetic embryos were generated and cultured in PZM-3 medium supplemented with various amounts of resveratrol (0, 0.05, 0.1, 0.5, 1.0 and 25 μM final concentrations). In the presence of 0.5 μM resveratrol a significantly higher percentage of parthenogenetic embryos reached the blastocyst stage by day 7 compared to non-treated control (43.5 ± 6.3% vs. 33.0 ± 5.4%; P<0.05). The total cell number of blastocysts also increased as a result of incubation with 0.5 μM resveratrol; the difference was statistically significant between treated and non-treated embryos on day 5 of culture (35.8 ± 0.9 vs. 32.1 ± 1.1; P<0.05). Resveratrol incubation affected the expression levels of apoptosis-related genes in parthenogenetic blastocysts: the level of Bax transcripts was similar but lower expression of Bcl-2 and Caspase-3 was observed in embryos treated with 0.5 μM resveratrol when compared to control blastocysts (P<0.05). The results of the TUNEL assay were similar in blastocysts developing with or without resveratrol supplementation. In addition, when embryos produced by in vitro fertilization were incubated with 0.5 μM resveratrol, the treatment led to higher frequencies of blastocyst formation (8.6% vs. 13.3%) and elevated total cell numbers (37.1 ± 2.4 vs. 43.2 ± 1.7) by the end of the 7-day culture period (P<0.05). The results indicate that 0.5 μM resveratrol during culture has a positive effect on early embryonic development of porcine embryos.
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  • Kazumasa HONDA, Takashi HIGUCHI
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 336-340
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: March 03, 2010
    JOURNAL FREE ACCESS
    Using urethane-anesthetized lactating rats, extracellular action potentials were recorded from single cells in the dorsomedial hypothalamus (DMH), which were identified as projecting to the supraoptic nucleus (SON). Sixty-two DMH cells were identified as projecting to the SON. Of these 53, 4 and 5 cells had ipsilateral, contralateral and bilateral projections, respectively. Two cells (1 ipsilaterally and 1 contralaterally projecting cell) showed bursting activities preceding milk ejection that were similar to those of oxytocin (OT) cells in the SON or paraventricular nucleus. Two ipsilaterally and 2 bilaterally projecting cells reduced their firing rates preceding milk ejection. The results suggest that some of the projections from the DMH to the SON are contralateral or bilateral and that these projections may contribute to synchronized activation of OT cells bilaterally distributed in the hypothalamus during milk-ejection reflex.
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  • Gakushi KITO, Shinya ARAMAKI, Koji TANAKA, Tomoki SOH, Nobuhiko YAMAUC ...
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 341-346
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: March 24, 2010
    JOURNAL FREE ACCESS
    The Deleted in Azoospermia-Like (DAZL) protein coded by Dazl gene is a germline-specific RNA-binding protein essential for gametogenesis in vertebrates, and the chicken Dazl gene has also been identified in primordial germ cells (PGCs). However, the temporal and spatial expression of chicken DAZL (cDAZL) and its molecular role in germ cell development remain enigmatic. Here, we investigated the subcellular distribution and expression of cDAZL at the various stages by using a polyclonal antibody raised against its C-terminal region and compared them with those of additional germline-specific proteins chicken vasa homologue (CVH) and chicken dead end homologue (CDH). Western blot analysis for cDAZL revealed a single band in the embryonic gonads and premature chicken testis, whereas no band was detected in the premature chicken ovary. Fluorescent immunohistochemistry revealed that cDAZL was present in the nucleus and cytoplasm of circulating PGCs. Cells positive for cDAZL and CVH coexisted in the embryonic gonads and premature chicken testis, in which they were distributed near the basement membrane of seminiferous tubules. Of interest, cDAZL was not found in the premature chicken ovary, whereas CVH and CDH were present in germ cells. Collectively, three germline-specific proteins are expressed in chicken germ cells, but their patterns of expression are temporally and spatially distinct.
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  • Leopoldo Mayer de FREITAS NETO, Maico Henrique Barbosa dos SANTOS, Cr ...
