Production of cloned goats by nuclear transfer of cumulus cells and long-term cultured fetal fibroblast cells into abattoir-derived oocytes

Dairy goats are ideal for the transgenic production of therapeutic recombinant proteins. The use of recombinant somatic cell lines for nuclear transfer (NT) allows the introduction of genes by transfection, increases the efficiency of transgenic animal production to 100%, and overcomes the problem of founder mosaicism. Although viable animals have been cloned via NT from somatic cells of 11 species, the efficiency has been extremely low. Both blastomere and somatic cell NT increased fetal loss and perinatal morbidity/mortality in cattle and sheep, but fetal loss and perinatal mortality appear to be relatively low in goats. In this study, we produced cloned goats by NT from cumulus cells and long-term cultured fetal fibroblast cells (FFCs) to abattoir-derived oocytes. NT embryos were constructed from electrofusion of cumulus cells (CCs), FFCs, or skin fibroblast cells (SFCs) with cytoplasts prepared from abattoir-derived ovaries. The NT embryos were activated with an optimized activating protocol (1 min exposure to 2.5 microM ionomycin followed by 2 hr incubation in 2mM 6-DMAP). Two viable cloned kids from CCs and one from long-term cultured FFCs (at passage 20-25) were born. Microsatellite analysis of 10 markers confirmed that all cloned offspring were derived from corresponding donor cells. To our knowledge, the production of cloned goat offspring using abattoir-derived oocytes receiving nuclei from CCs and long-term cultured FFCs has not been reported. The production of viable cloned animals after activation with reduced intensity of ionomycin and 6-DMAP treatment has also not been reported. Loss of cloned embryos was obvious after 45 and 90 days of pregnancy, and a lack of cotyledons, heart defects, and improperly closed abdominal wall were observed in the aborted fetuses and one cloned kid. The fusibility and in vitro developmental potential of embryos reconstructed from FFCs at passage 20-25 were significantly lower than those of embryos reconstructed from FFCs at passage 3-5, and the cloning efficiency of the long-term cultured cells was low (0.5%).     

由成年转基因山羊体细胞而来的克隆山羊

在已经获得的乳腺特异性表达人促红细胞生成素 (rhEPO)成年转基因山羊 (Caprahircus)的基础上 ,取其耳尖成纤维细胞和卵巢颗粒细胞 ,进行体外传代培养 ,然后将这种培养的转基因山羊的体细胞移入去核的处于第Ⅱ次减数分裂中期的卵母细胞中 ,并进行电融合 ,构建重构胚胎 ,重构胚胎在体内培养 6d ,再将发育至囊胚或桑椹胚的重构胚胎移入同步情期的寄母羊子宫内。结果 ,有 2只寄母羊妊娠并最终产下 2只成活的克隆山羊。她们分别来自同一成年母羊的耳尖成纤维细胞和卵巢颗粒细胞。克隆羊经PCR RFLP图谱分析显示 :以克隆羊组织DNA为模板的PCR产物与相应的提供体细胞的基因羊的PCR产物的酶切图谱完全一致 ;并且经PCR对外源hEPO基因检测表明 2只克隆山羊均携带hEPO外源基因。由此证明获得了转基因成年体细胞的克隆山羊     

Xenonuclear transplantation of buffalo (Bubalus bubalis) fetal and adult somatic cell nuclei into bovine (Bos indicus) oocyte cytoplasm and their subsequent development

Bovine oocyte cytoplasm has been shown to support the development of nuclei from other species up to the blastocyst stage. Somatic cell nuclei from buffalo fetal fibroblasts have been successfully reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts. The aim of this study was to compare the in vitro development of fetal and adult buffalo cloned embryos after the fusion of a buffalo fetal fibroblast, cumulus or oviductal cell with bovine oocyte cytoplasm. The fusion of oviductal cells with enucleated bovine oocytes was higher than that of fetal fibroblasts or cumulus cells (83% versus 77 or 73%, respectively). There was a significantly higher cleavage rate (P < 0.05) for fused nuclear transferred embryos produced by fetal fibroblasts and oviductal cells than for cumulus cells (84 or 78% versus 68%, respectively). Blastocyst development in the nuclear transferred embryos produced by fetal fibroblasts was higher (P < 0.05) than those produced either by cumulus or oviductal cells. Chromosome analysis of cloned blastocysts confirmed the embryo was derived from buffalo donor nuclei. This study demonstrates that nuclei from buffalo fetal cells could be successfully reprogrammed to develop to the blastocyst stage at a rate higher than nuclei from adult cells.     

