Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
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  • Kenichiro SAKAGUCHI, Atsushi IDETA, Yojiro YANAGAWA, Masashi NAGANO, S ...
    Article ID: 2018-007
    [Advance publication] Released: June 16, 2018
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    Here, we describe a simplified procedure for embryo production in the Japanese black cow that uses a single caudal epidural injection of follicle-stimulating hormone (FSH). First, we compared the efficiency of superovulation for in vivo embryo production between conventional multiple FSH treatment (control, n = 10) and single epidural administration (epidural, n = 5). The number of transferable blastocysts was similar between control and epidural groups (4.7 ± 3.5 and 9.0 ± 6.0, respectively). Next, we compared in vitro embryo production by ovum pick-up and in vitro fertilization (OPU-IVF) between control (n = 12) and epidural groups (n = 12). The rate of development to transferable blastocysts was higher in the epidural group than in the control (23.3 vs. 10.5%, P < 0.001). In conclusion, a single epidural administration of FSH can induce follicular development comparable to that of the conventional superovulation protocol and may improve the productivity of OPU-IVF.

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  • Lauro GONZÁLEZ-FERNÁNDEZ, Beatriz MACÍAS-GARCÍA, Violeta CALLE-GUISADO ...
    Article ID: 2018-021
    [Advance publication] Released: June 10, 2018
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    We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at room temperature with 1 μM CZ, 200 μM W-7, or 1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP). Then, the sperm were 1) directly evaluated, 2) centrifuged and washed prior to evaluation, or 3) diluted with PBS prior to evaluation. Direct evaluation showed an increase in high M540 fluorescence in spermatozoa treated with the inhibitors (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] and 71.4 ± 4.2 [W-7], mean % ± SD, P < 0.001); washing decreased the percentage of sperm showing high M540 fluorescence for W-7 (4.8 ± 2.2, mean % ± SD) but not for CZ (69.4 ± 3.9, mean % ± SD, P < 0.001), and dilution showed an increase in high M540 fluorescence for both CZ and W-7; 8-Br-cAMP did not induce a rise in sperm showing high M540 fluorescence. Therefore, special care must be taken when M540 is used in spermatozoa with CaM inhibitors.

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  • Álvarez-Rodriguez MANUEL, Vicente-Carrillo ALEJANDRO, Rodriguez-Martin ...
    Article ID: 2017-141
    [Advance publication] Released: June 08, 2018
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    Hyaluronan (hyaluronic acid, HA) apparently improves sperm survival in vitro and in vivo (oviduct), maintaining sperm motility and inducing capacitation, but not acrosome exocytosis, either by direct action as a macromolecule or via CD44 membrane receptors. This study explored ejaculated, liquid-extended pig spermatozoa to ascertain (i) the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor, using a specific monoclonal commercial antibody; (ii) whether the CD44 receptor changed location when exposed to bicarbonate, a capacitating trigger, in vitro; and (iii) whether the addition of HA, of molecular size comparable to that produced in the oviduct sperm reservoir (0.0625 to 2.0 mg/ml; 0 HA: control), to semen extenders would improve sperm liquid storage in vitro or cryosurvival post-freezing. Variables tested were sperm velocity and progressive motility (Qualisperm™), sperm viability and acrosome status, membrane integrity and early destabilization, mitochondrial activation, and superoxide production (flow cytometry). The CD44 receptor presence in ejaculated, liquid-stored AI boar spermatozoa, as confirmed by a porcine-specific monoclonal antibody, maintained its membrane location under in vitro capacitation-inducing conditions. HA exposure to 24-, 48-, or 72-h liquid-stored (17–20ºC) spermatozoa lowered sperm velocity in membrane-intact spermatozoa, but increased mitochondrial superoxide production. Finally, HA addition during cooling did not improve cryosurvival but did increase mitochondrial activation and membrane destabilization in surviving cells. These results confirm the existence of a CD44 receptor in pig spermatozoa, but the usefulness of adding HA for long-term storage or cryopreservation of liquid-stored, extended boar semen remains in question, thereby warranting further non-empirical analyses of HA-sperm membrane interactions.

