Nucleoli from growing oocytes inhibit the maturation of enucleolated, full-grown oocytes in the pig

In mammals, the nucleolus of full‐grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full‐grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non‐treated or actinomycin D‐treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re‐injection of nucleoli from growing oocytes (23%), but not when nucleoli from full‐grown oocytes were re‐injected into enucleolated, growing oocytes (49%). When enucleolated, full‐grown oocytes were injected with nucleoli from growing or full‐grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full‐grown oocytes injected with nucleoli from full‐grown oocytes matured to metaphase II (56%), whereas injection with growing‐oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing‐oocyte nucleolus is involved in the oocyte's meiotic arrest, and that the full‐grown oocyte nucleolus has lost the ability. Mol. Reprod. Dev. 78:426–435, 2011. © 2011 Wiley‐Liss, Inc.     

Enucleolation of porcine oocytes

Germinal vesicles (GVs) in immature mammalian oocytes contain prominent nucleoli whose role in the process of oocyte maturation is not fully understood. Here we report that the microsurgical removal of nucleoli from immature fully grown porcine oocytes permits germinal vesicle breakdown and chromosome condensation and the enucleolated oocytes mature up to the second metaphase. Interestingly, the enucleolation of growing oocytes which, although unable to mature, resulted in germinal vesicle breakdown and the formation of a cluster of condensed chromatin. These results indicate that the nucleolus in fully grown oocytes is dispensable at least for nuclear maturation. On the other hand, the results obtained in growing oocytes suggest the role of the nucleolus in the cell cycle regulation.     

Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer

Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.     

Development of bovine oocytes reconstructed with a nucleus from growing stage oocytes after fertilization in vitro

The developmental capacity of reconstructed bovine oocytes that contained nuclei from growing stage oocytes, 70-119 microm in diameter, was assessed after fertilization in vitro. Nuclei from growing stage oocytes of adult ovaries were transferred to enucleated, fully grown germinal vesicle (GV) stage oocytes. After culture in vitro, the reconstructed oocytes matured, forming the first polar body and MII plate. To supply the ability to form pronuclei, the resultant MII plate was transferred to enucleated MII oocytes, which were obtained by in vitro culture of cumulus-oocyte complexes. After fertilization in vitro, 11-15% of the reconstructed oocytes developed to morulae and blastocysts. To assess the ability to develop to term, a total of 27 late morulae and blastocysts were transferred to 19 recipient cows. Of the three cows that subsequently became pregnant, one recipient, who received two embryos derived from reconstructed oocytes with a nucleus from oocytes 100 to 109 microm in diameter, continued the pregnancy to Day 278 of gestation. This pregnancy, however, was unexpectedly a triplet pregnancy that included a set of identical twins and resulted in the premature birth of the calves, followed by death from lack of post-parturient treatment. These results show that bovine oocyte genomes are capable of supporting term development before the oocytes grow to their full size, which suggests that growing stage oocytes can be directly used as a source of maternal genomes.     

Development potential of bovine oocytes matured in vitro or in vivo

Bovine oocytes matured in vivo or in vitro were evaluated after sperm-oocyte incubation for frequency of sperm penetration, frequency of male pronuclei formation, and embryonic development. The frequency of sperm penetration was not different for in vitro matured oocytes (216/295, 73%) vs. in vivo matured oocytes (119/176, 70%). However, formation of male pronuclei was reduced (p less than 0.05) for oocytes matured in vitro (149/216, 69%) vs. in vivo (104/119, 88%). Early embryonic development was evaluated 48 h after the onset of sperm-egg incubations. In vitro matured and fertilized oocytes failed to develop to the 2-cell stage (3/88, 3%), whereas oocytes matured in vivo showed normal development (23/56, 40%) to the 2- and 4-cell stage. Development to the blastocyst stage was evaluated after 5 days in ovine oviducts (in vivo). Morulae and blastocysts were obtained only after in vitro fertilization from oocytes that were in vivo-matured (recovered from oviduct, 14/56, 25%; recovered from follicle, 36/80, 45%). Oocytes that were matured in vitro and fertilized in vitro failed to develop to morulae (0/33) in vivo.     

