Aspects réglementaires et organisationnels de l’activité d’autoconservation. Le point de vue du juriste

At the present time, legal texts in application of bioethics laws only briefly mention cryopreservation. The conditions of cryopreservation differ according to the type of tissue stored: while cryopreservation of oocytes and ovarian tissue corresponds to the field of biomedical research, semen cryopreservation corresponds to medically assisted procreation. Cryopreservation activity is more clearly defined in the draft revision of the bioethics law. Concerning the cryoconservation of ovarian tissue and oocytes, these difficulties result in particular in this activity’s mixed nature. It is located indeed, halfway between research and care. These two spheres of medical activity are subjected to their own distinct and exclusive, and their application is conditioned by the qualification of the implemented act. However, this qualification is dubious here, because of, in particular, the impossibility of determining which acts of sampling, cryoconservation and use might be concerned with a research protocol. Concerning the cryoconservation of sperm, the texts, first of all, seem to assimilate the activity of cryoconservation within an activity of assisted medical procreation. However, such assimilation would be equivalent to the impossibility of its implementation, because of the difficulty of respecting all of the legal conditions of assisted medical procreation. However, another more favourable interpretation of the provisions seems to be possible. Taking into consideration these uncertainties, contradictions and difficulties, the legislator intervened and devoted, by the widening of the indications of assisted medical procreation, the activity of cryoconservation in the project of revision of the laws of bioethics. He however did not solve all the difficulties. These cryoconserved elements can be restored only to the depositor. Indeed, the texts make obstacle to a delivery for a third party of the cryoconserved sperm, whatever the moment. They can also be used. This use can consist of assisted medical procreation, which is the first finality of cryoconservation — but it could only be implemented in respect of the whole of the legal conditions. It seems that the cryoconserved elements could also be used within the framework of research, whatever its nature (biomedical or not) and the moment of its implementation (while the person is alive or after his death). Its implementation should however be subjected to prior agreement and expressed while the person, whose elements were preserved, is alive. Such a use is expressly made possible in the project of revision of the laws of bioethics. Following a partial use or a lack of such a use, the destruction of the cryoconserved elements can be considered, as well as the continuation of the cryoconservation. However, these hypotheses raise difficulties that have not yet been resolved in the draft revision of the bioethics law.     

Factors influencing chromosome condensation and development of cloned rat embryos

Factors influencing premature chromosome condensation (PCC) in transferred rat nuclei have been examined. Chromosome condensation of rat cumulus cell nuclei did not occur when the cell nuclei were injected into enucleated rat oocytes. By contrast, chromosome condensation did occur after transfer to enucleated mouse oocytes or intact rat oocytes. In the first serial NT experiment, rat somatic cell nuclei were injected into enucleated mouse oocytes, and the reconstructed oocytes were activated by strontium chloride. From these reconstructed embryos, karyoplasts containing pronucleus-like vesicles were transferred into pronuclear zygote-derived cytoplasts by a DC pulse. Transfer of a total of 340 serial NT zygotes into recipient females, including 206 two-cell embryos, resulted in only seven implantation sites. In the second serial NT experiment, rat somatic cell nuclei were injected into intact rat oocytes; the recipient metaphase-plate was then aspirated under UV light from the NT oocytes in which PCC of injected nuclei was observed. After activation of the NT oocytes, karyoplasts were introduced into zygote-derived cytoplasts. Transfer of a total of 115 serial NT zygotes, including 37 two-cell embryos, resulted in four implantation sites but no live offspring. These results establish a mean of inducing chromosome condensation in rat oocytes and demonstrate that reconstructed rat zygotes can be prepared by serial NT procedures. Developmental competence of these embryos remains to be clarified.     

In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach

Development of new technology related to in vitro embryo production has allowed for the commercial use of this method of reproduction. In the present work, we evaluate the efficiency of this technology compared with conventional embryo production based on results obtained with a standard procedure, including the sexing of embryos. The donor animals were mature nonlactating dairy cows (n = 92) kept under a constant environment and feeding program in an ET center. Ultrasound guided transvaginal ovum pick-up following 48 h pre-treatment with FSH has been used for the IVF-IVC protocol. A total of 437 oocyte recovery sessions performed on 92 cows yielded 4145 oocytes, which were used in an IVF-IVC protocol. Using the conventional approach, 156 embryo collections on 49 cows yielded 1652 ova and embryos. All Quality 1 and 2 embryos were sexed by a PCR procedure, and embryos of the desired sex were transferred to synchronized recipients located at the center. The results obtained in the IVF protocol showed that 4 oocyte collections per cow performed within 60 d, yielded 38 oocytes, which resulted in 18.8 viable embryos, of which 7.05 were female. After transfer of the female embryos, an average of 3.8 recipients were pregnant at 60 d. One embryo collection under the conventional approach yielded an average of 1.2 female pregnancies, which was confirmed during the same 60-d time period. These results indicate that IVF procedures can effectively replace conventional embryo production methods when a predetermined number of pregnancies of known sex are needed within a short period of time.     

Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation

Conditions for the electroporation of mouse oocytes and preimplantation embryos have been optimised by following the incorporation of rhodamine labeled dextran. This procedure includes a step to weaken but not remove the zona pellucida that helps achieve good survival. This approach has been applied to introduce double-stranded RNA for c-mos into oocytes and green fluorescent protein (GFP) into transgenic GFP-expressing embryos at the 1- and 4-cell stages. In both cases we were able to observe sequence-specific interference with the expression of the target gene--a failure of oocytes to arrest at metaphase II and a loss in the green fluorescence of embryos by the morula or blastocyst stages. These effects could be observed in multiple oocytes or embryos allowed to develop together following electroporation.     

Production of live piglets following cryopreservation of embryos derived from in vitro-matured oocytes

We have successfully produced healthy piglets following cryopreservation of embryos derived from oocytes matured and fertilized in vitro. The appropriate timing of cryopreservation pretreatment (removal of cytoplasmic lipid droplets [delipation] and vitrification) was initially determined using parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. Viable embryos were obtained at the highest rate when embryos were delipated at the four- to eight-cell stages (Day 2 of embryo culture) and were vitrified approximately 15 h later (Day 3) by means of the minimum volume cooling method. After cryopreservation of embryos derived from oocytes matured and fertilized in vitro under the most appropriate conditions, 401 embryos were transferred to five recipient gilts, and the recipients all became pregnant. At autopsy of one of the recipients, which had received 47 embryos, eight fetuses (17.0%) were found. Three recipients each gave birth to two to four piglets (1.4%-6.0%). These results demonstrate that normal offspring can be produced from vitrified porcine embryos derived from IVM oocytes by a strategic combination of delipation and vitrification at the early cleavage stages. This approach has great potential in the reproduction of micromanipulated porcine embryos, such as cloned and sperm-injected embryos, produced from IVM oocytes.     

Cryopreservation of goat oocytes and in vivo derived 2- to 4-cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods

This study evaluated the efficiency and toxicity of two cryopreservation methods, solid-surface vitrification (SSV) and cryoloop vitrification (CLV), on in vitro matured oocytes and in vivo derived early stage goat embryos. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4% trehalose. Microdrops containing the oocytes were cryopreserved by dropping them on a cold metal surface that was partially immersed in liquid nitrogen. In the cryoloop method, oocytes were transferred onto a film of the CLV solution (20% DMSO, 20% EG, 10mg/ml Ficoll and 0.65 M sucrose) suspended in the cryoloop. The cryoloop was then plunged into the liquid nitrogen. In vivo derived embryos were vitrified using the same procedures. The SSV microdrops were warmed in a solution of 0.3M trehalose and those vitrified with CLV were warmed with incubation in 0.25 and 0.125 M sucrose. Oocytes and embryos vitrified by the SSV method had a significantly lower survival rate than the control (60 and 39% versus 100%, respectively; P<0.05), while the survival rate of CLV oocytes and embryos (89 and 88%, respectively) did not differ from controls. Cleavage and blastocyst rates of the surviving vitrified oocytes (parthenogenetically activated) and embryos (cultured for 9 days) were not significantly different (P>0.05) from the control nor did they differ between vitrification methods. Embryos vitrified with the CLV method gave rise to blastocysts (2/15). Our data demonstrated that the two vitrification methods employed resulted in acceptable levels of survival and cleavage of goat oocytes and embryos.     

The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33–34%) compared to that of 2-cell embryos (78–79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7–15% for oocytes and 79–80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.     

Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro

The mechanisms underlying the visual assessment and selection of immature oocytes resulting in optimum embryonic development following in vitro maturation, fertilization and culture (in vitro maturation (IVM)/in vitro fertilization (IVF)/in vitro embryo culture (IVC)) are unknown. Also, the reasons for the more frequent occurrence of cytoplasmic fragmentation in in vitro produced bovine embryos, resulting in poor survival following cryopreservation and decreased pregnancy rates following embryo transfer are not clear. The objectives of this study are: (1) to investigate whether differences in the quality of immature oocytes and embryo fragmentation are associated with apoptosis; and (2) to study the pattern of Bcl-2 and Bax expression in oocytes and embryos to help elucidate their potential roles in the regulation of apoptosis during development. Bovine oocytes were obtained from slaughterhouse ovaries and divided into four grades (grades I–IV) based on their morphology. Oocytes of different grades were cultured in serum-free medium for 48 h. Embryos were produced only from grade I oocytes (highest quality) via IVM, IVF and IVC procedures. The morphological analysis of apoptosis in oocytes and embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expression of Bcl-2 and Bax in oocytes and embryos of different qualities and stages was determined using western blotting. The results showed that the number of morphologically abnormal oocytes with shrinkage and/or fragmentation of the ooplasm, which are typical features of apoptosis, was significantly higher in grade IV oocytes (denuded oocytes, the lowest quality) than in grade I oocytes after 48 h in vitro culture (P<0.05). DNA fragmentation, a hallmark of the biochemical changes seen in apoptotic cell death, was observed in morphologically fragmented oocytes and embryos. The expression of Bcl-2 was high in good quality oocytes and embryos, low in fragmented embryos, and hardly detectable in denuded oocytes. In contrast, the expression of Bax was found in all types of oocytes and embryos with the highest expression in the denuded oocytes. This implies that the ratio of Bcl-2 to Bax may be used to gauge the tendency of oocytes and embryos towards either survival or apoptosis. Overall, our results show that apoptosis appears to be an underlying mechanism of bovine oocyte degeneration and embryo fragmentation. Interactions between the Bcl-2 family of proteins may play a critical role in pre-implantation embryo development. These findings could have important implications for improving IVF and related techniques.     