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower     

Improvement of the nuclear transfer efficiency by using the same genetic background of recipient oocytes as the somatic donor cells in goats

We have compared the effect of the genetic background of recipient oocytes on the in vitro and in vivo development of nuclear transfer reconstructed embryos in goats. Adult fibroblast cells from Boer goats were used as donor cells, and recipient oocytes were obtained from Boer goats and Boer cross-breeds (Boer♂×Huanghuai♀). Nuclear transfer reconstructed embryos were cultured in vitro, or transferred into recipient goats. The mitochondrial origin of 2 cloned Boer goats was investigated by analysing the D-loop region based on polymorphisms via DNA sequencing. There was no significant difference in the fusion rate and cleavage rate of reconstructed embryos (P>0.05), when using Boer and cross-breeding goat oocytes as recipient cytoplast respectively. However, in vitro morula development of reconstructed embryos from Boer oocytes was significantly higher than that of cross-breeding embryos (34.1% versus 19.1%, P<0.05). There was no significant difference in the rate of pregnancy and foetus loss between the 2 breeds. However, the live-birth rate was significantly higher with Boer goat oocyte recipients than the cross-breeds (3.1% versus 0.8%, P<0.05). Mitochondrial analysis showed that the 2 cloned goats were similar to their respective oocyte donor goats, and significantly different from the nucleus donor. In conclusion, genetic background of recipient oocytes affected in vitro and in vivo development of reconstructed embryos, with the homologous background of cytoplast and nuclear donor benefiting development of reconstructed embryos. The mitochondrial origin of the 2 cloned Boer goats came from recipient oocytes, not donors.     

完整的供体细胞膜及供体胞质对小鼠体细胞克隆胚发育的影响

利用显微注射和电融合的方法都可以成功地获得体细胞克隆小鼠, 由于电融合法操作耗时, 融合率低, 因而大多数克隆小鼠是采用注射方法。而注射法需要将供体细胞核从细胞中分离出来, 此分离操作有可能导致对DNA的损伤, 曾有人使用直径较粗的注射管进行完整的供体细胞注射, 这种方法操作相对简单而且对供体核没有损伤。为了研究这种方法在小鼠核移植中是否适用, 本实验使用完整的小鼠卵丘细胞作供体, 进行显微注射, 结果显示, 完整的卵丘细胞注入卵母细胞后, 无论在1小时或者6小时激活, 大部分的重构胚在2细胞期碎裂, 而去掉细胞膜的供体体细胞核注入卵母细胞后, 重构胚可以卵裂并进一步发育。卵母细胞去核后不注射供体也发生碎裂, 大部分的孤雌胚(不去核)在完整的卵丘细胞被注入后同样发生碎裂。在供体卵丘细胞刚破膜后即被注入卵胞质和供核被充分剥离后注入两种情况下获得的重构胚的体外发育中, 前者发育各期的比率显著低于后者。这些结果说明完整的卵丘细胞膜阻碍了卵胞质对体细胞核的重编程作用, 造成碎裂; 注入卵胞质的供体质膜和胞质成分影响了克隆胚的体外发育。     

Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation

We conducted this study to examine whether serum starvation in culture contributes to better development of bovine reconstructed oocytes and to evaluate which serum-starved somatic cell is the most effective for cloned calf production. In Experiment 1, donor cells of four different types (cumulus cells, ear fibroblasts, oviduct cells and uterine cells) were either serum-starved or not before fusion with enucleated oocytes, and reconstructed oocytes were further cultured for 168 h. Regardless of serum starvation, cumulus cells or ear fibroblasts yielded higher (P < 0.05) rates of fusion than other cells (62.6-69.3 versus 33.3-38.7%). In the serum-starved group, the first cleavage after reconstruction was significantly increased in cumulus cells and ear fibroblasts, compared with oviduct cells (93.4-94.3 versus 78.8-86.0%), and oocytes reconstructed with either of these yielded more blastocysts than oocytes reconstructed with oviduct or uterine cells (40.6-43.8 versus 20.3-19.0%). We observed a similar pattern in the non-starved group, but we found a significant increase in blastocyst formation was found only in cumulus cells compared with other donor cells (42.6 versus 15.4-27.7%). Overall comparison showed that serum starvation increased the rates of cleavage and development to the blastocyst stage, but we found a statistical significance only in the cleavage rate (80.0 versus 89.5%). In Experiment 2, we transferred randomly selected 59 blastocysts that were developed from oocytes reconstructed with serum-starved cells to 44 synchronised recipients. Of those recipients, 23 became pregnant on Day 60 after transfer (52.3%) and 12 (27.3%) delivered cloned calves. The mean gestation length and birth weight was 275 +/- 8 days and 39.6 +/- 15.6 kg, respectively. Although there was no significant difference among donor cells, blastocysts that were derived from oocytes reconstructed with ear fibroblasts yielded the highest rates of pregnancy (50.0%) and delivery (27.3%). In conclusion, serum starvation is effective for improving preimplantation development of oocytes reconstructed with cumulus or ear fibroblast cells and it may positively influence on obtaining better pregnancy outcome.     

单、双标记基因筛选的转人乳铁蛋白基因供核细胞生产克隆山羊效率的比较

为比较两种筛选标记基因生产转人乳铁蛋白(hLF)基因克隆山羊的效率,利用单(新霉素抗性基因,Neor)、双(新霉素抗性和绿色荧光蛋白基因,Neor/GFP)标记基因筛选转基因的供核细胞,并制作体细胞核移植转基因山羊。山羊胎儿成纤维细胞电转染单标记基因表达载体(pBLC14)或双标记基因表达载体(pAPLM),分别有58.8%(20/34)和86.7%(26/30)的抗性细胞株检测到外源基因;转染pAPLM的细胞传代培养后,仅有20%(6/30)株细胞在传代中所有细胞均能观察到荧光;分别以pBLC14和pAPLM的细胞株作为供核细胞进行体细胞核移植,共获得806枚重构胚胎,胚胎移植受体后35 d、60 d妊娠率分别为53.8%、26.9%和39.1%、21.7%,最终分别产下5只(1.9%)和7只(1.4%)克隆山羊;经PCR及Southern blotting检测,所有出生山羊均整合有外源基因。结果显示,以单、双标记基因筛选供核细胞,其重构胚融合率、怀孕率和克隆动物出生率差异不显著(P>0.05),Neor/GFP双标记基因能准确、有效地用于转基因供核细胞筛选。同时,结果也表明Neor/GFP双标记基因转染的体细胞作为供核细胞对体细胞克隆效率未出现不利影响。     

Effect of cytoplast on the development of inter-subspecies nuclear transfer reconstructed goat embryo

The aim of this study was to investigate effect of cytoplast on the development competence of reconstructed embryos derived from inter-subspecies somatic cell nucleus transfer (SCNT). First, the development potency of reconstructed embryos produced by transferring Boer goat fibroblast cell nucleus of different ages into enucleated Sannen goat ova was evaluated in order to determine which age of nuclear donor is favorable for the reconstructed embryos development. Secondly, the another component of reconstructed embryos, "cytoplast," was evaluated by comparing the effect of ovum cytoplast derived from Sannen male symbol x Boer female symbol descendant on the reconstructed embryos development to that of Sannen goat ovum cytoplast. The results revealed that the development rate of the reconstructed embryos derived from 2 months old Boer goat somatic cells was the highest, their gestation rate was up to 50%, and one viable male offspring was obtained. The cytoplast derived from the crossbreeding goats improves the development competence of reconstructed embryos, which birth rate was 5.5%. The genetic identification of offspring by using PCR-SSCP analysis confirmed that these cloned kids were derived from the donor. The results above reveal that the cytoplast of Sannen goat ovum could induce the dedifferentiation of somatic cell nuclei derived from Boer goat, but the reprogramming process of these reconstructed embryos seems incomplete, probably due to some incorrect processes happened after implantation. Relatedness components of nucleus donor in cytoplast of the crossbreeding goat may be helpful to induce the dedifferentiation of somatic cell nuclei completely and improve the development competence of the reconstructed embryos.     