Reprogramming of Round Spermatids by the Germinal Vesicle Cytoplasm in Mice

The birthrate following round spermatid injection (ROSI) remains low in current and evidence suggests that factors in the germinal vesicle (GV) cytoplasm and certain substances in the GV such as the nucleolus might be responsible for genomic reprogramming and embryonic development. However, little is known whether the reprogramming factors in GV oocyte cytoplasm and/or nucleolus in GV are beneficial to the reprogramming of round spermatids and development of ROSI embryos. Here, round spermatids were treated with GV cytolysates and injected this round spermatid alone or co-injected with GV oocyte nucleolus into mature metaphase II oocytes. Subsequent embryonic development was assessed morphologically and by Oct4 expression in blastocysts. There was no significant difference between experimental groups at the zygote to four-cell development stages. Blastocysts derived from oocytes which were injected with cytolysate treated-round spermatid alone or co-injected with nucleoli injection yielded 63.6% and 70.3% high quality embryos, respectively; comparable to blastocysts derived by intracytoplasmic sperm injection (ICSI), but higher than these oocytes which were co-injected with lysis buffer-treated round spermatids and nucleoli or injected with the lysis buffer-treated round spermatids alone. Furthermore, the proportion of live offspring resulting from oocytes which were co-injected with cytolysate treated-round spermatids and nucleoli or injected with cytolysate treated-round spermatids alone was higher than those were injected with lysis buffer treated-round spermaids, but comparable with the ICSI group. Our results demonstrate that factors from the GV cytoplasm improve round spermatid reprogramming, and while injection of the extra nucleolus does not obviously improve reprogramming its potential contribution, although which cannot be definitively excluded. Thus, some reprogramming factors are evidently present in GV oocyte cytoplasm and could significantly facilitate ROSI technology, while the nucleolus in GV seems also having a potential to improve reprogramming of round spermatids.     

Effects of beta-mercaptoethanol on formation of pronuclei and developmental competence of swamp buffalo oocytes

This study was conducted to determine the effect of supplementing maturation medium with beta-mercaptoethanol (betaME) on pronuclei formation and developmental competence of swamp buffalo oocytes. Buffalo oocytes were matured in TCM199 medium either with 10mM betaME or without betaME supplementation for 24h. In Experiment 1, oocytes were fixed and stained for cytological evaluation after in vitro fertilization (IVF). In Experiment 2, presumptive zygotes were cultured and their developmental competency was assessed. It was found that betaME significantly improved the proportion of oocytes that exhibited synchronous pronuclei formation (31.8+/-5.1% versus 17.9+/-3.3%, P<0.05). There were no significant differences between oocytes matured with or without betaME in their capability of developing into blastocyst-stage embryos (3.0+/-1.3% versus 1.8+/-0.9%). However, blastocysts produced from oocytes matured in the presence of betaME appeared to develop faster than those from oocytes matured in the absence of betaME (P<0.05). Cavitation of embryos from oocytes matured in the presence of betaME occurred at 156 hpi, whereas those matured in the absence of betaME occurred at 180 hpi. Although in vitro production of blastocysts did not increase by addition of betaME to maturation medium, quality of blastocysts produced from oocytes matured in the presence of betaME was improved. This study provides information for further investigations on optimizing a system for in vitro production of swamp buffalo embryos.     