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 347-350
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: March 15, 2010
    JOURNAL FREE ACCESS
    The objective of this study was to identify the migration period of the genital tubercle and the period of visualization of external genital structures in fetuses of the Dorper breed of sheep derived from natural mating and from fresh, frozen and vitrified embryo transfer. Transrectal ultrasound was performed using a double-frequency linear transducer (6.0 and 8.0 MHz) to monitor 130 ewe fetuses distributed in the four treatments regarding embryo origin. The accuracy of the ultrasound was 100% in this experiment. The fetuses originated from controlled natural mating (NM) and from fresh (FrE), frozen (FE) and vitrified (VE) embryo transfer, with embryos collected 7 days after breeding. Migration of the genital tubercle occurred earlier (P<0.05) in NM (42.21 ± 2.86 days) than in FrE (43.98 ± 3.00 days), FE (44.97 ± 1.83 days) and VE (44.58 ± 1.97days). Visualization of the scrotal bag, prepuce and vulva occurred, respectively, earlier (P<0.05) in NM (45.22 ± 1.25, 45.95 ± 1.53 and 45.01 ± 1.03 days) than in FrE (48.91 ± 1.92, 48.52 ± 1.41 and 47.41 ± 1.41 days), FE (49.97 ± 1.08, 49.18 ± 2.00 and 47.64 ± 1.82 days) and VE (50.12 ± 1.66, 49.27 ± 1.61 and 47.93 ± 1.92 days). The results show that fetal sexing can be accomplished from the 50th day onward in fetuses produced by natural mating and from the 55th day onward in fetuses derived from fresh, frozen and vitrified embryos. It can also be concluded that real-time ultrasonography is a reliable tool for fetal sex determination in sheep taking into account both the location of the genital tubercle and the identification of external genital structures.
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  • Hajime YOSHIOKA, Michie ITO, Yasuyuki TANIMOTO
    Article type: -Original Article-
    2010 Volume 56 Issue 3 Pages 351-355
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: March 24, 2010
    JOURNAL FREE ACCESS
    The objective of this study was to investigate the efficiency of estrus detection and determination of the time of ovulation in Japanese Black cows by a real-time radiotelemetric pedometer. A herd of Japanese Black cows was housed in a loose-housing barn. This pedometer system and a pressure-sensing radiotelemetric device, which detects standing events, were concurrently compared in regard to their availability for detection of estrus events. Pedometer estrus and standing events were observed for all cows (n=20), and the duration of increased walking activity was 15.8 ± 0.9 h, which was longer (P<0.0001) than the duration of standing events (9.0 ± 1.3 h). Ovulation was observed 30.2 ± 0.6 h after the onset of pedometer estrus and 29.0 ± 0.6 h after the first standing event. There was a high correlation (r=0.89, P<0.0001) between the intervals from the onset of pedometer estrus and onset of standing event to ovulation. The animals of the pedometric group (n=20) were inseminated 10 to 18 h after the onset of pedometer estrus and had a higher conception rate (90.0%, P<0.05) than those inseminated according to the a.m. /p.m. method (58.4%, n=24). In conclusion, the pedometer system is an excellent tool for precise estrus detection and insemination at the appropriate time, which ensure a high pregnancy rate in Japanese Black cows.
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Technology Report
  • Buko OGAWA, Satoshi UENO, Naoki NAKAYAMA, Hitomi MATSUNARI, Kazuaki NA ...
    Article type: -Technology Report-
    2010 Volume 56 Issue 3 Pages 356-361
    Published: 2010
    Released on J-STAGE: June 29, 2010
    Advance online publication: March 24, 2010
    JOURNAL FREE ACCESS
    The aim of the present study was to investigate whether a combination of cytoplasmic lipid removal (delipation) and treatment by a microtubule stabilizer, paclitaxel, would lead to efficient cryopreservation of porcine in vitro matured (IVM) oocytes at the meiosis II (MII) stage. Vitrification and subsequent re-warming and culture of 109 untreated oocytes produced only 9 blastocysts (8.3%). On the other hand, the post-vitrification blastocyst rate was significantly improved (21/113, 18.6%, P<0.05) when oocytes were treated with 1 μM paclitaxel. Oocyte delipation also significantly increased the post-vitrification blastocyst rate compared with the untreated group (15/37, 40.5%, P<0.05). The delipation-and-paclitaxel group exhibited a significantly higher blastocyst rate (34/75, 45.3%, P<0.05) than the paclitaxel group, although it was not significantly higher than that for the delipation group. In transfer experiment, a total of 109 (18.6%) parthenogenetic blastocysts were obtained from 586 oocytes vitrified with the delipation-and-paclitaxel treatment. Transfer of 72 blastocysts to two recipients resulted in 14 (19.4%) somite stage fetuses. In conclusion, we demonstrated for the first time that by removing cytoplasmic lipid droplets from oocytes and performing a microtubule stabilization procedure, vitrified porcine IVM MII-stage oocytes could efficiently develop to the blastocyst stage while retaining the ability to develop into fetuses.
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