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  • Jianning YU, Leyan YAN, Zhe CHEN, Hui LI, Huanxi ZHU, Rong CHEN, ZhenD ...
    Article ID: 2018-001
    [Advance publication] Released: June 08, 2018
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    Treatment of fetal rat and embryonic chicken with exogenous glucocorticoids induces premature differentiation of growth hormone (GH) secreting cells. The effect of corticosterone (CORT) on somatotroph differentiation was mostly studied in pituitary cells in vitro. Currently, there is no evidence for glucocorticoid-mediated induction of somatotroph differentiation during pituitary development in bird species other than chicken. In this study, we sought to find out if in ovo injection of corticosterone into developing goose embryos could induce premature increase of GH in somatotrophs. On embryonic day (e) 15, the albumen of fertile goose eggs was injected with 300 μl of 0.9% saline, 300 μl 5 × 10–8M CORT, or 300 μl 5 × 10–6 M CORT. Embryos were allowed to develop until e20 and e28 and isolated pituitaries were subjected to quantitative real-time PCR and immunocytochemistry to detect GH mRNA and protein, respectively. At e20 and e28, blood from chorioallantoic vessels was subjected to radioimmunoassay for analysis of plasma GH protein. In ovo administration of exogenous corticosterone brought about a 2.5-fold increase in the expression of GH mRNA and increased the in situ expression of GH protein in goose pituitary cells, and enhanced plasma GH levels compared to that of the respective controls at e20. These findings prove that administration of glucocorticoid could stimulate the expression of GH in somatotrophs during goose embryonic development. Our results suggest the probable involvement of membrane glucocorticoid receptor in the corticosterone mediated expression of GH. Together with previously published data, our results suggests that corticosterone mediated induction of GH expression during embryonic development is relatively conserved among different vertebrates.

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  • Maiko KOBAYASHI, Takamasa KOYAMA, Yasuhiro YASUTOMI, Tadashi SANKAI
    Article ID: 2017-164
    [Advance publication] Released: May 31, 2018
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    We attempted to elucidate female reproduction in long-tailed macaques (Macaca fascicularis). These monkeys have a non-seasonal menstruation cycle, which makes them suitable subjects for studies in a variety fields including medical science and regenerative medicine. We analyzed individual breeding data including time of menarche, start of regular menstruation, and first pregnancy. These three events are related to the maturation of female long-tailed macaques. All research subjects were female long-tailed macaques bred at the Tsukuba Primate Research Center. The study comprised 45 females; we included time of menstruation, male-female cohabitation, and first pregnancy in their growth records. We extracted age and weight data relating to menarche, start of regular menstruation, and first pregnancy from these records. In the two years typically required from menarche to first pregnancy, the body weight increased by approximately 500 g (21% of the weight at menarche); it is clear that there is a significant physical change after menarche. Our findings suggest that female monkeys are not necessarily mature enough for pregnancy at menarche. Therefore, the use of the word “maturity” in terms of fecundity may be more accurate after the start of regular menstruation. This is what we term “adolescence” in the developmental process. Therefore, M. fascicularis monkeys are candidates for an animal model of human adolescence.

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  • Masaki TAKASU, Masami MAEDA, Julio ALMUNIA, Kotono NAKAMURA, Noriko NI ...
    Article ID: 2017-107
    [Advance publication] Released: May 27, 2018
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    In microminipigs, estrus induction with abortion treatment, which is typically performed between 25 and 40 days after mating, is not always successful. Thus, the authors hypothesized that it may be more difficult to induce estrus by treating microminipigs approximately 40 days after mating. Accordingly, in this study, estrus induction was performed with abortion treatment in four microminipigs as follows: 0.3 mg of cloprostenol, a prostaglandin F2-alpha analog, was administered (day 0); after 24 h, 0.15 mg of cloprostenol and 250 IU of equine chorionic gonadotrophin were administered intramuscularly and simultaneously (day 1); after 96 h, 120 IU of human chorionic gonadotropin was injected intramuscularly (day 4). These treatments were compared at two different stages of pregnancy: early treatment (26.5 ± 0.7 days) and late treatment (38.3 ± 0.8 days). In the early treatment, all four microminipigs exhibited estrus on day 5, whereas in the late treatment, estrus was observed clearly in only two pigs on day 6 and slightly in 1 pig on day 10, whereas it was unclear in 1 pig. These results suggest that it is difficult to induce estrus with abortion treatment in microminipigs at approximately 40 days after mating.