BH4 peptide derived from Bcl‐xL and Bax‐inhibitor peptide suppresses apoptotic mitochondrial changes in heat stressed bovine oocytes

Mitochondria play an important role in the integration and transmission of cell death signals mediated by the Bcl‐2 family proteins. Experiments were conducted to determine whether the anti‐apoptotic peptides BH4 domain of Bcl‐xL (TAT‐BH4) and Bax inhibitor peptide (BIP) suppresses heat stress (HS) injury in oocytes by reduction of apoptotic‐like events. Cumulus–oocyte complexes (COCs) were matured at 39°C (control) or 41°C (HS) for 21 hr then placed in maturation medium containing 0 or 100 µM BIP in water and 0 or 1 µM TAT‐BH4 in dimethyl sulfoxide (DMSO), or a combination of both peptides (BIP + BH4). Peptide effects on embryo development, DNA fragmentation, mitochondrial membrane potential (ΔΨm), and mitochondrial DNA (mtDNA) copy number were measured. All groups were fertilized and cultured in vitro at 39°C for 8 days. Compared to control, HS‐treated oocytes induced a decrease in embryo development (P < 0.05), increase in proportion of TUNEL‐positive chromatin in oocytes and blastocysts (P < 0.05), and loss of oocyte ΔΨm (P < 0.001). In the presence of BIP or BIP + BH4, development of HS‐treated oocytes into blastocysts was increased (P < 0.05). Conversely, COCs matured with TAT‐BH4 at 41°C showed reduced embryonic development (P < 0.05). Exposure of HS‐treated to each or both peptides resulted in a reduction of TUNEL frequency in oocytes and blastocysts cells derived from these oocytes (P < 0.05). The loss of ΔΨm in HS‐treated oocytes was not restored by exposure to BIP + BH4 and there was no effect in mtDNA copy number. In conclusion, the present results show that HS‐induced apoptosis in bovine oocytes involves Bax and BH4 domain‐dependent pathways. Mol. Reprod. Dev. 76: 637–646, 2009. © 2008 Wiley‐Liss, Inc.     

Investigation of means to improve rates of fertilization in in vitro matured/in vitro fertilized bovine oocytes

Experiments were conducted with 5,979 oocytes to determine whether detaching some of the cumulus cells from oocytes either before or after maturation would improve the fertilization rate and proportion of oocytes that developed to expanded blastocysts. Oocytes were aspirated from ovaries of slaughtered cows and matured, fertilized and cultured in vitro. Pipetting immature oocytes before maturation to detach some of the cumulus, with all cumulus cells left in the maturation wells, significantly increased fertilization rates, especially of oocytes that initially had a full cumulus investment. In further experiments, pipetting oocytes either before or after maturation to detach most of the cumulus, or treating with hyaluronidase after maturation to disperse the cumulus, significantly increased fertilization rates and proportions of oocytes developing to expanded blastocysts.     

Colcemid‐treatment of heifer oocytes enhances nuclear transfer embryonic development,establishment of pregnancy and development to term

Four experiments were designed to examine the effects of colcemid, a microtubule assembly inhibitor, on the development of bovine nuclear transfer (NT) embryos in vitro and in vivo. Recipient oocytes matured at different times were exposed to colcemid. Approximately 80–93% of the exposed oocytes, with or without the first polar body (PB1), developed obvious membrane projections. In Experiment 1, oocytes matured for either 14–15 or 16–17 hr, treated with colcemid and used as recipient cytoplasm for NT resulted in over 40% blastocyst development. In Experiment 2, oocytes matured for 16–17 hr were treated with either 0.2 or 0.4 µg/ml colcemid for 2–3 or 5–6 hr, respectively. The percentages of blastocyst development (39–42%) were not statistically different among the different colcemid treatment groups, but were both higher (P < 0.05) than the control group (30%). Colcemid concentrations and length of colcemid treatment of oocytes did not affect their ability to support NT embryo development to the blastocyst and hatched blastocyst stages. Results from Experiment 3 indicate that semi‐defined medium increases morula and blastocyst development of NT embryos derived fromcolcemid‐treated oocytes under 5% CO₂ in air atmosphere. In addition, cell numbers of blastocysts in colcemid‐treated groups were numerically higher than the control groups. After embryo transfer, higher (P < 0.05) pregnant rates were obtained from the colcemid‐treated group than the nontreated group. Five of 40 recipients (12.5%) which received embryos from colcemid‐treated oocytes delivered healthy calves, significantly higher than those recipients (3.3%) that received embryos derived from nontreated oocytes. Mol. Reprod. Dev. 76: 620–628, 2009. © 2009 Wiley‐Liss, Inc.     

Fertilization following intracytoplasmic sperm injection of in vivo and in vitro matured oocytes from an Australian marsupial, the tammar wallaby (Macropus eugenii)

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm-oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.e. development to the two-pronuclei stage) of in vivo and in vitro matured oocytes following ICSI and sham injection was assessed at 17-19 h after injection. Fertilization occurred in 48% (45/93) of in vivo matured oocytes that were injected with spermatozoa. Significantly fewer sham-injected oocytes (6/82, P < 0.005) and uninjected control oocytes (5/84, P < 0.005) formed two pronuclei. In a direct comparison, the numbers of in vivo and in vitro matured oocytes that formed two pronuclei after ICSI were 22/28 (78.6%) and 23/40 (57.6%), respectively, which are not significantly different. There was also no significant difference in the nuclear response of in vivo and in vitro matured oocytes to sham injection. The numbers of oocytes forming a single pronucleus after sham injection were 10/24 (41.7%) and 24/37 (64.9) for in vivo and in vitro matured oocytes, respectively. Immature germinal-vesicle-stage oocytes were unable to decondense sperm injected during ICSI or to form pronuclei. These results demonstrate that both in vitro and in vivo matured tammar wallaby oocytes can be fertilized by ICSI. The success of ICSI not only offers the opportunity for fundamental analysis of marsupial fertilization but could, in conjunction with development of appropriate culture conditions and embryo transfer technologies, contribute to increased production of offspring from rare or valuable marsupials.     

Oogenesis of microlecithal oocytes in the viviparous teleost Heterandria formosa

Viviparous teleosts exhibit two patterns of embryonic nutrition: lecithotrophy (when nutrients are derived from yolk that is deposited in the oocyte during oogenesis) and matrotrophy (when nutrients are derived from the maternal blood stream during gestation). Nutrients contained in oocytes of matrotrophic species are not sufficient to support embryonic development until term. The smallest oocytes formed among the viviparous poeciliid fish occur in the least killifish, Heterandria formosa, these having diameters of only 400 μm. Accordingly, H. formosa presents the highest level of matrotrophy among poeciliids. This study provides histological details occurring during development of its microlecithal oocytes. Five stages occur during oogenesis: oogonial proliferation, chromatin nucleolus, primary growth (previtellogenesis), secondary growth (vitellogenesis), and oocyte maturation. H. formosa, as in all viviparous poeciliids, has intrafollicular fertilization and gestation. Therefore, there is no ovulation stage. The full‐grown oocyte of H. formosa contains a large oil globule, which occupies most of the cell volume. The oocyte periphery contains the germinal vesicle, and ooplasm that includes cortical alveoli, small oil droplets and only a few yolk globules. The follicular cell layer is initially composed of a single layer of squamous cells during early previtellogenesis, but these become columnar during early vitellogenesis. They are pseudostratified during late vitellogenesis and reduce their height becoming almost squamous in full‐grown oocytes. The microlecithal oocytes of H. formosa represent an extreme in fish oogenesis typified by scarce yolk deposition, a characteristic directly related to matrotrophy. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Birth of normal calves after intracytoplasmic sperm injection of bovine oocytes: a methodological approach

