in vitro development of porcine enucleated oocytes reconstructed by the transfer of porcine fetal fibroblasts and cumulus cells

In the pig little information is available on cytoplasmic events during the reprogramming of oocytes reconstructed with somatic nuclei. The present study was conducted to determine the developmental potential of porcine cumulus cells (CC) and fetal fibroblasts (FF) after they were transferred into enucleated oocytes. Non-quiescent FF were fused to the enucleated oocytes using electrical pulse, whereas CC were directly injected into the oocytes. Transferred nuclei from both CC and FF underwent premature chromosome condensation (PCC), nuclear swelling and pronucleus formation. The remodeled oocytes developed to the mitotic and 2-cell stage at 18 to 24 h after nuclear transfer. The pattern of nuclear remodeling was similar regardless of the sources of karyoplasts or nuclear transfer methods. However, using FF, 24% of nuclear transferred embryos developed to the morula or blastocyst stage, whereas only 8% of those using CC developed to the morula or blastocyst stage. These results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of nuclear transferred embryos to the blastocyst stage.     

人-山羊异种核移植胚胎发育的初步研究

以体外分离培养的人胚胎成纤维细胞为核供体,经血清饥饿培养后,通过显微操作技术移入山羊去核卵母细胞中,采用化学方法激活重组胚.通过体外培养观察,2-细胞胚胎发育率可达51.33%,4-细胞发育率为31.42%,但发育至桑椹胚阶段的胚胎数目大大减少,仅为9.73%.虽然目前尚未能获得异种核移植囊胚,但实验结果说明山羊成熟卵母细胞可以支持人体细胞核完成重编程,人-山羊异种体细胞核移植重组胚可在体外完成其早期发育.     

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear transplantation in early pig embryos

Nuclear transfer was evaluated in early porcine embryos. Pronuclear stage embryos were centrifuged, treated with cytoskeletal inhibitors, and subsequently enucleated. Pronuclei containing karyoplasts were placed in the perivitelline space of the enucleated zygote and fused to the enucleated zygote with electrofusion. The resulting pronuclear exchange embryos were either monitored for cleavage in vitro (9/13 cleaved and contained 2 nuclei after 24 h, 69%) or for in vivo development. In vivo development after 3 days resulted in 14/15 (93%) of the embryos transferred cleaving to the greater than or equal to 4-cell stage and after 7 days 6/16 (38%) reaching the expanded blastocyst stage. A total of 56 pronuclear exchange embryos were allowed to go to term, and 7 piglets were born. A similar manipulation procedure was used to transfer 2-, 4- or 8-cell nuclei to enucleated, activated meiotic metaphase II oocytes. Enucleation was effective in 74% (36/49) of the contemporary oocytes. Activation was successful in 81% (37/46) of nonmanipulated but pulsed oocytes versus 13% (4/31) of control oocytes (p less than 0.01). After 6 days in vivo, 9% (1/11) of the 2-cell nuclei, 8% (7/83) of the 4-cell nuclei, and 19% (11/57) of the 8-cell nuclei transferred to enucleated, activated meiotic metaphase II oocytes resulted in development to the compact morula or blastocyst stage (p less than 0.01). A total of 88 nuclear transfer embryos were transferred to recipient gilts for continued development. A single piglet was born after the transfer of a 4-cell nucleus to an enucleated, activated metaphase II oocyte and subsequent in vivo development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.     

Expression of enhanced green fluorescent protein (EGFP) and neomycin resistant (Neo(R)) genes in porcine embryos following nuclear transfer with porcine fetal fibroblasts transfected by retrovirus vector

In this study, we demonstrated expression of enhanced green fluorescent protein (EGFP) and neomycin resistant (Neo(R)) genes in porcine embryos following nuclear transfer from porcine fetal fibroblasts (PFFs) transduced with the EGFP and Neo(R) genes by retrovirus-mediated infection. Nuclear transfer of the nonstarved transfected PFF into enucleated oocytes was accomplished by cell to cell fusion. Out of 188 porcine eggs reconstructed by nuclear transfer, 116 (61.7%) eggs cleaved and 25 (13.3%) developed to morula and blastocyst stages. Of these 25 morulae and blastocysts, 25 (100%) embryos emitted green fluorescence. Expression of the both EGFP and Neo(R) genes was detected as early as the 2-cell stage. As determined by EGFP gene expression, mosaicism was not observed in any embryo. These results suggest that porcine oocytes reconstructed by nuclear transfer with transfected PFFs can successfully develop to the blastocyst stage. In addition, this approach might be applicable to the production of transgenic pigs with complex genetic modifications.     

