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1.
Nuclear Replacement of In Vitro‐Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method
N Maedomari K Kikuchi T Nagai M Fahrudin H Kaneko J Noguchi M Nakai M Ozawa T Somfai LV Nguyen J Ito N Kashiwazaki 《Reproduction in domestic animals》2010,45(4):659-665
The objective of the present study was to establish a method for nuclear replacement in metaphase‐II (M‐II) stage porcine oocytes. Karyoplasts containing M‐II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro‐matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona‐free M‐II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2–77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2–32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0–15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri‐Fusion) is an effective method for producing M‐II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development. 相似文献
2.
Kou HIRAGA Yumi HOSHINO Kentaro TANEMURA Eimei SATO 《The Journal of reproduction and development》2013,59(4):405-408
Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were
evaluated as a novel marker for in vitro maturation (IVM) of oocytes with
high developmental competence. Porcine oocytes were cultured in TCM-199, which is a
complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2
classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those
that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes
exhibited the class II pattern. To investigate the relation between the distribution of
lipid droplets and the developmental rate of the oocyte, the developmental rates of class
I and class II oocytes were compared after in vitro fertilization (IVF).
Class II oocytes showed a significantly higher rate of blastocyst development than class I
oocytes. These results suggest that porcine oocytes with high developmental competence can
be selected based on the localization patterns of lipid droplets. 相似文献
3.
Morikawa M Seki M Kume S Endo T Nishimura Y Kano K Naito K 《The Journal of reproduction and development》2007,53(6):1151-1157
A high cyclic adenosine monophosphate (cAMP) level in fully-grown immature oocytes prevents meiotic resumption. In Xenopus, inhibitory cAMP is synthesized within oocytes depending on a stimulatory alpha-subunit of G-protein (Gsalpha). In the present study, we examined whether ooplasmic Gsalpha is involved in meiotic arrest of porcine oocytes. First, we studied the presence of Gsalpha molecules in porcine oocytes by immunoblotting, and this suggested the presence of reported isoforms (45 and 48 kDa) not only in cumulus cells but also in porcine oocytes. Then we injected an anti-Gsalpha antibody into porcine immature oocytes and found that inhibition of ooplasmic Gsalpha functions significantly promoted germinal vesicle breakdown of the oocytes, whose spontaneous meiotic resumption was prevented by 3-isobutyl-l-methylxanthine (IBMX) treatment. Although cyclin B synthesis and M-phase promoting factor (MPF) activation were largely prevented until 30 h of culture in IBMX-treated oocytes, injection of anti-Gsalpha antibody into these oocytes partially recovered cyclin B synthesis and activated MPF activity at 30 h. These results suggest that meiotic resumption of porcine oocytes is prevented by ooplasmic Gsalpha, which may stimulate cAMP synthesis within porcine oocytes, and that synthesized cAMP prevents meiotic resumption of oocytes through the signaling pathways involved in MPF activation. 相似文献
4.
Ayano OI Hidetaka TASAKI Yasuhisa MUNAKATA Koumei SHIRASUNA Takehito KUWAYAMA Hisataka IWATA 《The Journal of reproduction and development》2015,61(3):191-197
In this study, we examined the effects of reconstructed oocyte–granulosa cell complexes (OGCs) on the development of porcine oocytes derived from early antral follicles (EAFs; 0.5–0.7 mm in diameter). When denuded oocytes were cocultured with granulosa cells derived from other EAFs, the oocytes and granulosa cells aggregated to form OGCs after 2 days of culture. After 14 days of culture, we compared cell number, oocyte diameter, and oocyte chromatin configuration in unmanipulated (natural) OGCs, reconstructed OGCs, and OGCs collected from antral follicles (AFs, 3.0–6.0 mm in diameter). The diameters of oocytes from reconstructed OGCs grown in vitro were not different from those of oocytes from natural OGCs, although they were significantly smaller than those of oocytes from antral follicle (AF) OGCs. Oocyte chromatin configuration did not differ among the 3 OGC groups, but the oocyte nuclear maturation rate was lower in the reconstructed OGCs and higher in
the AF OGCs. However, when the in vitro culture period for the reconstructed OGCs was extended by 2 days, the nuclear maturation rate of oocytes from reconstructed OGCs was similar to that of oocytes from natural OGCs. In addition, blastocysts were successfully obtained from oocytes from reconstructed OGCs. In conclusion, we established an innovative culture method that allows oocytes and granulosa cells from EAFs to reaggregate as reconstructed OGCs, which yield oocytes with the ability to develop to the blastocyst stage. 相似文献
5.