Intracytoplasmic sperm injection (ICSI) is advantageous when only very few spermatozoa are available for insemination. Bovine spermatozoa were injected individually into matured oocytes using a piezo electric actuator. Spermatozoa were "immobilized", by scoring their tails immediately before injection, or "killed", by repeated freezing and thawing. About 4 h after ICSI, the oocytes with two polar bodies (activated by sperm injection) were selected and treated 5 min with 7% ethanol before further culture. When examined 19-21 h after ICSI, nearly 90% of the oocytes were fertilized normally (two pronuclei and two polar bodies) irrespective of the sperm treatment (immobilization or killing) prior to ICSI, but subsequent preimplantation embryo development was much superior (cleavage 72%: blastocysts 20%) after ICSI with immobilized spermatozoa than by using killed spermatozoa (cleavage 28%; blastocysts 1%). Ethanol activation of bovine oocytes with two polar bodies 4 h after ICSI improved the cleavage (33% versus 72%) and blastocyst (12% versus 20%) rates markedly (P < 0.05). Five normal calves were born after transplantation of ten blastocysts to ten surrogate cows. These results show that piezo-ICSI using immobilized spermatozoa, combined with ethanol treatment of sperm-injected oocytes, is an effective method to produce bovine offspring.     

Pronuclear synchronization and nuclear morphology of mature and in vitro matured oocytes in the rat: An ultrastructural study

Summary The objective of this study was to evaluate synchronous and asynchronous pronucleus (PN) formation and the related patterns of juxtapositional nucleolus (n) formation in immature (prophase I [PI] and metaphase I [MI]) and mature (metaphase II [MII]) oocytes after fertilization, both ultrastructurally and at the level of light microscope. A single dose of 15 IU gonadotrophin was injected subcutaneously to twenty four 26-wk-old, female Wistar rats to induce ovulation. Human chorionic gonadotrophin (4 IU) was administered 40 h later, and after 4–6 h the ovaries were dissected, and the oocytes were aspirated. A total of 214 rat oocytes were classified according to a maturation index as follows: group I, 80 PI oocytes; group II, 50 MI oocytes; and group III, 84 MII oocytes. Immature oocytes were in vitro matured for 18–36 h. Spermatozoa were acquired by microepididymal sperm aspiration and processed using swim-up technique. Intracytoplasmic sperm injection was performed on mature oocytes after 2 h of incubation and on in vitro matured (IVM) oocytes 4 h after maturation. Pronuclear synchronization [both pronucleases (PNs) centrally located, equal sized, with equal numbers and sizes of juxtapositional nucleoli (Nn)] was observed in fertilized oocytes. Asynchronous PN formation (diversity between male and female PNs, related to dimensions, localization, and the number of Nn) in groups I, II, and III was found in 75, 86, and 47% of preembryos, respectively. There was a significant difference of synchronous pronuclear formation between mature and IVM oocytes (P<0.05). In IVM oocytes, asynchronous PN formation is high, and juxtapositional pronucleolar patterns are observed to be low by transmission electron microscope (TEM).     

Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection

Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2 g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to 9.1) or oocytes recovered (4.8 to 5.0) per mare per aspiration session. Of 314 oocytes recovered, 180 (57%) matured in culture. Thirty-six mature oocytes were subjected to ICSI; 33% formed blastocysts (63% per mare per aspiration session). In Experiment 2, the preovulatory follicle was aspirated every 14 d for three to four cycles. Prostaglandin F_2α was given on Days 6 and 7 after aspiration. A follicle ≥25 mm in diameter was present on Day 13, the day of deslorelin administration, in 23 of 24 cycles, and ovulatory response (granulosa expansion) was seen in 24 of 25 follicles aspirated. Blastocyst development after ICSI was 41% per injected oocyte, or an estimated 33% per mare per aspiration session. We concluded that both aspiration of immature follicles and aspiration of the preovulatory follicle can be performed effectively every 14 d without monitoring ovarian follicular growth. As performed in these separate experiments, aspiration of immature follicles provided more blastocysts per aspiration session.     

Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.     