In vivo aging of oocytes influences the behavior of nuclei transferred to enucleated rabbit oocytes

The present study examined nuclear remodeling in rabbit nuclear transfer (NT) embryos formed from metaphase II (MII) oocytes aged in vivo until 19 hr postcoitum (hpc), enucleated, and fused at 22–26 hpc with 32-cell morula blastomeres by means of electric fields, which also induced recipient oocyte activation. Post-activation events observed during the first hour following the fusion/activation pulse were studied in terms of chromatin, lamins, and micro-tubules, and revealed that transferred nuclei underwent premature chromosomes condensation (PCC) in only one-third of NT embryos and remained in interphase in others. Recipient oocytes were mostly not activated by manipulations performed before the fusion/activation pulse. The persistance of transferred nuclei in interphase resulted from the rapid progression of recipient oocytes to interphase after activation, suggesting that the cytoplasmic state of MII oocytes aged in vivo was poised for the approach to interphase. Studying micro-tubular organization in MII oocytes before nuclear transfer manipulations, we found that 19 hpc MII oocytes aged in vivo differed from 14 hpc MII oocytes (freshly ovulated) and from 19-hpc MII oocytes aged in vitro (collected at 14 hpc and cultured for 5 hr), notably by the presence of microtubule asters and tubulin foci or only tubulin foci dispersed throughout the cytoplasm. When PCC was avoided, remodeling of the transferred nucleus was well advanced 1 hr after nuclear transfer, and NT embryos developed better to the blastocyst stage. Mol. Reprod. Dev. 46:325–336, 1997. © 1997 Wiley-Liss, Inc.     

用小鼠血液淋巴细胞构建核移植重构胚的研究

本实验用小鼠血液淋巴细胞为核供体进行了核移植研究。用淋巴细胞分离液(比重1.088)分离出小鼠血液中的淋巴细胞,直接用作核移植供体细胞,采用胞质内注射法成功构建的重构胚经常规培养2h后,SrCl_2激活处理6h,然后添加mM16培养液和小鼠输卵管上皮细胞饲养层共培养。把发育至早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加ES细胞培养液继续培养。对孵化出的内细胞团进行消化,然后接种培养。结果显示,小鼠血液淋巴细胞可以支持体细胞核移植重构胚的发育,核移植重构胚2-细胞率41.03%(128/312),桑葚胚和囊胚发育率分别为9.29%(29/312),1.92%(6/312)。重构囊胚在小鼠胎儿成纤维细胞饲养层上分离出2个内细胞团,分离率为0.64%(2/312)。实验证实利用小鼠血液淋巴细胞进行体细胞核移植是可行的,可用于深入研究。     

用小鼠血液淋巴细胞构建核移植重构胚的研究

本实验用小鼠血液淋巴细胞为核供体进行了核移植研究。用淋巴细胞分离液(比重1．088)分离出小鼠血液中的淋巴细胞,直接用作核移植供体细胞,采用胞质内注射法成功构建的重构胚经常规培养2h后,SrCl2激活处理6h,然后添加mM16培养液和小鼠输卵管上皮细胞饲养层共培养。把发育至早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加ES细胞培养液继续培养。对孵化出的内细胞团进行消化,然后接种培养。结果显示,小鼠血液淋巴细胞可以支持体细胞核移植重构胚的发育,核移植重构胚2-细胞率41．03％(128／312),桑葚胚和囊胚发育率分别为9．29％(29／312),1．92％(6／312)。重构囊胚在小鼠胎儿成纤维细胞饲养层上分离出2个内细胞团,分离率为0．64％(2／312)。实验证实利用小鼠血液淋巴细胞进行体细胞核移植是可行的,可用于深入研究。     

