Co-transfer of parthenogenotes and single porcine embryos leads to full-term development of the embryos

Kawarasaki, Tatsuo; Otake, Masayoshi; Tsuchiya, Seiko; Shibata, Masatoshi; Matsumoto, K.; Isobe, Naoki

doi:10.1016/j.anireprosci.2008.03.022

Cited by 27 publications

(29 citation statements)

References 33 publications

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“…Our previous study [31] suggested that porcine fertilized oocytes produced by ICSI have lower viability than conventionally in vitro -fertilized oocytes [30]. Co-transfer of parthenogenotes with a single embryo was proven to be sufficient for maintenance of pregnancy and development of the embryo [42]. Through co-transfer, we were able to obtain live piglets from 4 recipient gilts in each immersion-time group; these results clearly indicate that our vitrification protocols with 10 or 20 min of exposure to cryoprotectant are effective for cryopreservation of testicular tissues, and allow the full developmental competence of germ cells to be maintained.…”

Section: Discussionmentioning

confidence: 99%

Generation of Live Piglets for the First Time Using Sperm Retrieved from Immature Testicular Tissue Cryopreserved and Grafted into Nude Mice

et al. 2013

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Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230−350 (day 0 = grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230−350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.

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Section: Discussionmentioning

confidence: 99%

Generation of Live Piglets for the First Time Using Sperm Retrieved from Immature Testicular Tissue Cryopreserved and Grafted into Nude Mice

et al. 2013

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“…However, the production efficiency of normal offspring remains low. Particularly, in pigs, a large number of embryos with high quality is needed to produce cloned offspring, because pigs have a unique reproductive feature necessitating several embryos are needed to establish and maintain pregnancy [27,28]. On the other and DNA (red).…”

Section: Discussionmentioning

confidence: 99%

Acetylation Level of Histone H3 in Early Embryonic Stages Affects Subsequent Development of Miniature Pig Somatic Cell Nuclear Transfer Embryos

Yamanaka

Sugimura

Wakai³

et al. 2009

J. Reprod. Dev.

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Abstract.Successful cloning by somatic cell nuclear transfer (SCNT) requires a reprogramming process in which the epigenetic state of a differentiated donor nucleus must be converted into an embryonic totipotent state. However, this epigenetic reprogramming is incomplete in SCNT embryos, causing low production efficiency. Recently, it has been reported that trichostatin A (TSA), an inhibitor of histone deacetylase, potentially enhances cloning efficiency. The aim of the present study was to optimize the TSA treatment for miniature pig SCNT embryos and investigate the effect of the acetylation level of histone on developmental competence of SCNT embryos. In order to optimize the TSA treatment, we examined the developmental competence of SCNT embryos under various exposure times (0-50 h) and concentrations (0-500 nM). Treatment with 5 nM TSA for 15 and 20 h beginning at the start of activation significantly increased the blastocyst formation rate (34.6 and 32.4 vs. 18.2%, respectively) and mean cell number (57.0 ± 2.7 and 56.6 ± 2.7 vs. 43.5 ± 2.1, respectively) as compared with the non-treated group (0 h). We then investigated the acetylation levels of histone H3 in SCNT embryos treated with or without TSA (TSA (+) or TSA (-)) as compared with in vitrofertilized (IVF) embryos. The acetylation levels of the TSA (-) SCNT embryos at the pseudo-pronuclear and 2-cell stages were significantly lower than those of the IVF embryos at the same developmental stages. In contrast, the acetylation levels of the TSA (+) SCNT embryos were similar to those of the IVF embryos. There was no difference in the acetylation levels of all groups at the blastocyst stage. Our data therefore suggests that the acetylation level of histone H3 at the pseudo-pronuclear and 2-cell stages is positively correlated with subsequent development of SCNT embryos, which may be an important event for the vital development of SCNT embryos in miniature pigs. Key words: Embryo development, Histone acetylation, Miniature pig, Nuclear transfer, Trichostatin A (J. Reprod. Dev. 55: [638][639][640][641][642][643][644] 2009) lthough many studies have been performed to improve the developmental competence of miniature pig SCNT embryos [1][2][3][4], the overall efficiency remains low. To obtain sufficient developmental competence for SCNT embryos to develop to term, the differentiated cell nucleus should be subjected to epigenetic reprogramming processes including chromatin remodeling and DNA methylation during preimplantation development. However, reprogramming of a differentiated nucleus to an embryonic state is reportedly delayed and incomplete in SCNT embryos [5]. In fact, previous studies have demonstrated that epigenetic modifications such as DNA methylation and histone acetylation are abnormally reprogrammed in SCNT embryos during early development [6,7]. Incomplete reprogramming of epigenetic modifications causes abnormal gene expression including imprinted genes [8], resulting in deleterious effects on the development of SCNT embryos [9].Histone acety...

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“…To evaluate in vivo embryonic developmental ability to term, we transferred sperm-injected oocytes at 4 h after electrical stimulation to the oviducts of synchronized recipient gilts. In order to assist pregnancy, in some trials we co-transferred parthenogenetic oocytes without injection of spermatozoa but stimulated with an electrical pulse (King et al 2002, Kawarasaki et al 2009). Two out of 23 recipient gilts gave birth (Supplementary Table 1, see section on supplementary data given at the end of this article).…”

Section: Resultsmentioning

confidence: 99%

“…Although reports of the viability of transferred ICSI embryos in pigs are limited, the viability seems to be lower than that of conventionally fertilized oocytes. Co-transfer of parthenogenetic oocytes may be an aid to successful implantation (Kawarasaki et al 2009). Porcine parthenogenotes can be implanted and can remain viable during the early stage of pregnancy, but they cannot develop beyond the 29th day after transfer (Kure-bayashi et al 2000).…”

Section: Resultsmentioning

confidence: 99%

Production of viable piglets for the first time using sperm derived from ectopic testicular xenografts

Nakai¹,

Kaneko²,

Somfai³

et al. 2010

Reproduction

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Xenografting of testicular tissue into immunodeficient mice is known to be a valuable tool for facilitating the development of immature germ cells present in mammalian gonads. Spermatogenesis in xenografts and/or in vitro embryonic development to the blastocyst stage after ICSI of xenogeneic sperm has already been reported in large animals, including pigs; however, development of the embryos to term has not yet been confirmed. Therefore, in pigs, we evaluated the in vivo developmental ability of oocytes injected after ICSI of xenogeneic sperm. Testicular tissues prepared from neonatal piglets, which contain seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 133 and 280 days after xenografting, morphologically normal sperm were recovered, and a single spermatozoon was then injected into an in vitro matured porcine oocyte. After ICSI, the oocytes were electrostimulated and transferred into estrus-synchronized recipients. Two out of 23 recipient gilts gave birth to six piglets. Here, we describe for the first time that oocytes fertilized with a sperm from ectopic xenografts have the ability to develop to viable offspring in large mammals.

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Co-transfer of parthenogenotes and single porcine embryos leads to full-term development of the embryos

Cited by 27 publications

References 33 publications

Generation of Live Piglets for the First Time Using Sperm Retrieved from Immature Testicular Tissue Cryopreserved and Grafted into Nude Mice

Generation of Live Piglets for the First Time Using Sperm Retrieved from Immature Testicular Tissue Cryopreserved and Grafted into Nude Mice

Acetylation Level of Histone H3 in Early Embryonic Stages Affects Subsequent Development of Miniature Pig Somatic Cell Nuclear Transfer Embryos

Production of viable piglets for the first time using sperm derived from ectopic testicular xenografts

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