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  • Takashi FUJII, Hiroki HIRAYAMA, Shigeo FUKUDA, Soichi KAGEYAMA, Akira ...
    Article ID: 2017-166
    [Advance publication] Released: May 24, 2018
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    Artificial insemination with cryopreserved semen is a well-developed technique commonly used for controlled reproduction in cattle. However, despite current technical advances, cryopreservation continues to damage bull spermatozoa, resulting in a loss of approximately 30 to 50% of viable spermatozoa post thawing. To further improve the efficiency of cryopreservation of bull spermatozoa, understanding the molecular mechanisms underlying the cryobiological properties that affect cryoinjuries during cryopreservation process of bull spermatozoa is required. In this study, we examined the expression and localization of aquaporin (AQP) 3 and AQP7 in fresh, cooled, and frozen–thawed bull spermatozoa. Furthermore, we investigated the relevance of AQP3 and AQP7 to motility and to membrane integrity in frozen–thawed bull spermatozoa. Western blotting against AQP3 and AQP7 in bull spermatozoa revealed bands with molecular weights of approximately 42 kDa and 53 kDa, respectively. In immunocytochemistry analyses, immunostaining of AQP3 was clearly observed in the principal piece of the sperm tail. Two immunostaining patterns were observed for AQP7 ―pattern 1: diffuse staining in head and entire tail, and pattern 2: diffuse staining in head and clear staining in mid-piece. Cooling and freeze–thawing did not affect the localization pattern of AQP7 and the relative abundances of AQP3 and AQP7 evaluated by Western blotting. Furthermore, we demonstrated that the relative abundances of AQP3 and AQP7 varied among ejaculates, and they were positively related to sperm motility, particularly sperm velocity, post freeze-thawing. Our findings suggest that AQP3 and AQP7 are possibly involved in the tolerance to freeze-thawing in bull spermatozoa, particularly in the sperm’s tail.

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  • Mohammed. A. ABDEL-GHANI, Kenichiro SAKAGUCHI, Chihiro KANNO, Yojiro Y ...
    Article ID: 2018-004
    [Advance publication] Released: May 24, 2018
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    We investigated the effects of pre-maturational (pre-IVM) culture on the developmental competence of small-sized bovine oocytes (110 and < 115 µm). Oocytes were cultured with 3-isobutyl-1-methylxanthine (IBMX) for 0, 5, or 10 h and subjected to in vitro maturation, fertilization, and culture. The cleavage rate (73%) of small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (61 and 62%, respectively). The blastocyst rate (16%) of embryos derived from small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (9 and 8%, respectively). In addition, small-sized oocytes with 5 h pre-IVM had a higher mean cell number in blastocysts (134.1 ± 34.8) than those with 0 and 10 h pre-IVM (100.2 ± 17.2 and 107.8 ± 23.7, respectively). In conclusion, the pre-IVM of small-sized oocytes with IBMX for 5 h improved the developmental competence of bovine oocytes, as well as the quality of blastocysts.

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  • Saffet OZTURK, Fatma UYSAL
    Article ID: 2018-026
    [Advance publication] Released: May 18, 2018
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    Spermatogenesis is briefly defined as the production of mature spermatozoa from spermatogonial stem cells at the end of a strictly regulated process. It is well known that, to a large extent, transcriptional activity ceases at mid-spermiogenesis. Several mRNAs transcribed during early stages of spermatogenesis are stored as ribonucleoproteins (RNPs). During the later stages, translational control of these mRNAs is mainly carried out in a time dependent-manner by poly(A)-binding proteins (PABPs) in cooperation with other RNA-binding proteins and translation-related factors. Conserved PABPs specifically bind to poly(A) tails at the 3′ ends of mRNAs to regulate their translational activity in spermatogenic cells. Studies in this field have revealed that PABPs, particularly poly(A)-binding protein cytoplasmic 1 (Pabpc1), Pabpc2, and the embryonic poly(A)-binding protein (Epab), play roles in the translational regulation of mRNAs required at later stages of spermatogenesis. In this review article, we evaluated the spatial and temporal expression patterns and potential functions of these PABPs in spermatogenic cells during spermatogenesis. The probable relationship between alterations in PABP expression and the development of male infertility is also reviewed.