体细胞来源及培养代数对核移植重构胚发育的影响

为探讨体细胞来源及培养代数对核移植重构胚发育的影响,实验采用电融合法将小鼠2—细胞胚胎卵裂球、胚胎干细胞(ES)、胎儿成纤维细胞、耳成纤维细胞、尾尖成纤维细胞、睾丸支持细胞和精原细胞以及不同培养代次的胎儿成纤维细胞进行了核移植。结果显示：2—细胞胚胎卵裂球供核重构胚发育最好,囊胚率为7．4％;ES细胞重构胚虽然发育率低,但仍有囊胚出现,比例为0．7％;胎儿成纤维细胞重构胚最高发育阶段为桑椹胚,比例为0．2％;精原细胞重构胚只能发育到8-细胞阶段,比例为0．3％;其他几类细胞重构胚则仅能发育至4-细胞阶段。不同培养代数的胎儿成纤维细胞重构胚除第3代外都可发育到8-细胞阶段,且发育率差异不显著,但第一代细胞重构胚2-细胞发育率(40．7％)显著低于2、3和4代细胞重构胚。结果表明：不同分化程度的细胞核移植后,重新编程的难易程度是不一样的,分化程度越高则重新编程越难;未调整细胞周期的ES细胞由于多数处于S期,所以重构胚发育率很低;体外培养传代有利于体细胞核移植后重新编程。     

Viability of mouse half-embryos in vitro and in vivo

Mouse embryos at the 2-, 4-, 8-cell, and morula stage were divided in half by using microsurgical procedures and were either grown in vitro up to the blastocyst stage or transferred at the late morula stage into the uteri of pseudopregnant recipients. A relatively high percentage of the half embryos from 2-cell (70%), 4-cell (75%), 8-cell (93%), or morula stage embryos (75%) developed into blastocysts in vitro. However, the overall development in vivo of half embryos was low, as 3%, 13%, 8%, and 1% of half embryos from the 2-cell, 4-cell, 8-cell, and morula stages, respectively, developed into live fetuses. Embryos which were divided in half at different stages developed at different rates in vitro. This determined the stage of embryonic development at the time of transfer, which might have interacted with the stage of pseudopregnancy of the recipients to influence embryo survival in vivo.     

Germinal vesicle material is essential for nucleus remodeling after nuclear transfer

Successful cloning by nuclear transfer has been reported with somatic or embryonic stem (ES) cell nucleus injection into enucleated mouse metaphase II oocytes. In this study, we enucleated mouse oocytes at the germinal vesicle (GV) or pro-metaphase I (pro-MI) stage and cultured the cytoplasm to the MII stage. Nuclei from cells of the R1 ES cell line were injected into both types of cytoplasm to evaluate developmental potential of resulting embryos compared to MII cytoplasmic injection. Immunocytochemical staining revealed that a spindle started to organize 30 min after nucleus injection into all three types of cytoplasm. A well-organized bipolar spindle resembling an MII spindle was present in both pro-MI and MII cytoplasm 1 h after injection with ES cells. However, in the mature GV cytoplasm, chromosomes were distributed throughout the cytoplasm and a much bigger spindle was formed. Pseudopronucleus formation was observed in pro-MI and MII cytoplasm after activation treatment. Although no pronucleus formation was found in GV cytoplasm, chromosomes segregated into two groups in response to activation. Only 8.1% of reconstructed embryos with pro-MI cytoplasm developed to the morula stage after culture in CZB medium. In contrast, 53.5% of embryos reconstructed with MII cytoplasm developed to the morula/blastocyst stage, and 5.3% of transferred embryos developed to term. These results indicate that GV material is essential for nucleus remodeling after nuclear transfer.     