Keisuke KOYAMA Sung-Sik KANG Weiping HUANG Yojiro YANAGAWA Yoshiyuki TAKAHASHI Masashi NAGANO 《The Journal of reproduction and development》2014,60(2):136-142
The objective of this research was to clarify the aging-related changes in in
vitro-matured bovine oocytes. Firstly, we examined the fertilization and
embryonic development of bovine oocytes after 22 and 30–34 h of in vitro
maturation (IVM). The oocytes after 30–34 h of IVM (penetrated by sperm at around 40 h
after starting IVM) showed a lower developmental rate to blastocysts (P<0.01), although
normal fertilization rates were similar regardless of IVM duration. In the next
experiment, reactive oxygen species (ROS), mitochondrial activity and ATP content in
oocytes after 20, 30 and 40 h of IVM were examined. The lowest level of ROS was found in
the group subjected to 30 h of IVM. The mitochondrial activity and ATP content in the
group subjected to 40 h of IVM were higher than in the group subjected to 20 h of IVM
(P<0.01), and those in the group subjected to 30 h of IVM showed intermediate values.
Thereafter, the mitochondrial activities at 3 days after in vitro
fertilization in embryos derived from the oocytes subjected to 22 and 34 h of IVM were
evaluated. In the group subjected to 34 h of IVM, high-polarized mitochondria were
frequently observed at the periphery of blastomeres. The present results suggest that high
mitochondrial activity observed in oocytes after prolonged IVM culture and localization of
high-polarized mitochondria at the periphery of blastomeres during early embryonic
development may be associated with the low developmental competence in aged bovine
oocytes. 相似文献
6.
Microtubule assembly crucial to bovine embryonic development in assisted reproductive technologies
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Shinichi Hochi 《Animal Science Journal》2016,87(9):1076-1083
Centrosome integrity and microtubule network are crucial to the events around fertilization, including pronuclear development, migration and fusion, and the first mitotic division. The present review highlights the importance of bull spermatozoal centrosomes to function as a microtubule‐organizing center for successful fertilization and the subsequent embryonic development. Spermatozoal centrosomes need to be blended with ooplasmic pericentriolar materials accurately to nucleate and organize the sperm aster. Dysfunction of the spermatozoal centrosomes is associated with fertilization failure, which has been overcome with supplemental stimuli for oocyte activation following intracytoplasmic sperm injection in humans. Even though the spermatozoal centrosomes are functionally intact, abnormal sperm aster formation was frequently observed in vitrified‐warmed bovine oocytes, with delayed pronuclear development and migration. Treatment of the post‐warm oocytes with Rho‐associated coiled‐coil kinase inhibitor or α‐tocopherol inhibited the incidence of the abnormal aster formation, resulting in higher blastocyst yields following in vitro fertilization and culture. Thus, understanding of centrosomal function made it possible to improve the performance of advanced reproductive technologies. 相似文献
7.
Seon-Hyang KIM Ming-Hui ZHAO Shuang LIANG Xiang-Shun CUI Nam-Hyung KIM 《The Journal of reproduction and development》2015,61(4):261-268
Cathepsin B, a lysosomal cysteine protease of the papain family, has recently been implicated in the quality and developmental competence of bovine preimplantation embryos. In this study, to determine whether inhibition of cathepsin B activity can improve porcine oocyte maturation and early embryo developmental competence, we supplemented in vitro maturation or embryo culture media with E-64, a cathepsin B inhibitor. Cathepsin B activity was high in poor quality germinal vesicle stage oocytes, but no differences in mRNA expression or protein localization were observed between good and poor quality oocytes, which were categorized based on morphology. Following treatment with 1 μM E-64, cathepsin B activity sharply decreased in 4-cell and blastocyst stage embryos. E-64 had no effect on cell number but significantly (P < 0.05) increased blastocyst formation and decreased the number of apoptotic cells in blastocysts. It also significantly (P < 0.05)
enhanced mitochondrial membrane potential in blastocysts, reducing the release of cytochrome c and resulting in decreased expression of Caspase-3 and Caspase-9. In conclusion, inhibition of cathepsin B activity in porcine parthenotes using 1 μM E-64 resulted in attenuation of apoptosis via a reduction in the release of cytochrome c from mitochondria. 相似文献
8.
Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system
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Ruth Appeltant Tamás Somfai Kazuhiro Kikuchi Dominiek Maes Ann Van Soom 《Animal Science Journal》2016,87(4):503-510
Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system. 相似文献
9.
