著者

Shinya AYABE Kenichi NAKASHIMA Atsushi YOSHIKI

出版者

THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2018-128, (Released:2018-12-06)

被引用文献数

23

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based genome editing technology has enabled manipulation of the embryonic genome. Unbiased whole genome sequencing comparing parents to progeny has revealed that the rate of Cas9-induced mutagenesis in mouse embryos is indistinguishable from the background rate of de novo mutation. However, establishing the best practice to confirm on-target alleles of interest remains a challenge. We believe that improvement in editing strategies and screening methods for founder mice will contribute to the generation of quality-controlled animals, thereby ensuring reproducibility of results in animal studies and advancing the 3Rs (replacement, reduction, and refinement).

著者

Ayumi HASEGAWA Keiji MOCHIDA Narumi OGONUKI Michiko HIROSE Toshiko TOMISHIMA Kimiko INOUE Atsuo OGURA

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.63, no.6, pp.539-545, 2017 (Released:2017-12-15)

参考文献数

29

被引用文献数

7 14

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In embryo transfer experiments in mice, pseudopregnant females as recipients are prepared by sterile mating with vasectomized males. Because only females at the proestrus stage accept males, such females are selected from a stock of animals based on the appearance of their external genital tract. Therefore, the efficiency of preparing pseudopregnant females largely depends on the size of female colonies and the skill of the operators who select females for sterile mating. In this study, we examined whether the efficiency of preparing pseudopregnant females could be improved by applying an estrous cycle synchronization method by progesterone (P4) pretreatment, which significantly enhances the superovulation outcome in mice. We confirmed that after two daily injections of P4 (designated Days 1 and 2) in randomly selected females, the estrous cycles of most females (about 85%) were synchronized at metestrus on Day 3. When P4-treated females were paired with vasectomized males for 4 days (Days 4–8), a vaginal plug was found in 63% (20/32) of the females on Day 7. After the transfer of vitrified-warmed embryos into their oviducts, 52% (73/140) of the embryos successfully developed into offspring, the rate being comparable to that of the conventional embryo transfer procedure. Similarly, 77% (24/31) of females became pregnant by fertile mating with intact males for 3 days, which allowed the scheduled preparation of foster mothers. Thus, our estrous cycle synchronization method may omit the conventional experience-based process of visually observing the vagina to choose females for embryo transfer. Furthermore, it is expected that the size of female stocks for recipients can be reduced to less than 20%, which could be a great advantage for facilities/laboratories undertaking mouse-assisted reproductive technology.

著者

Islam M. SAADELDIN WooJae CHOI Bego ROIBAS DA TORRE BongHan KIM ByeongChun LEE Goo JANG

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.58, no.4, pp.425-431, 2012 (Released:2012-08-30)

参考文献数

36

被引用文献数

9 10

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The temporal progressive increase of interferon tau (IFNτ) secretion from the bovine trophoblast is a major embryonic signal of establishing pregnancy. Here, we cultured and isolated bovine trophoblast cells (BTs) from IVM/IVF oocytes and in vitro produced blastocysts, used them, for the first time, as donor cells for nuclear transfer and compared them with adult fibroblasts (AFs) as donor cells. BTs were reprogrammed in enucleated oocytes to blastocysts with similar efficiency to AFs (14.5% and 15.6% respectively, P≤0.05). The levels of IFNτ, CDX2 and OCT4 expression in IVF-, BT- and AF-derived blastocysts were analyzed using reverse transcription polymerase chain reaction and reverse transcription quantitative polymerase chain reaction (RT-PCR and RT-qPCR). IVF-produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid, expanded and hatching blastocysts. RT-PCR and RT-qPCR studies showed that IFNτ expression was higher in BT-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and BT-derived blastocysts showed a progressive increase in IFNτ expression as blastocyst development advanced when it compared with AF-derived blastocysts. OCT4 was inversely related with IFNτ expression, while CDX2 was found to be directly related with IFNτ temporal expression. Persistence of high expression of IFNτ and CDX2 was found to be higher in BT-derived embryos than in IVF- or AF-derived embryos. In conclusion, using BTs expressing IFNτ as donor cells for bovine NT could be a useful tool for understanding the IFNτ genetics and epigenetics.