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell-cycle synchronized in G0/G1 using serum starvation (serum-starved) was examined. Oocyte-karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum-starved group (79 vs. 56% respectively; P < 0.05). Development to the 4-cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum-starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum-starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum-starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty-five nuclear transfer embryos reconstructed using serum-starved cells were transferred to 15 temporarily mated recipients for 3-4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4-cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred.     

Developmental regulation of an snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning.

The appearance and stabilization of a core protein epitope of the snRNP is developmentally regulated during pig embryogenesis. The epitope recognized by the monoclonal antibody Y12 is present in the germinal vesicle of mature oocytes and interphase nuclei of late 4-cell stage (24 to 30 hours post cleavage to the 4-cell stage) to blastocyst stage embryos. There was no antibody localization within pronuclei, or nuclei of 2-cell or early 4-cell stage embryos. Zygotes or 2-cell stage embryos cultured in the presence of alpha-amanitin to the late 4-cell stage showed no immunoreactivity, whereas control embryos had immunoreactivity. Thus antibody localization was correlated with RNA synthesis and RNA processing that begins by 24 hours post cleavage to the 4-cell stage. A final experiment showed no detectable immunoreactivity in 16-cell stage nuclei that had been transferred to enucleated activated meiotic metaphase II oocytes. Since immunoreactivity is associated with active RNA synthesis and RNA processing, it suggests that the 16-cell stage nucleus, which is RNA synthetically active, does not process RNA after nuclear transfer to an enucleated activated meiotic metaphase II oocyte.     

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells

Fetal-derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1-cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2- to 4-cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1-cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine-treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells.     

In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro

The objective of this study was to compare the development of porcine embryos from the 2- and 4-cell stages to the blastocyst stage after in vivo or in vitro fertilization and in vivo or in vitro culture. Early-stage embryos were collected either from superovulated gilts 36 h after the second mating or after in vitro fertilization (IVF) of in vivo-matured oocytes, both followed by in vitro culture to the blastocyst stage. Blastocysts collected from superovulated donors served as controls. In the first experiment, a total of 821 2- and 4-cell embryos derived from in vivo-fertilized oocytes was cultured either in medium NCSU 23, modified Whittens' medium or modified KRB for 5 d. Significantly (P < 0.05 and P < 0.001) more embryos overcame the 4-cell block and developed to the blastocyst stage in medium NCSU 23 than in the 2 other culture media. Hatching was only observed in medium NCSU 23. In the second experiment, embryos derived from in vivo-matured oocytes fertilized in vitro were cultured in medium NCSU 23. Of 1869 mature oocytes 781 (41.8%) cleaved within 48 h after in vitro fertilization. A total of 715 embryos was cultured to the morula and blastocyst stages, and 410 (57.3%) overcame the developmental block stage, with 358 embryos (50.1%) developing to the morula and blastocyst stages. None of the embryos hatched, and the number of nuclei was significantly (P < 0.05) lower compared with that of in vivo-fertilized embryos (18.9 +/- 9.8 vs 31.2 +/- 5.8). In the third experiment, 156 blastocysts derived from in vitro fertilization and 276 blastocysts derived from in vivo fertilization and in vitro culture were transferred into synchronized recipients, while 164 blastocysts were transferred immediately after collection into 6 recipients, resulting in a pregnancy rate of 83.3%, with 35 piglets (on average 7.0) born. From the in vitro-cultured embryos, 58.3% (7/12) of the recipients remained pregnant at Day 35 after transfer, but only 33.3% maintained pregnancy to term, and 14 piglets (on average 3.5) were born. In contrast, the transfer of embryos derived from in vitro-fertilized oocytes did not result in pregnancies. It is concluded that 1) NCSU 23 is superior to modified Whittens' medium and modified KRB and 2) blastocysts derived from in vitro fertilization have reduced viability as indicated by the lower number of nuclei and failure to induce pregnancy upon transfer into recipients.     

Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT-PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1-cell, 2-cell, 6-8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK-L, PFK-M, and PFK-P. PFK-L and PFK-P were not detected in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrated that PFK was not detected in 8-cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH-A and LDH-B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH-C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH-C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer.