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Journal of Reproduction and Development

Diqi Yang, Lei Wang, Pengfei Lin, Tingting Jiang, Nan Wang, Fan Zhao, Huatao Chen, Keqiong Tang, Dong Zhou, Aihua Wang, Yaping Jin
With granulosa and theca cells, the ovaries are responsible for producing oocytes and secreting sex steroids such as estrogen and progesterone. Endoplasmic reticulum stress (ERS) plays an important role in follicle atresia and embryo implantation. In this study, goat granulosa cells were isolated from medium-sized (4-6 mm) healthy follicles. Primary granulosa cells were immortalized by transfection with human telomerase reverse transcriptase (hTERT) to establish a goat granulosa cell line (hTERT-GGCs). These hTERT-GGCs expressed hTERT and had relatively long telomeres at passage 50...
October 15, 2016: Journal of Reproduction and Development
Hanchao Gao, Chengjiang Zhao, Xi Xiang, Yong Li, Yanli Zhao, Zesong Li, Dengke Pan, Yifan Dai, Hidetaka Hara, David K C Cooper, Zhiming Cai, Lisha Mou
Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs...
October 8, 2016: Journal of Reproduction and Development
Michiko Nakai, Junya Ito, Shun-Ichi Suzuki, Dai-Ichiro Fuchimoto, Shoichiro Senbon, Misae Suzuki, Junko Noguchi, Hiroyuki Kaneko, Akira Onishi, Naomi Kashiwazaki, Kazuhiro Kikuchi
In pigs, the efficiency of embryo production after intracytoplasmic sperm injection (ICSI) is still low because of frequent failure of normal fertilization, which involves formation of two polar bodies and two pronuclei. To clarify the reasons for this, we hypothesized that ICSI does not properly trigger sperm-induced fertilization events, especially intracellular Ca(2+) signaling, also known as Ca(2+) oscillation. We also suspected that the use of in vitro-matured oocytes might negatively affect fertilization events and embryonic development of sperm-injected oocytes...
September 30, 2016: Journal of Reproduction and Development
Michiko Nakai, Shun-Ichi Suzuki, Junya Ito, Dai-Ichiro Fuchimoto, Shoichiro Sembon, Junko Noguchi, Akira Onishi, Naomi Kashiwazaki, Kazuhiro Kikuchi
In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used...
September 30, 2016: Journal of Reproduction and Development
Mei Rong, Atsushi Matsuda, Yasushi Hiraoka, Jibak Lee
Cohesins containing a meiosis-specific α-kleisin subunit, RAD21L or REC8, play roles in diverse aspects of meiotic chromosome dynamics including formation of axial elements (AEs), assembly of the synaptonemal complex (SC), recombination of homologous chromosomes, and cohesion of sister chromatids. However, the exact functions of individual α-kleisins remain to be elucidated. Here, we examined the localization of RAD21L and REC8 within the SC by super-resolution microscopy, 3D-SIM. We found that both RAD21L and REC8 were localized at the connection sites between lateral elements (LEs) and transverse filaments (TFs) of pachynema with RAD21L locating interior to REC8 sites...
September 26, 2016: Journal of Reproduction and Development
Shin-Ichi Kashiwabara, Satsuki Tsuruta, Keitaro Okada, Yutaro Yamaoka, Tadashi Baba
The testis-specific cytoplasmic poly(A) polymerase PAPOLB/TPAP is essential for spermatogenesis. Although this enzyme is responsible for poly(A) tail extension of a subset of mRNAs in round spermatids, the stability and translational efficiency of these mRNAs are unaffected by the absence of PAPOLB. To clarify the functional importance of this enzyme's adenylation activity, we produced PAPOLB-null mice expressing a polyadenylation-defective PAPOLB mutant (PAPOLB(D114A)), in which the catalytic Asp at residue 114 was mutated to Ala...
September 18, 2016: Journal of Reproduction and Development
Masaya Yamanaka, Kazuhisa Tomita, Shu Hashimoto, Hiroshi Matsumoto, Manabu Satoh, Hiromi Kato, Yoshihiko Hosoi, Masayasu Inoue, Yoshiharu Nakaoka, Yoshiharu Morimoto
Density gradient centrifugation (DGC) and swim-up techniques have been reported for semen preparation in assisted reproductive techniques in humans. We investigated whether semen preparation using a combination of DGC and swim-up techniques could effectively decrease morphologically abnormal human sperms at the ultrastructural level. Semen samples were obtained from 16 infertile males and fractionated by swim-up following DGC. Ultrastructural abnormalities of sperms obtained from original semen, lower layer of swim-up following DGC, and upper layer of swim-up following DGC were analyzed by transmission electron microscopy...