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2–3 h, was significantly lower than that of the 3–6 h groups (31.0%), while not significantly different among 3–4 h (P < 0.05), 4–5 h, and 5–6 h groups (P≥0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.     

Preimplantation and postimplantation development of rat embryos cloned with cumulus cells and fibroblasts

In this report we demonstrate the successful in vitro culture of fertilised embryos from 1-cell to blastocyst stage, albeit in a strain-dependent fashion. We report procedures for the enucleation of rat oocytes; nuclear transfer by injection of nuclei (NT) from adult rat cumulus cells, rat primary embryonic fibroblasts and genetically modified rat fibroblasts; and activation resulting in advanced preimplantation development. Blastocyst stage rat embryos were obtained after in vitro culture of nuclear transfer zygotes at similar frequencies with each of these nuclear donor cell types. Transfer of NT embryos to surrogate mothers leads to implantation of 24% of the zygotes. These results suggest that the nuclei of cultured rat cells, even following genetic modification, can be reprogrammed to support early embryonic development, which is a prerequisite to cloning the rat.     

Cloned goats (Capra hircus) from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MII chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female dining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5μ mol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no signifi     

Cloned goats (Capra hircus) from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MⅡchromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female Jining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5 μmol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no significant differences in development in vitro between the cloned embryos derived from donor cells precooled at 4℃ for 24 h and nonprecooled donor cells. The cleavage rates, 4-cell development, and blastocyst development of reconstructed embryos were 72.48% (79/109), 53.16% (42/79), and 19.05% (8/42) in precooled group; 68.5% (211/308), 59.72% (126/211), and 17.46% (22/126) in nonprecooled group, respectively. Eighteen cloned 4-cell embryos derived from precooled donor cells were transferred and one cloned kid was born. Eighty-four cloned 4-cell embryos derived from nonprecooled donor cells were transferred and no offspring were produced. Of 18 cloned morale from nonprecooled donor cells transferred, one kid was born. The results of microsatellite DNA analyses indicated that the two cloned kids were from the same donor fibroblast cell line derived from an adult goat ear skin.     

Generation of cloned goats (Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle.

The neomycin-resistant gene (neo(r)) is probably the most commonly used selectable marker gene in gene targeting and gene transfection research. In this study, the neo(r) gene construct was introduced into in vitro cultured goat foetal fibroblast cells (IV-5), and the cells were selected with 900 microg/ml G418. The G418-resistant colonies were analysed by neo-specific PCR, karyotyping and anti-intermediate filament proteins antibody (anti-vimentin) staining. Cell cycle analysis of the neo(r) positive foetal fibroblast cell colony (IV-5.1) cultured in a variety of cell cycle-arresting medium indicated that 74.2% of cells cultured in serum-deprived medium for 3 days and 71.7% of cells grown to confluence were at G0/G1 stage of cell cycle, respectively, in comparison to 61.6% of cells in normal culture (cycling) medium. Nocodazole treatment for 17 hr in vitro culture could increase the number of cells at G2/M stage of cell cycle from 20.3% (in cycling medium) to 39.7%. In total, one early pregnancy was observed by B ultra-sound scanning in a surrogate transferred with cloned embryos from IV-5.1 cells at M stage (cells were cultured in nocodazole medium). Seven cloned goats, including two that miscarried at a late stage, were derived from the IV-5.1 cell clone cultured in starved medium (G0). Indeed, one surrogate receiving three blastocysts reconstituted from the starved donor cells, gave birth to three live cloned goats, all of which are healthy and doing well. PCR, Southern blot and G418 resistance in vitro of fibroblast cells from cloned goats confirmed that all cloned goats are positive for neo(r) transgene. This study demonstrates that a foreign gene, such as the neo-resistant gene, can be introduced into goat foetal fibroblast cells, and that the resulting transgenic cells are capable of being cloned to produce 100% transgenic animals.     