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  • Toshinori OIKAWA, Tomoko ITAHASHI, Risa YAJIMA, Takashi NUMABE
    Article ID: 2018-023
    [Advance publication] Released: May 07, 2018
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    Intracytoplasmic sperm injection (ICSI) was expected to enable more efficient use of sperm from sires with preferable genetic traits and result in a generation containing a larger number of offspring with superior genetic characteristics in livestock. However, the efficiency of the early development of embryos produced by ICSI is still far from satisfactory in cattle. The present study aimed to investigate the effects of the treatment of cryopreserved sperm with glutathione (GSH) on the early development of embryos produced by ICSI in Japanese Black cattle. Moreover, the disulfide bond state and mitochondrial function were investigated in the sperm treated with GSH to confirm the effectiveness of the abovementioned treatment. We also investigated the effect of 7% ethanol activation treatment on the developmental ability of ICSI embryos using GSH-treated sperm. There was no effect on the blastocyst rate from the activation treatment. When sperm-injected oocytes were cultured in vitro, the treatment with GSH significantly improved the early development of embryos. Specifically, the rates of embryos reaching the 4–8-cell stage and blastocyst stage were significantly higher in ICSI with GSH-treated sperm (71.4% and 31.0%, respectively) than that with the control sperm (36.6% and 7.0%, respectively). Moreover, the GSH-treated sperm treatment significantly decreased the number of disulfide bonds in the sperm head (as shown by monobromobimane staining) and enhanced the mitochondrial function in the sperm middle piece (as shown by Rhodamine 123 staining and the adenosine triphosphate-dependent bioluminescence assay). Based on these results, we suggest that the treatment of cryopreserved sperm with GSH might contribute to the improvement of ICSI techniques for the production of blastocysts in Japanese Black cattle.

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  • Kento MIURA, Shogo MATOBA, Narumi OGONUKI, Takafumi NAMIKI, Junya ITO, ...
    Article ID: 2018-053
    [Advance publication] Released: May 05, 2018
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    In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/μl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ–AID–EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ–AID–EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ–AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.

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  • Shinnosuke HONDA, Yuka MIKI, Yuya MIYAMOTO, Yu KAWAHARA, Satoshi TSUKA ...
    Article ID: 2018-024
    [Advance publication] Released: May 03, 2018
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    Oog1, an oocyte-specific gene that encodes a protein of 425 amino acids, is present in five copies on mouse chromosomes 4 and 12. In mouse oocytes, Oog1 mRNA expression begins at embryonic day 15.5 and almost disappears by the late two-cell stage. Meanwhile, OOG1 protein is detectable in oocytes in ovarian cysts and disappears by the four-cell stage; the protein is transported to the nucleus in late one-cell to early two-cell stage embryos. In this study, we examined the role of Oog1 during oogenesis in mice. Oog1 RNAi-transgenic mice were generated by expressing double-stranded hairpin Oog1 RNA, which is processed into siRNAs targeting Oog1 mRNA. Quantitative RT-PCR revealed that the amount of Oog1 mRNA was dramatically reduced in oocytes obtained from Oog1-knockdown mice, whereas the abundance of spermatogenesis-associated transcripts (Klhl10, Tekt2, Tdrd6, and Tnp2) was increased in Oog1 knockdown ovaries. Tdrd6 is involved in the formation of the chromatoid body, Tnp2 contributes to the formation of sperm heads, Tekt2 is required for the formation of ciliary and flagellar microtubules, and Klhl10 plays a key role in the elongated sperm differentiation. These results indicate that Oog1 down-regulates the expression of spermatogenesis-associated genes in female germ cells, allowing them to develop normally into oocytes.

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  • Shunsuke SAKAI, Natsumi HAGIHARA, Mariko KUSE, Koji KIMURA, Kiyoshi OK ...
    Article ID: 2018-051
    [Advance publication] Released: April 30, 2018
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    Heat stress (HS) negatively affects reproduction in cattle; however, its effect on endocrine function in bovine endometrial cells remains unclear. In this study, we examined the effects of HS on the production of prostaglandin (PG)E2 and PGF2α in the cultured bovine endometrial epithelial and stromal cells separately. To evaluate the effect of HS on endocrine function, the cells were cultured at 38.5°C (control) or 40.5°C (HS). After treatment, PGE2 and PGF2α levels were measured via enzyme immunoassay (EIA) and mRNA expressions of enzymes involved in PG synthesis were examined via quantitative reverse transcription polymerase chain reaction (RT-PCR). HS did not influence the production of PGE2 or PGF2α in the epithelial cells; however, HS significantly enhanced the production of both PGE2 and PGF2α in the stromal cells (P < 0.05). In addition, HS significantly increased phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), and carbonyl reductase 1 (CBR1) mRNA expression in the stromal cells (P < 0.05). The overall results suggest that HS induces mRNA expression of enzymes involved in PG synthesis, resulting in the upregulation of PGE2 and PGF2α production in the stromal cells, but not in the epithelial cells. The HS-induced increase of PGE2 and PGF2α secretion in bovine endometrial stromal cells may disrupt the normal estrous cycle and cause infertility in cows during summer.

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  • Min Jung KIM, Hyun Ju OH, Yoo Bin CHOI, Sanghoon LEE, Erif Maha Nugrah ...
    Article ID: 2017-112
    [Advance publication] Released: April 26, 2018
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    This study was conducted to investigate whether the treatment of dog to pig interspecies somatic cell nuclear transfer (iSCNT) embryos with a histone deacetylase inhibitor, to improve nuclear reprogramming, can be applied to dog SCNT embryos. The dog to pig iSCNT embryos were cultured in fresh porcine zygote medium-5 (PZM-5) with 0, 1, or 10 µM suberoylanilide hydroxamic acid (SAHA) for 6 h, then transferred to PZM-5 without SAHA. Although there were no significant differences in cleavage rates, the rates of 5-8-cell stage embryo development were significantly higher in the 10 µM group (19.5 ± 0.8%) compared to the 0 µM groups (13.4 ± 0.8%). Acetylation of H3K9 was also significantly higher in embryos beyond the 4-cell stage in the 10 µM group compared to the 0 or 1 µM groups. Treatment with 10 µM SAHA for 6 h was chosen for application to dog SCNT. Dog cloned embryos with 0 or 10 µM SAHA were transferred to recipients. However, there were no significant differences in pregnancy and delivery rates between the two groups. Therefore, it can be concluded that although porcine oocytes support nuclear reprogramming of dog fibroblasts, treatment with a histone deacetylase inhibitor that supports nuclear reprogramming in dog to pig iSCNT embryos is not sufficient for reprogramming in dog SCNT embryos.

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  • Satoshi TSUKAMOTO, Takayuki TATSUMI
    Article ID: 2018-039
    [Advance publication] Released: April 26, 2018
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    During oogenesis, oocytes accumulate a large set of proteins derived from the maternal genome. These proteins, known as maternal proteins, are not only required for oocyte maturation and fertilization, but also implicated in subsequent embryonic development. However, most maternal proteins are degraded and their amino acid components are utilized for newly synthesized proteins from the embryonic genome. This process is known as the oocyte-to-embryo transition; because it occurs over a short period, mechanisms involving massive degradation of maternal proteins have been proposed. Intracellular protein degradation mechanisms can be broadly classified into two types. The first is the ubiquitin–proteasome system, a highly selective pathway in which ubiquitylated proteins are degraded by proteasomes. The second mechanism is autophagy, which involves lysosome-mediated degradation of cytoplasmic components. In this review, we describe recent advances in the understanding of autophagy, focusing on its role in early embryonic development.