September 11, 2016: Journal of Reproduction and Development
Fang Zeng, Zhihua Huang, Yujuan Yuan, Junsong Shi, Gengyuan Cai, Dewu Liu, Zhenfang Wu, Zicong Li
Xist is an X-linked gene responsible for cis induction of X chromosome inactivation. Studies have indicated that Xist is abnormally activated in the active X chromosome in cloned mouse embryos due to loss of the maternal Xist-repressing imprint following enucleation during somatic cell nuclear transfer (SCNT). Inhibition of Xist expression by injecting small interfering RNA (siRNA) has been shown to enhance the in vivo developmental efficiency of cloned male mouse embryos by more than 10-fold. The purpose of this study was to investigate whether a similar procedure can be applied to improve the cloning efficiency in pigs...
August 29, 2016: Journal of Reproduction and Development
Hiroyuki Imai, Wataru Fujii, Ken Takeshi Kusakabe, Yasuo Kiso, Kiyoshi Kano
Alterations in ploidy tend to influence cell physiology, which in the long-term, contribute to species adaptation and evolution. Polyploid cells are observed under physiological conditions in the nerve and liver tissues, and in tumorigenic processes. Although tetraploid cells have been studied in mammalian cells, the basic characteristics and alterations caused by whole genome duplication are still poorly understood. The purpose of this study was to acquire basic knowledge about the effect of whole genome duplication on the cell cycle, cell size, and gene expression...
August 29, 2016: Journal of Reproduction and Development
Yuko Toishi, Nobuo Tsunoda, Kouiti Kume, Kentaro Nagaoka, Gen Watanabe, Kazuyoshi Taya
A novel chemiluminescent enzyme immunoassay system, PATHFAST, for the measurement of estradiol in horses was evaluated. The concentrations of estradiol in the whole blood and serum of mares were measured using PATHFAST and the estradiol concentrations measured by PATHFAST were compared with those measured by a time-resolved fluoro-immunoassay (FIA). To monitor physiological changes, serum estradiol concentrations in mares were measured using PATHFAST throughout the gestation period. The serum estradiol concentrations correlated highly with those in whole blood samples...
August 20, 2016: Journal of Reproduction and Development
Nelson Fijamo Mesquita, Renata Maculan, Luciana França Smith Maciel, Nathalia Alves, Rodirgues Rafaela DE Carvalho, Gabriel Miranda Moreira, José Camisão DE Souza
The aim of this study was to investigate possible relationships between morphometric characteristics of the bovine reproductive tract and measures of fertility, such as antral follicle counts (AFCs) and the number and quality of recovered cumulus oophorus complexes (COCs). First, the genital tracts of 360 abattoir Zebu/Holstein crossbred cows were studied. Rima vulvae length (RL), vulvar width (VW), ovarian size, pelvic fat, number of aspirated COCs, and AFCs were recorded. An index of COC quality (CQI) was established, which weighed overall gamete quality based on the IETS classification...
August 19, 2016: Journal of Reproduction and Development
Tayita Suttirojpattana, Tamas Somfai, Satoko Matoba, Takashi Nagai, Rangsun Parnpai, Masaya Geshi
We assessed the effect of pretreating sperm with dithiobutylamine (DTBA) to improve embryo development by intracytoplasmic sperm injection (ICSI) in cows. Acridine Orange staining revealed that when applied at different concentrations (2.5, 5, and 10 mM) and exposure times (5 min, 20 min, 1 h, and 2 h), DTBA reduced disulfide bonds in spermatozoa with the highest efficacy at 5 mM for 5 min. DTBA enhanced the percentage of spermatozoa with free protamine thiol groups compared with untreated spermatozoa (control) (P < 0...
August 14, 2016: Journal of Reproduction and Development
Yubyeol Jeon, Junchul David Yoon, Lian Cai, Seon-Ung Hwang, Eunhye Kim, Eunsong Lee, Eui Bae Jeung, Sang-Hwan Hyun, Woo Suk Hwang
Zinc supplementation (0.8 µg/ml) in in vitro maturation (IVM) medium significantly enhances oocyte quality. In this study, we compared the development of somatic cell nuclear transfer (SCNT) embryos produced from conventional IVM (control) and zinc-supplemented IVM oocytes. A total of 1206 and 890 SCNT embryos were produced using control and zinc-supplemented oocytes, respectively, and then were transferred to 11 and 8 recipients, respectively. Five control recipients and three zinc-supplemented recipients became pregnant...