著者

Yolanda SEGOVIA Noemí VICTORY Irene PEINADO Laura M GARCÍA-VALVERDE Magdalena GARCÍA Jon AIZPURUA Ana MONZÓ María José GÓMEZ-TORRES

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2017-009, (Released:2017-04-30)

被引用文献数

11

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The development of an effective program that combines in vitro maturation (IVM) and cryopreservation for immature oocytes would represent a novel advance for in vitro fertilization (IVF), especially as a means to preserve the fertility of women in unique situations. The aim of this study was to analyze the ultrastructural characteristics of human oocytes, obtained after controlled ovarian stimulation, to determine whether IVM is best performed before or after vitrification. To this end, we analyzed the following features in a total of 22 MII oocytes: size, zona pellucida and perivitelline space, mitochondria number, M-SER (mitochondria-smooth endoplasmic reticulum) aggregates and M-V (mitochondria-vesicle) complexes, the number of cortical granules and microvilli, and the presence of vacuolization using transmission electron microscopy (TEM). Each oocyte presented a rounded shape, with an intact oolemma, and was surrounded by a continuous zona pellucida and perivitelline space. Statistical analysis comparing oocytes vitrified before or after IVM indicated that there were no significant differences between examined characteristics.

著者

Dimas Arya ABDILLAH Onalenna KEREILWE Raihana Nasrin FERDOUSY Risa SAITO Hiroya KADOKAWA

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2021-126, (Released:2022-01-27)

被引用文献数

2

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Coronavirus disease (COVID-19), the ongoing global pandemic, is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Recent evidence shows that the virus utilizes angiotensin-converting enzyme 2 (ACE2) as a spike protein receptor for entry into target host cells. The bovine ACE2 contains key residues for binding to the spike protein receptor-binding domain. This study evaluated the hypothesis that bovine gonadotroph expresses ACE2, and spike protein suppresses luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from cultured bovine anterior pituitary (AP) cells. ACE2 mRNA expression and ACE2 protein expression were detected in the bovine AP cells using reverse transcription PCR and western blot analysis. Immunofluorescence microscopy analysis with the anti-ACE2 antibody revealed the co-localization of ACE2 and gonadotropin-releasing hormone (GnRH) receptor on the gonadotroph plasma membrane. Approximately 90% of GnRH receptor-positive cells expressed ACE2, and approximately 46% of ACE2-positive cells expressed the GnRH receptor. We cultured bovine AP cells for 3.5 days and treated them with increasing concentrations (0, 0.07, 0.7, or 7 pM) of recombinant spike protein having both S1 and S2 regions. The spike protein (0.07–7 pM) suppressed both basal and GnRH-induced LH secretion (P < 0.05). Spike protein (0.7–7 pM) suppressed GnRH-induced (P < 0.05), but not basal FSH secretion. In contrast, pre-treatment with ERK 1/2/5 inhibitor (U0126) partially restored the GnRH-induced LH and FSH secretion from the spike protein suppression. Collectively, the results indicate that gonadotrophs express ACE2, a receptor for coronavirus 2 spike protein, which in turn suppresses LH and FSH secretion from AP cells.

著者

Ewelina WARZYCH Paulina LIPINSKA

出版者

THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2019-102, (Released:2019-12-02)

被引用文献数

56

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Oocyte quality is affected by many factors, among which the environment of growth and maturation seems to be crucial. Studies show that well balanced oocyte energy metabolism has a significant impact on several elements of cytoplasmic and nuclear maturation as well as further embryo developmental competence. Therefore homeostasis between metabolism of glucose and fatty acids in the oocyte is being widely described nowadays. This review aims to discuss the follicular (in vivo) or maturation media (in vitro) environments with regard to glucose and fatty acid metabolism, as the main sources of the energy for the oocyte. A great emphasis is given on the balance between those two metabolic pathways and its further impact on female fertility.