Effects of cumulus cells on sperm penetration of bovine oocytes in protein-free medium

The present study was conducted to examine the effects of cumulus cells on sperm capacitation, acrosome reaction and penetration of bovine oocytes in vitro in a protein-free medium. In vitro matured oocyte-cumulus complexes (OCCs) and denuded oocytes were co-incubated with spermatozoa in the medium with or without bovine serum albumin (BSA). Higher fertilization rates were obtained in the OCCs (92 and 89%, respectively) than denuded oocytes (57 and 6%, respectively) in the medium with or without BSA (P<0.01). Higher proportion of the denuded oocytes were fertilized in the medium with BSA (57%) than without BSA (6%; P<0.01). These results suggest that the cumulus cells are more effective for increasing fertilization rate than BSA (P<0.05). Both the percentages of capacitated and acrosome-reacted spermatozoa incubated for 4 h with isolated cumulus cells were not significantly different in the medium without cumulus cells in the presence or absence of BSA. The denuded oocytes were inseminated with isolated cumulus cells taken from OCCs matured with or without hormones, follicle stimulating hormone (FSH) and estradiol-17beta (E(2)), and from immature OCCs in a protein-free medium. Presence of the cumulus cells matured with hormones enhanced sperm penetration of denuded oocytes more effectively (81%) than either of the cells matured without hormones (41%) or the immature cells (26%; P<0.01). The conditioned medium of cumulus cells matured with hormones was not effective for sperm penetration of denuded oocytes (2%), while a high proportion (82%) of the oocytes were fertilized when they were inseminated with isolated cumulus cells (P<0.01). In conclusion, the presence of cumulus cells matured with FSH and E(2) was effective for sperm penetration but not for sperm capacitation or acrosome reaction.     

Production of cloned pigs by whole-cell intracytoplasmic microinjection

Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.     

体细胞克隆山羊微卫星DNA分析

用10对山羊微卫星DNA多态性引物对2只体细胞克隆济宁青山羊、青山羊供体细胞、受体奶山羊母羊以及具有亲缘关系的3只对照济宁青山羊进行微卫星DNA分析.结果表明有5对山羊微卫星DNA多态性引物,即SR-CRSP1, SR-CRSP5, SR-CRSP6, SR-CRSP7和SR-CRSP24,扩增产物有明显的多态性.扩增产物经过6%变性聚丙烯酰胺凝胶电泳后银染,结果2只体细胞克隆山羊的微卫星DNA指纹与供体细胞完全相同,而且不同于其受体母亲也不同于其他所有同品种不同个体的对照青山羊.证明体细胞克隆山羊基因组来源于供体细胞.     

Developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles

The objective of this article was to study the developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles. M-199 medium was conditioned for 24 hr with cumulus-denuded oocytes (DOs), oocytectomized complexes (OOXs), or mural granulosa cells (MGCs) from goat follicles of different sizes. Mouse OOXs and eCG were added to culture drops of the conditioned medium and cumulus expansion was scored at 18 hr of culture to assess CEEF production. While mouse OOXs did not expand, goat OOXs underwent full cumulus expansion when cultured in nonconditioned eCG-supplemented M-199 medium. When cultured in nonconditioned medium containing 10% follicular fluid (FF) from goat medium (2-4 mm) and small (0.8-1.5 mm) follicles, 71-83% mouse OOXs expanded; but expansion rates decreased (P < 0.05) at either lower or higher FF concentrations. FF from large (5-6 mm) follicles did not support mouse OOX expansion at any concentrations. While medium conditioned with DOs from follicles of all the three sizes supported expansion of 80-90% mouse OOXs, medium conditioned with mature DOs had no effect. While cumulus cells from follicles of all the three sizes secreted CEEF in the absence of gonadotropins, MGCs from large follicles became gonadotropin dependent for CEEF production. Both FSH and LH stimulated CEEF production by large follicle MGCs, but FSH had a shorter half-life than LH to expand mouse OOXs. Few meiosis-incompetent goat oocytes from small follicles underwent cumulus expansion when cultured in medium conditioned with goat DOs or cocultured with goat COCs from medium follicles. It is concluded that (1) goat cumulus expansion is independent of the oocyte; (2) the limited CEEF activity in FF from large follicles was due mainly to the inability of MGCs in these follicles to secret the factor in absence or short supply of gonadotropins; (3) the cumulus expansion inability of the meiosis incompetent goat oocytes was due to the inability of their cumulus cells to respond to rather than to produce CEEF.