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  • Mizuki SAKAI, Kaito MASAKI, Shota AIBA, Masaaki TONE, Seiji TAKASHIMA
    Article ID: 2018-015
    [Advance publication] Released: April 16, 2018
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    Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells (SSCs). Although GDNF is indispensable for the maintenance of SSCs, the role of FGF2 in the testis remains to be elucidated. To clarify this, the expression dynamics and regulatory mechanisms of Fgf2 and Gdnf in the mouse testes were analyzed. It is well known that Sertoli cells express Gdnf, and its receptor is expressed in a subset of undifferentiated spermatogonia, including SSCs. However, we found that Fgf2 was mainly expressed in the germ cells and its receptors were expressed not only in the cultured spermatogonial cell line, but also in testicular somatic cells. Aging, hypophysectomy, retinoic acid treatment, and testicular injury induced distinct Fgf2 and Gdnf expression dynamics, suggesting a difference in the expression mechanism of Fgf2 and Gdnf in the testis. Such differences might cause a dynamic fluctuation of Gdnf/Fgf2 ratio depending on the intrinsic/extrinsic cues. Considering that FGF2-cultured spermatogonia exhibit more differentiated phenotype than those cultured with GDNF, FGF2 might play a role distinct from that of GDNF in the testis, despite the fact that both factors are self-renewal factor for SSC in vitro.

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  • Kenya IMAIMATSU, Wataru FUJII, Ryuji HIRAMATSU, Kento MIURA, Masamichi ...
    Article ID: 2017-161
    [Advance publication] Released: April 14, 2018
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    Mammalian zygote-mediated genome editing via the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system is widely used to generate genome-modified animals. This system allows for the production of loss-of-function mutations in various Y chromosome genes, including Sry, in mice. Here, we report the establishment of a CRISPR-Cas9-mediated knock-in line of Flag-tag sequences into the Sry locus at the C-terminal coding end of the Y chromosome (YSry-flag). In the F1 and successive generations, all male pups carrying the YSry-flag chromosome had normal testis differentiation and proper spermatogenesis at maturity, enabling complete fertility and the production of viable offspring. To our knowledge, this study is the first to produce a stable Sry knock-in line at the C-terminal region, highlighting a novel approach for examining the significance of amino acid changes at the naive Sry locus in mammals.

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  • Takehito KANEKO
    Article ID: 2018-035
    [Advance publication] Released: April 14, 2018
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    Many types of mutant and genetically engineered strains have been produced in various animal species. Their numbers have dramatically increased in recent years, with new strains being rapidly produced using genome editing techniques. In the rat, it has been difficult to produce knockout and knock-in strains because the establishment of stem cells has been insufficient. However, a large number of knockout and knock-in strains can currently be produced using genome editing techniques, including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Microinjection technique has also contributed widely to the production of various kinds of genome edited animal strains. A novel electroporation method, the “Technique for Animal Knockout system by Electroporation (TAKE)” method, is a simple and highly efficient tool that has accelerated the production of new strains. Gamete preservation is extremely useful for maintaining large numbers of these valuable strains as genetic resources in the long term. These reproductive technologies, including microinjection, TAKE method, and gamete preservation, strongly support biomedical research and the bio-resource banking of animal models. In this review, we introduce the latest reproductive technologies used for the production of genetically engineered animals, especially rats, using genome editing techniques and the efficient maintenance of valuable strains as genetic resources. These technologies can also be applied to other laboratory animals, including mice, and domestic and wild animal species.

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  • Kazuki KANSAKU, Yasuhisa MUNAKATA, Nobuhiko ITAMI, Koumei SHIRASUNA, T ...
    Article ID: 2018-012
    [Advance publication] Released: April 03, 2018
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    This study examined the concentration of cell-free mitochondrial DNA (cf-mtDNA) in porcine follicular fluid (FF) and explored whether the cfDNA level in the culture medium could reflect mitochondrial dysfunction in cumulus cell-oocyte complexes (COCs). cfDNA concentration was higher in the fluid of small-sized follicles, compared to that in larger follicles. The length of cfDNA ranged from short (152 bp) to long (1,914 bp) mtDNA in FF, detected by polymerase chain reaction (PCR). cfDNA concentration in FF significantly correlated with the mtDNA copy number in FF but not with the number of one-copy gene (nuclear DNA) in FF. When the COCs were treated with the mitochondrial uncoupler, namely carbonyl cyanide m-chlorophenyl hydrazone (CCCP), for 2 h and incubated for 42 h, subsequent real-time PCR detected significantly higher amount of cf-mtDNA, compared to nuclear cfDNA, in the spent culture medium. The mtDNA number and viability of cumulus cells and oocytes remained unchanged. When the oocytes were denuded from the cumulus cells following CCCP treatment, PCR detected very low levels of cfDNA in the spent culture medium of the denuded oocytes. In contrast, CCCP treatment of granulosa cells significantly increased the amount of cf-mtDNA in the spent culture medium, without any effect on other markers, including survival rate, apoptosis of cumulus cells, and lactate dehydrogenase levels. Thus, cf-mtDNA was present in FF in a wide range of length, and mitochondrial dysfunction in COCs increased the active secretion of cf-mtDNA in the cultural milieu.

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  • Yoshie A. FUJIOKA, Asuka ONUMA, Wataru FUJII, Koji SUGIURA, Kunihiko N ...
    Article ID: 2018-006
    [Advance publication] Released: March 23, 2018
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    Vertebrate oocytes arrested at the first meiotic prophase must proceed to the second meiotic metaphase (MII) before fertilization. This meiotic process requires the precise control of protein degradation. Part of the protein degradation in oocytes is controlled by members of the ubiquitin-conjugating enzyme family, UBE2C and UBE2S, which are known to participate in mono-ubiquitination and poly-ubiquitination, respectively. Although UBE2 enzymes have been well studied in mitosis, their contribution to mammalian oocyte meiosis is relatively unknown and has been studied only in mice. Here, we investigated the contribution of UBE2C and UBE2S to porcine oocyte maturation using an RNA injection method. Overexpression of UBE2S prevented MII arrest of oocytes and led to the formation of a pronucleus (PN) at 48 h of culture. This effect was also observed for prolonged cultures of UBE2C-overexpressing oocytes, suggesting the effectiveness of poly-ubiquitination in the rapid escape from M-phase in porcine oocytes. Although the inhibition of either UBE2C or UBE2S by antisense RNA (asRNA) injection had no effect on oocyte maturation, asRNA-injected oocytes showed inhibited PN formation after parthenogenetic activation. These results indicated that ubiquitination of certain factors by UBE2S and UBE2C plays a role in the escape from MII arrest in porcine oocytes. Further investigations to identify the factors and how mono- and/or poly-ubiquitination contributes to protein degradation could provide a better understanding of UBE2 roles in oocyte maturation.

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  • Rebecca R. PAYTON, Louisa A. RISPOLI, Kimberly A. NAGLE, Cedric GONDRO ...
    Article ID: 2017-160
    [Advance publication] Released: March 18, 2018
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    Hyperthermia during estrus has direct consequences on the maturing oocyte that carries over to the resultant embryo to compromise its ability to continue in development. Because early embryonic development is reliant upon maternal transcripts and other ooplasmic components, we examined impact of heat stress on bovine oocyte transcripts using microarray. Oocytes were matured at 38.5ºC for 24 h or 41.0ºC for the first 12 h of in vitro maturation; 38.5ºC thereafter. Transcriptome profile was performed on total (adenylated + deadenylated) RNA and polyadenylated mRNA populations. Heat stress exposure altered the abundance of several transcripts important for mitochondrial function. The extent to which transcript differences are coincident with functional changes was evaluated by examining reactive oxygen species, ATP content, and glutathione levels. Mitochondrial reactive oxygen species levels were increased by 6 h exposure to 41.0ºC while cytoplasmic levels were reduced compared to controls (P < 0.0001). Exposure to 41.0ºC for 12 h increased total and reduced glutathione levels in oocytes at 12 h but reduced them by 24 h (time × temperature P < 0.001). ATP content was higher in heat-stressed oocytes at 24 h (P < 0.0001). Heat-induced increases in ATP content of matured oocytes persisted in early cleavage-stage embryos (8- to 16-cell embryos; P < 0.05) but were no longer apparent in blastocysts (P > 0.05). Collectively, results indicate that direct exposure of maturing oocytes to heat stress may alter oocyte mitochondrial processes/function, which is inherited by the early embryo after fertilization.