著者

Ryuki SHIMADA Kei-ichiro ISHIGURO

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2023-010, (Released:2023-03-17)

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In mouse fetal gonads, germ cell development is accompanied by changes in cell cycle mode in response to external signals and intrinsic mechanisms of cells. During fetal development, male germ cells undergo G0/G1 arrest, while female germ cells exit the mitotic cell cycle and enter meiosis. In fetal testes, NANOS2 and CYP26B1 force germ cells to stay in G0/G1 arrest phase, preventing them from entering the meiotic cell cycle. In the fetal ovary, external signals, such as RA, BMP, and WNT, promote the competency of female germ cells to enter the meiotic cell cycle. MEIOSIN and STRA8 ensure the establishment of the meiotic cell cycle by activating meiotic genes, such that meiotic entry coincides with the S phase. This review discusses germ cell development from the viewpoint of cell cycle regulation and highlights the mechanism of the entry of germ cells into meiosis.

著者

Charley-Lea POLLARD Zamira GIBB Aleona SWEGEN Christopher G. GRUPEN

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2022-052, (Released:2022-09-27)

被引用文献数

6

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Oocyte quality is the limiting factor in female fertility. It is well known that maternal nutrition plays a role in reproductive function, and manipulating nutrition to improve fertility in livestock has been common practice in the past, particularly with respect to negative energy balance in cattle. A deficiency in nicotinamide adenine dinucleotide (NAD+) production has been associated with increased incidences of miscarriage and congenital defects in humans and mice, while elevating NAD+ through dietary supplements in aged subjects improved oocyte quality and embryo development. NAD+ is consumed by Sirtuins and poly-ADP-ribose polymerases (PARPs) within the cell and thus need constant replenishment in order to maintain various cellular functions. Sirtuins and PARPs play important roles in oocyte maturation and embryo development, and their activation may prove beneficial to in vitro embryo production and livestock breeding programs. This review examines the roles of NAD+, Sirtuins and PARPs in aspects of fertility, providing insights into the potential use of NAD+-elevating treatments in livestock breeding and embryo production programs.

著者

Charley-Lea POLLARD Zamira GIBB Azelle HAWDON Aleona SWEGEN Christopher G. GRUPEN

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.67, no.5, pp.319-326, 2021 (Released:2021-10-29)

参考文献数

54

被引用文献数

6

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In vitro maturation (IVM) is an important reproductive technology used to produce embryos in vitro. However, the developmental potential of oocytes sourced for IVM is markedly lower than those matured in vivo. Previously, NAD+-elevating treatments have improved oocyte quality and embryo development in cattle and mice, suggesting that NAD+ is important during oocyte maturation. The aim of this study was to examine the effects of nicotinic acid (NA), nicotinamide (NAM) and nicotinamide mononucleotide (NMN) on oocyte maturation and subsequent embryo development. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation medium supplemented with NA, NAM and resveratrol or NMN. Mature oocytes were artificially activated and presumptive zygotes cultured for 7 days. Additionally, oocytes were matured without treatment then cultured for 7 days with NMN. Supplementing the IVM medium with NA improved maturation and blastocyst formation while NAM supplementation improved cleavage rates compared with untreated controls. Supplementing the IVM or embryo culture media with NMN had no effect on maturation or embryo development. The results show that supplementing the maturation medium with NA and NAM improved maturation and developmental potential of porcine oocytes.

著者

Midori YOSHIZAWA Masaru TAKADA Satoshi NAKAMOTO Takashi MURAMATSU Akira OKAMOTO

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.39, no.2, pp.115-122, 1993 (Released:2008-05-15)