August 2, 2016: Journal of Reproduction and Development
Yoshihisa Uenoyama, Vutha Pheng, Hiroko Tsukamura, Kei-Ichiro Maeda
Kisspeptin, encoded by KISS1/Kiss1 gene, is now considered a master regulator of reproductive functions in mammals owing to its involvement in the direct activation of gonadotropin-releasing hormone (GnRH) neurons after binding to its cognate receptor, GPR54. Ever since the discovery of kisspeptin, intensive studies on hypothalamic expression of KISS1/Kiss1 and on physiological roles of hypothalamic kisspeptin neurons have provided clues as to how the brain controls sexual maturation at the onset of puberty and subsequent reproductive performance in mammals...
July 29, 2016: Journal of Reproduction and Development
Xiaonan Qi, Huatao Li, Xia Cong, Xin Wang, Zhongling Jiang, Rongfeng Cao, Wenru Tian
Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis...
July 29, 2016: Journal of Reproduction and Development
Kohei Umezu, Yuuki Hiradate, Toshinori Oikawa, Hirotoshi Ishiguro, Takashi Numabe, Kenshiro Hara, Kentaro Tanemura
Recently, the conception rates after artificial insemination have been pointed out to decline continuously. To overcome this problem, the control of frozen and thawed sperm quality is required. However, the mechanism of bovine sperm functional regulation is still largely unknown. In mammals, the ejaculated sperm are capable of showing fertilizing ability during migration in the female reproductive organs. It is well known that these female organs secrete several factors contributing to sperm capacitation. We previously reported that neurotensin (NT) secreted from the oviduct and cumulus cells enhanced sperm capacitation and acrosome reaction in mice...
August 25, 2016: Journal of Reproduction and Development
Natsuko Emura, Nobuyuki Sakurai, Kazuki Takahashi, Tsutomu Hashizume, Ken Sawai
Oct-4, a member of the POU family of transcription factors, is a key factor that regulates the segregation of the inner cell mass (ICM) and the trophectoderm (TE) during the transition from morula to blastocyst in mice. However, little is known about its role in porcine early embryogenesis. To determine the function of OCT-4 in the ICM and TE segregation of porcine embryos, we studied the developmental morphology of porcine embryos using RNA interference technology. Our experiments demonstrated that when 1-cell stage embryos were co-injected with the small interfering RNA (siRNA)for targeted knockdown of OCT-4 (OCT-4-siRNA) and tetramethylrhodamine isothiocyanate (TRITC)-dextran conjugate (Dx), they failed to form blastocysts...
August 25, 2016: Journal of Reproduction and Development
Keisuke Edashige
The permeability of the plasma membrane to water and cryoprotectants is one of the most important factors for determining suitable conditions for vitrification of mammalian oocytes and embryos. In mouse oocytes and early stage embryos, water and cryoprotectants move slowly, principally by simple diffusion. In contrast, in morulae (and probably blastocysts), water, glycerol, and ethylene glycerol move rapidly, principally by facilitated diffusion via aquaporin 3, and DMSO moves rapidly via channels other than aquaporin 3...
August 25, 2016: Journal of Reproduction and Development
Jun-Ichi Sumitomo, Chihiro Emori, Yuta Matsuno, Mizuki Ueno, Kurenai Kawasaki, Takaho A Endo, Katsuyuki Shiroguchi, Wataru Fujii, Kunihiko Naito, Koji Sugiura
This study tested the hypothesis that oocyte-derived paracrine factors (ODPFs) regulate miRNA expression in mouse granulosa cells. Expression of mmu-miR-322-5p (miR-322) was higher in mural granulosa cells (MGCs) than in cumulus cells of the Graafian follicles. The expression levels of miR-322 decreased when cumulus cells or MGCs were co-cultured with oocytes denuded of their cumulus cells. Inhibition of SMAD2/3 signaling by SB431542 increased miR-322 expression by cumulus-oocyte complexes (COCs). Moreover, the cumulus cells but not the MGCs in Bmp15(-/-)/Gdf9(+/-) (double-mutant) mice exhibited higher miR-322 expression than those of wild-type mice...
August 25, 2016: Journal of Reproduction and Development
Koh-Ichi Hamano, Tae Kawanishi, Atsuko Mizuno, Makoto Suzuki, Yuji Takagi
Transient Receptor Potential Vanilloid (TRPV) 4 is one of the temperature-sensitive ion channels involved in temperature receptors, and it is known to be activated from 35 to 40ºC. Here we analyzed sperm motility function of Trpv4 knockout (KO) mouse in temperature-gradient conditions to elucidate the thermotaxis of mouse sperm and the involvement of TRPV4 in thermotaxis. The sperm were introduced at the vertical column end of a T-shaped chamber filled with medium in a plastic dish, and we measured the number of sperm that arrived at both ends of the wide column where we had established a temperature gradient of approx...
August 25, 2016: Journal of Reproduction and Development
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