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  • Dong-Mei SU, Ying FENG, Lin WANG, Yi-Lun WU, Ren-shan GE, Xue MA
    Article ID: 2017-102
    [Advance publication] Released: March 06, 2018
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

    Leydig cells are the main endogenous testosterone synthesis cells in the body. Testosterone is an essential hormone in males that affects metabolism, emotion, and pubertal development. However, little is known about the development of Leydig cells and relationship between fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). The aims of this study were to investigate the effect of (FLCs) on ALC development. Our study showed that FLCs in neonatal rat testis can be eliminated by 100 mg/kg ethane dimethane sulfonate (EDS) treatment without affecting the health of newborn rats. Immunohistological results showed that eliminating FLCs led to early re-generation of the ALC population (progenitor Leydig cells [PLCs] and ALCs) accompanied at first by increased and then by decreased serum testosterone, indicating that ALCs which appeared after neonatal EDS treatment were degenerated or had attenuated functions. Our results showed that FLCs were eliminated 4 days after EDS treatment, the ALC population regenerated by 21 days, and serum testosterone levels dramatically decreased at 56 days. Collectively, our results indicate that the ablation of FLCs in neonatal rat results in abnormal development of ALCs. Our study further indicates that abnormal development of Leydig cells in the fetal stage leads to steroid hormone disorders, such as testosterone deficiency, in the adult stage. Therefore, studies of Leydig cell development are important for understanding the pathogenesis of testosterone deficiency or pubertas praecox.

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  • Hiroya KADOKAWA, Kiran PANDEY, Kereilwe ONALENNA, Asrafun NAHAR
    Article ID: 2017-153
    [Advance publication] Released: March 06, 2018
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

    Estrone (E1) and estriol (E3) are considered “weak” estrogens, which exert suppressive effects through estrogen receptors α and β. However, recent studies have demonstrated that E1 and E3, as well as estradiol (E2), suppress gonadotropin-releasing hormone-induced luteinizing hormone secretion from bovine gonadotrophs via G-protein-coupled receptor 30, which is expressed in various reproductive organs. Currently, there is a lack of fundamental knowledge regarding E1 and E3, including their blood levels. In addition, xenoestrogens may remain in the body over long time periods because of enterohepatic circulation. Therefore, it is time to reconsider the roles of endogenous estrogens and xenoestrogens for reproduction.

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  • Satoshi SUGIMURA, Tadayuki YAMANOUCHI, Maria Grazia PALMERINI, Yutaka ...
    Article ID: 2018-009
    [Advance publication] Released: March 02, 2018
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

    The administration of follicle-stimulating hormone (FSH) prior to oocyte retrieval improves oocyte developmental competence. During bovine embryo production in vitro, however, oocytes are typically derived from FSH-unprimed animals. In the current study, we examined the effect of pre-in vitro maturation (IVM) with cAMP modulators, also known as the second messengers of FSH, on the developmental competence of oocytes derived from small antral follicles (2–4 mm) of FSH-unprimed animals. Pre-IVM with N6,2ʹ-O-dibutyryladenosine 3′,5′-cyclicmonophosphate (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) for 2 h improved the blastocyst formation in oocytes stimulated by FSH or amphiregulin (AREG). Furthermore, pre-IVM enhanced the expression of the FSH- or AREG-stimulated extracellular matrix-related genes HAS2, TNFAIP6, and PTGS2, and epidermal growth factor (EGF)-like peptide-related genes AREG and EREG. Additionally, pre-IVM with dbcAMP and IBMX enhanced the expression of EGFR, and also increased and prolonged cumulus cell-oocyte gap junctional communication. The improved oocyte development observed using the pre-IVM protocol was ablated by an EGF receptor phosphorylation inhibitor. These results indicate that pre-IVM with cAMP modulators could contribute to the acquisition of developmental competence by bovine oocytes from small antral follicles through the modulation of EGF receptor signaling and oocyte-cumulus/cumulus-cumulus gap junctional communication.

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  • Charlie HUVENEERS, Nicholas M. OTWAY, Megan T. STORRIE, Robert G. HARC ...
    Article ID: 20144
    [Advance publication] Released: February 23, 2009
    JOURNALS FREE ACCESS ADVANCE PUBLICATION
    This article was retracted. See the Notification.
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