Fuminori TANIHARA Michiko NAKAI Hiroyuki KANEKO Junko NOGUCHI Takeshige OTOI Kazuhiro KIKUCHI 《The Journal of reproduction and development》2013,59(4):385-392
In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm
penetration or prevention of polyspermy is not well understood. In the present study, we
investigated the effects of the ZP on sperm penetration during in vitro
fertilization (IVF). We collected in vitro-matured oocytes with a first
polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two
treatments (pronase and mechanical pipetting), and the effects of these treatments on
sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per
oocyte) were evaluated. There was no evident difference in the parameters between the two
groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes
using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+
oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the
sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation).
The proportions of oocytes penetrated by sperm increased significantly with time in both
groups; however, the number of penetrated sperm per oocyte did not increase in ZP−
oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and
prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5
h group than in the control group. These results suggest that the ZP and oolemma are not
competent factors for prevention of polyspermy in our present porcine IVF system. However,
it appears that ZP removal is one of the possibilities for reducing polyspermic
penetration in vitro in pigs. 相似文献
10.
Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species‐specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly demonstrated that porcine sperm attached to the zona and underwent the acrosome reaction. Equine spermatozoa displayed a similar binding affinity, but unlike the porcine spermatozoa even penetrated the zp and were taken up into the oocyte after a longer period of co‐incubation. Considering these new results the dogma of a strict species specificity of sperm zona interactions under in vitro conditions has to be reconsidered. 相似文献
11.
Masahiko Hirose Maki Kamoshita Katsuyoshi Fujiwara Tsubasa Kato Ayaka Nakamura Richard J. H. Wojcikiewicz Jan B. Parys Junya Ito Naomi Kashiwazaki 《Animal Science Journal》2013,84(10):693-701
Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm‐penetrated oocytes were observed to some extent (30–40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two‐cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP3R1), the channel responsible for Ca2+ release during IVF in porcine oocytes. Localization of IP3R1 close to the plasma membrane and total expression level of IP3R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified‐warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP3R1 by the vitrification procedure. 相似文献
12.
Shun TAKEO Daichi SATO Koji KIMURA Yasunori MONJI Takehito KUWAYAMA Ryoka KAWAHARA-MIKI Hisataka IWATA 《The Journal of reproduction and development》2014,60(2):92-99
The aim of the present study was to address the effect of resveratrol-mediated
upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the
developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic
fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in
TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the
effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1);
fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt
number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and
the time required for proteinase to dissolve the zona pellucida following in
vitro fertilization (as a marker of zona pellucida hardening), as well as on
the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1,
the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res
treatment improved the ratio of normal fertilization and the total cell number of
blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured
oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane
potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening
improved the distribution and exocytosis of cortical granules after in
vitro fertilization. In conclusion, Res improved the quality of oocytes by
improving mitochondrial quantity and quality. In addition, Res added to the maturation
medium enhanced SIRT1 protein expression in oocytes and improved fertilization via
reinforcement of the mechanisms responsible for blockage of polyspermic fertilization. 相似文献
13.
A. Krogences E. Ropstad T Nilsen
. Pedersen M. Forsberg 《Acta veterinaria Scandinavica》1993,34(2):211
In vitro maturation of oocytes has been described for several species, but to our knowledge this is the first attempt to mature oocytes from reindeer in vitro. Reindeer are seasonal breeders with their main period of breeding activity starting in the beginning of October. Little knowledge exists about in vivo oocyte maturation, fertilization and development of the early embryonic stages after fertilization. The aim of the present study was to examine whether reindeer oocytes collected out of breeding season could be matured in vitro in conventional medias used for bovine oocytes. 相似文献
14.
Yuta ISHIZUKA Toru TAKEO Satohiro NAKAO Hidetaka YOSHIMOTO Yumiko HIROSE Yuki SAKAI Yuka HORIKOSHI Shiori TAKEUJI Shuuji TSUCHIYAMA Naomi NAKAGATA 《The Journal of reproduction and development》2014,60(6):454-459
Hyaluronidase is generally used to remove cumulus cells from mouse oocytes before oocyte cryopreservation, intracytoplasmic sperm injection or DNA injection. In general, use of cumulus-free mouse oocytes decreases in vitro fertilizing ability compared with cumulus-surrounded oocytes. The effect of hyaluronidase exposure on the quality of mouse oocytes is not fully understood. Here, we investigated the effect of hyaluronidase exposure time on the fertilization rate of fresh and vitrified mouse oocytes and their subsequent developmental ability in vitro. We found that the fertilization rate decreased with hyaluronidase treatments. This reduction in the fertilization rate following treatment with hyaluronidase was fully reversed by removal of the zona pellucida. In addition, oocytes treated with hyaluronidase for 5 min or longer had a reduced capacity to develop to the morula and blastocyst stage. The survival, fertilization, and
developmental rates of vitrified-warmed oocytes were also reduced by longer exposure to hyaluronidase. In conclusion, these results suggest that prolonged exposure to hyaluronidase decreases the quality of mouse oocytes and shorter hyaluronidase treatment times may help achieve a stable and high fertilization rate in fresh and cryopreserved oocytes. 相似文献
15.