Protein phosphorylation is essential for formation of male pronucleus in bovine oocytes

This study examined the association between the morphological and protein phosphorylation events following sperm penetration of in vitro matured and in vitro fertilized bovine oocytes. Oocytes were labeled with [³²P]‐orthophosphate at 3 hr intervals from 3 to 18 hr of following insemination. The phosphorylation of protein complexes of 23 kDa and 18 kDa specifically increased with the formation of male and female pronuclei. In addition, oocytes were treated with 6‐Dimethylaminopurine (6‐DMAP) or Okadaic acid (OA) at 0, 3, 6, and 9 hr respectively following insemination. Although the formation of female pronucleus was not affected by 6‐DMAP, the male pronuclear formation was completely inhibited by the presence of 6‐DMAP at 0 and 3 hr of post insemination. The formation of both pronuclei was inhibited by the presence of OA at any time following insemination. These results suggest that the male pronuclear formation is associated with protein phosphorylation and that the formation of the male and the female pronuclei may involve different factors in bovine zygotes since they respond differently to the kinase modulations. Mol. Reprod. Dev. 52:43–49, 1999. © 1999 Wiley‐Liss, Inc.     

Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes.

The introduction of mammalian artificial chromosomes (ACs) into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. Bovine oocytes aspirated from 2-5 mm size follicles were matured in vitro for 22 hr. Mature oocytes were fertilized in vitro with frozen- thawed bull spermatozoa. Artificial chromosome carrying either beta-galactosidase (Lac-Z) gene or green fluorescence protein (GFP) gene were isolated by flow cytometry. A single chromosome was microinjected into one of the two pronuclei of bovine zygotes. Sham injected zygotes served as controls. Injected zygotes were cultured in G 1.2 medium for 7 days. Hatched blastocysts were cultured on blocked STO cell feeder layer for attachment and outgrowth of ICM and trophectoderm cells. The results showed a high zygote survival rate following LacZ-ACs microinjection (74%). However, the blastocyst development rate after 7 days of culture was significantly lower than that of sham injected zygotes (7.5 vs. 22%). Embryonic cells positive for Lac-Z gene were detected by PCR in three of nine outgrowth colonies. In addition, GFP gene expression was observed in 15 out of 85 (18%) embryos at the arrested 2-cell stage to blastocyst stage. Six blastocysts successfully outgrew, three outgrowths were GFP positive for up to 3 weeks in culture. We conclude that the methodology for artificial chromosome delivery into bovine zygotes could lead to viable blastocyst development, and reporter gene expression could be sustained during pre-implantation development.     

Specific activation requirements of in vitro-matured sheep oocytes following vitrification-warming

Oocyte vitrification and assisted oocyte activation have increasingly important roles in assisted reproductive technology. Yet, an important area of concern with matured oocyte cryobiology is that elements of oocytes intimately involved in metaphase‐II arrest may be modified by cryopreservation. By comparing different cellular characteristics of unvitrified, vitrified‐warmed, and unvitrified‐activated oocytes, the present study investigated how vitrification‐warming process may affect developmental competence of in vitro‐matured sheep oocytes following parthenogenetic activation. Structural, ultrastructural, and molecular analyses indicated that the characteristics of vitrified‐warmed oocytes vastly differed from fresh oocytes, instead resembling unvitrified‐activated oocytes. For unvitrified oocytes, the highest blastocyst yield (41.8 ± 0.6%) was achieved using the maximum ionomycin concentration (5 µM), and importantly, the duration of ionomycin treatment was not of utmost importance at this concentration. In contrast, the maximum blastocyst yield of vitrified‐warmed oocytes (28.4 ± 1.4%) was achieved with a minimal duration of ionomycin treatment (1 min), and further extending the duration dramatically reduced developmental potential of vitrified‐warmed oocytes. These results suggested that vitrified‐warmed oocytes may need an activation protocol different from unvitrified oocytes. In this respect, unvitrified oocytes were more sensitive to the concentration rather than the duration of ionomycin treatment when compared with vitrified oocytes, which were sensitive to the treatment duration. These results may provide a platform to improve the potential applications of vitrified oocytes in medicine and agriculture. Mol. Reprod. Dev. 79:434–444, 2012. © Wiley Periodicals, Inc.