参考文献数

33

被引用文献数

10 14

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Superovulated eggs in (BALB/c×C57BL/6) F1 and ICR (outbred in a closed colony) female mice were fertilized in vitro with spermatozoa obtained from caudal epididymides of ICR males. Air-dried chromosome preparations were made from colcemid-primed 1-cell eggs and stained by the C-banding method. The fertilization rate was lower in F1 eggs than in ICR eggs (88 vs. 92%, P<0.02). The incidence of metaphase ("syngamy") eggs was higher in F1 eggs, and the incidence of pronuclear and late prometaphase ("pre-syngamy") eggs was higher in ICR eggs (P<0.001, P<0.005), showing delayed progress of the first cleavage in the ICR eggs. Developmental rates from 2-cell to 4-cell stage and from morula to blastocyst stage were significantly lower in ICR eggs (P<0.001) than in F1 eggs. These results show that a delay of embryo development had already appeared before male and female genomes fused (pre-syngamy). The incidence of triploidy, which might be caused by dispermy, was higher in F1 eggs, in both the pronuclear and mitotic stages (P<0.001). A little aneuploidy and structural aberration of chromosomes occurred in both F1 and ICR eggs, and no significant difference was observed between F1 and ICR eggs. The sex ratio at the first cleavage stage in both F1 and ICR eggs showed no significant deviation from equality.

著者

Ryoji KANEGI Shingo HATOYA Kazuto KIMURA Kyohei YODOE Toshiya NISHIMURA Kikuya SUGIURA Noritoshi KAWATE Toshio INABA

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2022-038, (Released:2023-10-26)

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Induced pluripotent stem (iPS) cells are generated from somatic cells and can differentiate into various cell types. Therefore, these cells are expected to be a powerful tool for modeling diseases and transplantation therapy. Generation of domestic cat iPS cells depending on leukemia inhibitory factor has been reported; however, this strategy may not be optimized. Considering that domestic cats are excellent models for studying spontaneous diseases, iPS cell generation is crucial. In this study, we aimed to derive iPS cells from cat embryonic fibroblasts retrovirally transfected with mouse Oct3/4, Klf4, Sox2, and c-Myc. After transfection, embryonic fibroblasts were reseeded onto inactivated SNL 76/7 and cultured in a medium supplemented with basic fibroblast growth factor. Flat, compact, primary colonies resembling human iPS colonies were observed. Additionally, primary colonies were more frequently observed in the KnockOut Serum Replacement medium than in the fetal bovine serum (FBS) medium. However, enhanced maintenance and proliferation of iPS-like cells occurred in the FBS medium. These iPS-like cells expressed embryonic stem cell markers, had normal karyotypes, proliferated beyond 45 passages, and differentiated into all three germ layers in vitro. Notably, expression of exogenous Oct3/4, Klf4, and Sox2 was silenced in these cells. However, the iPS-like cells failed to form teratomas. In conclusion, this is the first study to establish and characterize cat iPS-like cells, which can differentiate into different cell types depending on the basic fibroblast growth factor.

著者

Madalitso CHELENGA Yojiro YANAGAWA Seiji KATAGIRI Masashi NAGANO

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.69, no.4, pp.214-217, 2023 (Released:2023-08-11)

参考文献数

21

被引用文献数

1

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In this study, we evaluated the effects of pre-maturational culture (pre-IVM) on the developmental competence of bovine oocytes derived from an 8-day in vitro growth (IVG) culture system. IVG oocytes were subjected to 5 h pre-IVM before in vitro maturation, followed by in vitro fertilization (IVF). The proportion of oocytes that progressed to the germinal vesicle breakdown stage was similar between groups with and without pre-IVM. Although metaphase II oocytes and cleavage rates after IVF were similar regardless of pre-IVM culture, the blastocyst rate was significantly higher in the group with pre-IVM (22.5%) than without pre-IVM (11.0%, P < 0.05). In conclusion, pre-IVM culture improved the developmental competence of bovine oocytes derived from an 8-day IVG system.

著者

Junko NOGUCHI

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.63, no.1, pp.13-16, 2017 (Released:2017-02-16)

参考文献数

19

被引用文献数

4 6

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The impact of deer overabundance is a worldwide problem. Along with habitat expansion and population increase, damage by sika deer to the forest ecosystem and agriculture has become a serious issue in Japan. Deer also transmit a number of diseases and parasites to humans and livestock. The overabundance of deer is a result of their strong fecundity, and therefore the present situation should, in theory, be tackled by experts in reproductive biology.