Budiyanto AGUNG Takeshige OTOI Dai-ichiro FUCHIMOTO Shoichiro SENBON Akira ONISHI Takashi NAGAI 《The Journal of reproduction and development》2013,59(2):103-106
This study was conducted to assess the fertilization and development of porcine oocytes
matured in a solo follicular fluid (pFF) using different in vitro culture
systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs),
and pFF were collected from the follicles of ovaries. The pFF was used as a maturation
medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and
antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs
(5.2 × 106 cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a
35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes
were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The
total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN)
formation, cleavage, and development to the blastocyst stage of oocytes after insemination
were significantly higher (P<0.01) in the rotating culture system than in the static
culture system. In conclusion, compared with the static culture system, the rotating
culture system is adequate for the production of developmentally competent porcine oocytes
when MpFF is used as a maturation medium. 相似文献
16.
Rentsenkhand SAMBUU Mitsuhiro TAKAGI Zhao NAMULA Masahiro NII Masayasu TANIGUCHI Seiich UNO Emiko KOKUSHI Chenga TSHERING Regiane Rodrigues dos SANTOS Johanna FINK‐GREMMELS Takeshige OTOI 《Animal Science Journal》2013,84(1):28-34
The effects of in vitro exposure of porcine spermatozoa to zearalenone (ZEN) and α‐zearalenol (α‐ZOL) were studied by evaluating several parameters of an in vitro fertilization (IVF) system. For this purpose, boar spermatozoa cultured with semen storage medium containing 0 (control), 10 and 1000 µg/L of ZEN and α‐ZOL for 1 week at 5°C were used for IVF of in vitro matured oocytes. Overall, there were no significant differences in the rates of total penetration, monospermic fertilization, and polyspermic fertilization of oocytes inseminated with spermatozoa from the different groups. Similarly, ZEN and α‐ZOL at 10 and 1000 µg/L did not have detrimental effects on the cleavage and development to blastocysts of oocytes after in vitro fertilization. Although the motility, viability, and plasma membrane integrity of spermatozoa significantly decreased after 3 weeks of storage compared to non‐stored spermatozoa (P < 0.05), ZEN and α‐ZOL at the evaluated concentrations did not exert detrimental effects on the above parameters, even after 3 weeks of storage. These results indicate that prolonged exposure of boar spermatozoa to ZEN and α‐ZOL up to 1000 µg/L under reduced metabolic conditions does not affect their in vitro function. 相似文献
17.
Misa HOSOE Nao YOSHIDA Yutaka HASHIYADA Hidetoshi TERAMOTO Toru TAKAHASHI Sueo NIIMURA 《The Journal of reproduction and development》2014,60(4):268-273
Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in
vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum
replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the
rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%,
0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of
Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes
and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin
and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured
with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater
in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and
blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and
CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in
those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine
oocytes. 相似文献
18.
Seizo HAMANO 《The Journal of reproduction and development》2021,67(3):167
This review discusses the production and sale of fertile oocytes for in vitro fertilization technology, calf production through transplantation and delivery, and the current circulation of calves produced by in vitro production (IVP) embryos. 相似文献
19.
The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p < 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 µsec (19 ± 2% vs. 77 ± 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 µsec, although this was still lower than the rate of fusion in the CCCs (33 ± 1% vs. 80 ± 2%). The rates of cleavage (57 ± 5%) and blastocyst formation (1 ± 1%) in the DCC-derived embryos did not differ from those (55 ± 6% and 3 ± 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 ± 2%) showed higher levels of blastocyst formation (p < 0.01) than CCC-derived autologous SCNT embryos (1 ± 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts. 相似文献
20.
The objective was to investigate the RNA synthesis in porcine blastomere nuclei upon transplantation into in vitro matured enucleated oocytes. Nuclei from 2- to 8-cell porcine embryos were introduced into the ooplasm of in vitro matured and enucleated porcine oocytes by electrofusion, and the resultant reconstructed embryos were cultured in vitro. Before fusion or at different intervals after this event embryos were incubated with [3H]-uridine, fixed, and histologically processed for autoradiography in order to detect RNA synthesis. About two thirds of the embryos were considered to depict normal development. All blastomeres displayed pronounced RNA synthesis before fusion, at 3 and 9 h after fusion the synthesis decreased or ceased, and at 24-49 h some embryos resumed synthesis at the 1- to 2-cell stage. 相似文献