著者

Charley-Lea POLLARD Ashleigh YOUNAN Aleona SWEGEN Zamira GIBB Christopher G. GRUPEN

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2021-130, (Released:2022-03-28)

被引用文献数

8

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Treatments that elevate NAD+ levels have been found to improve oocyte quality in mice, cattle, and pigs, suggesting that NAD+ is vital during oocyte maturation. This study aimed to examine the influence of different NAD+ biosynthetic pathways on oocyte quality by inhibiting key enzymes. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation system supplemented with 2-hydroxynicotinic acid (2-HNA, nicotinic acid phosphoribosyltransferase; NAPRT inhibitor), FK866 (nicotinamide phosphoribosyltransferase; NAMPT inhibitor), or gallotannin (nicotinamide mononucleotide adenylyltransferase; NMNAT inhibitor) and their respective NAD+ pathway modulators (nicotinic acid, nicotinamide, and nicotinamide mononucleotide, respectively). Cumulus expansion was assessed after 22 hr of maturation. At 44 h, maturation rates were determined and mature oocytes were fixed and stained to assess spindle formation. Each enzyme inhibitor reduced oocyte maturation rate and adversely affected spindle formation, indicating that NAD+ is required for meiotic spindle assembly. Furthermore, NAMPT and NMNAT inhibition reduced cumulus expansion, whereas NAPRT inhibition affected chromosomal segregation. Treating oocytes with NAD+ pathway inhibitors in combination with nicotinamide mononucleotide or nicotinic acid improved spindle parameters compared with the inhibitors alone. These results indicate that the salvage pathway plays a vital role in promoting oocyte meiotic progression, while the Preiss-Handler pathway is essential for spindle assembly.

著者

桑原 慶紀 吉田 幸洋 中村 清 伊藤 茂

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.41, no.5, pp.j55-j60, 1995 (Released:2010-10-20)

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著者らは独自に開発した装置を用いてヤギ胎仔の長時間子宮外保育を試みてきた.すなわち,帝王切開で娩出したヤギ胎仔の臍帯血管にカテーテルを挿入し,ECMO(Extracorporeal Membrane Oxygenation)回路に接続する.胎仔は人工羊水中に置かれ,胎児循環を保持し,低酸素環境下で保育される.これまでの著者らの成績では10日間の保育が限度で,慢性循環不全を主たる原因として死亡しており,保育状況・期間ともに不満足であった.最近著者らは胎仔の体内環境の変動をできるだけ少なくし,水分貯留傾向と関係すると思われる飲水行動を抑制する目的で子宮外保育期間中,胎仔にminor tranquilizerと筋弛緩剤を投与した.その結果,保育期間中,胎仔の状態は極めて安定しており,水分貯留傾向も認められず,約20日間というこれまでにない長期間保育に成功した.さらに,子宮外保育からの離脱を試みたところ,レスピレーターによる人工換気は必要としたものの良好な換気が得られ,保育期間中に肺の機能的成熟が進行したことも確認できた.以上により,臍帯動静脈A-VEC MOが未熟動物の長期にわたる保育法として有効であることが証明されたが,長期間の体動抑制による未熟動物の筋力の発達成熟におよぼす影響などの解決すべき問題があり,正常新生仔への移行にはさらなる改善が必要である.

著者

Yukina OSHIMO Arisa MUNETOMO Fumie MAGATA Yuta SUETOMI Shuhei SONODA Yukari TAKEUCHI Hiroko TSUKAMURA Satoshi OHKURA Fuko MATSUDA

出版者

The Society for Reproduction and Development

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.67, no.1, pp.15-23, 2021 (Released:2021-02-15)

参考文献数

56

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Kisspeptin neurons located in the hypothalamic preoptic area (POA) are suggested to be responsible for the induction of the gonadotropin-releasing hormone (GnRH) surge and the following luteinizing hormone (LH) surge to regulate female mammals’ ovulation. Accumulating evidence demonstrates that the preovulatory level of estrogen activates the POA kisspeptin neurons (estrogen positive feedback), which in turn induces a GnRH/LH surge. This study aimed to derive a cell line from goat POA kisspeptin neurons as an in vitro model to analyze the estrogen positive feedback mechanism in ruminants. Neuron-derived cell clones obtained by the immortalization of POA tissue from a female Shiba goat fetus were analyzed for the expression of kisspeptin (KISS1) and estrogen receptor α (ESR1) genes using quantitative real-time reverse transcription-polymerase chain reaction and three cell clones were selected as POA kisspeptin neuron cell line candidates. One cell line (GP64) out of the three clones showed significant increase in the KISS1 level by incubation with estradiol for 24 h, indicating that the GP64 cells mimic endogenous goat POA kisspeptin neurons. The GP64 cells showed immunoreactivities for kisspeptin and estrogen receptor α and retained a stable growth rate throughout three passages. Further, intracellular calcium levels in the GP64 cells were increased by the KCl challenge, indicating their neurosecretory ability. In conclusion, we generated a new KISS1-expressing cell line derived from goat POA. The current GP64 cell line could be a useful model to elucidate the estrogen positive feedback mechanism responsible for the GnRH/LH surge generation in ruminants.

著者

Kyoji YAMADA Toshihiko NAKAO Naoki ISOBE

出版者

THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.49, no.5, pp.381-388, 2003 (Released:2003-10-22)

参考文献数

37

被引用文献数

17 16

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The aim of this study was to examine whether the nutritional state of cows peripartum was associated with the recovery of ovarian function and conception rates after synchronization of ovulation and fixed-time artificial insemination (OVSYNCH/TAI). The effect of the interval in days from calving to the first ovulation on conception rates after OVSYNCH/TAI was also investigated. Conception rates of cows after OVSYNCH/TAI (n=39) were 43.6%. The conception rates of cows with a body condition score (BCS) of 2.75-3.25 at 30 d postpartum and on the day of OVSYNCH treatment were significantly higher than in cows with a BCS ≤2.5 (P<0.05). The percentage of cows establishing ovarian cyclicity before 55 d postpartum in cows with a BCS of 2.75-3.25 at 30 d postpartum and on the day of OVSYNCH treatment were significantly higher than in cows with a BCS ≤2.5 (P<0.05). The conception rates after OVSYNCH/TAI in cows which recovered ovarian cyclicity within 34 d postpartum were significantly higher than in cows with first ovulation ≥56 d (P<0.05). These results indicated that the nutritional state in cows peripartum influenced the conception rates after OVSYNCH/TAI and the postpartum ovarian cyclicity and also suggested that the conception rates after OVSYNCH/TAI decreased in cows with delayed recovery of ovarian cyclicity.

著者

Shiro YAMASHITA Yuhei KOGASAKA Yuuki HIRADATE Kentaro TANEMURA Yutaka SENDAI

出版者

THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2019-088, (Released:2019-11-24)

被引用文献数

9

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Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 μm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 μm) as compared to those from the control group (178.9 ± 9.0 μm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.

著者

Kento MIURA Shogo MATOBA Narumi OGONUKI Takafumi NAMIKI Junya ITO Naomi KASHIWAZAKI Atsuo OGURA

出版者

THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

pp.2018-053, (Released:2018-05-05)

被引用文献数

9

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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.

著者

Arata HONDA Atsuo OGURA

出版者

日本繁殖生物学会

雑誌

Journal of Reproduction and Development (ISSN:09168818)

巻号頁・発行日

vol.63, no.5, pp.435-438, 2017 (Released:2017-10-18)

参考文献数

29

被引用文献数

8

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Although the laboratory rabbit has long contributed to many paradigmatic studies in biology and medicine, it is often considered to be a “classical animal model” because in the last 30 years, the laboratory mouse has been more often used, thanks to the availability of embryonic stem cells that have allowed the generation of gene knockout (KO) animals. However, recent genome-editing strategies have changed this unrivaled condition; so far, more than 10 mammalian species have been added to the list of KO animals. Among them, the rabbit has distinct advantages for application of genome-editing systems, such as easy application of superovulation, consistency with fertile natural mating, well-optimized embryo manipulation techniques, and the short gestation period. The rabbit has now returned to the stage of advanced biomedical research.