Ovum pick up, in vitro embryo production, and pregnancy rates from a large-scale commercial program using Nelore cattle (Bos indicus) donors

The objective was to clarify in vitro production of bovine embryos in Brazil. Data from 656 ovum pick-up/in vitro production (OPU/IVP) procedures, performed on 317 Nelore (Bos indicus) donors, without hormone stimulation or control of ovarian follicular waves, were analysed. Donors were subjected to OPU from one to nine times (no specific schedule), with < 15 d between consecutive procedures. There were 20,848 oocytes, of which 15,747 (75.53%) were considered viable, 5,446 embryos were obtained, 5,398 embryos were immediately transferred, resulting in 1,974 pregnancies (36.57%) at Day 30 and 1,788 (33.12%) pregnancies at Day 60. The average number of total and viable oocytes produced per OPU session was (mean ± SEM) 30.84 ± 0.88 and 23.35 ± 0.7 (average of 8.1 ± 0.3 embryos and 3.0 ± 0.1 pregnancies per OPU-IVP procedure). Since oocyte production varied widely among donor, they were designated as very high, high, intermediate, and low, with 58.94 ± 2.04, 32.61 ± 0.50, 22.13 ± 0.50, and 10.26 ± 0.57 oocytes, respectively, produced by 78, 80, 79, and 80 donors. The number of viable oocytes recovered ranged from 0 to 128; since donors with numerous viable oocytes produced many viable embryos and pregnancies, oocyte production was useful for donor selection. However, there was no significant effect of the number of OPU sessions per donor on mean numbers of oocytes produced. In conclusion, we confirmed field reports of high oocyte production by some Nelore donors and demonstrated individual variation in oocyte yield, which was associated with embryo production and pregnancy rates.     

Pregnancy rate after the transfer of sheep embryos originated from randomly chosen oocytes matured and fertilized in vitro

Randomly chosen sheep oocytes isolated from 2- to 5-mm follicles of hormonally nonstimulated slaughtered females were matured and fertilized in vitro. Using heparin for the induction of ram sperm capacitation, a fertilization rate close to 80% was recorded. After the transfer of 29 embryos cultured to the 2- to 4-cell stage to 4 recipients, each delivered 1 lamb. In another experiment, 34 2-cell embryos stage were transferred (1 to each oviduct) to 17 synchronized recipients; 8 pregnancies were established and each of 5 recipients delivered a single lamb. The remaining 3 recipients aborted at the third month of gestation. These results show that sheep embryos can be produced in vitro from randomly chosen oocytes and by using relatively simple procedures. However, the viability of the embryos was low, with approximately only 15% developing to term after transfer at the 2-cell stage.     

Nuclear transfer in cattle: Effect of nuclear donor cells,cytoplast age,co-culture,and embryo transfer

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20–22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8–10 hr or 18–20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18–20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen or IVF ambryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology. © 1993 Wiley-Liss, Inc.     

Cloning of bovine embryos by multiple nuclear transfer

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.     

Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques

In this study the solubility to alpha-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time +/- SE = 24.1+/-0.9 min, n = 28; and class II, 46.7+/-2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3+/-3.4 min, n = 18; ICSI: 48.9+/-2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0+/-2.7 min, n = 13; unfertilised ICSI: 43.0+/-2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5+/-2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.     

Successful in vitro fertilization of in vivo matured oocytes aspirated laparoscopically from red deer hinds (Cervus elaphus)

Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of oocytes derived from abattoir material. For application of IVF technology to captive endangered species, however, noninvasive techniques are required which allow repeated collection of oocytes from live females. The aim of this study was to develop a method for embryo production from mature oocytes collected laparoscopically from red deer hinds. Follicular development was synchronized in red deer hinds by the insertion of intravaginal progesterone-releasing devices for 10 d, and ovarian stimulation was induced with 1000 IU, i.m. PMSG 48 h before progesterone device removal. Oocytes were harvested by laparoscopy under xylazine/ketamine sedation 24 h after progesterone device removal and then co-incubated with frozen-thawed red deer spermatozoa for 24 h. In Experiment 1, oocytes and embryos were fixed and stained at different developmental timepoints. Their external morphological changes (cumulus expansion, extrusion of the second polar body and cytokinesis) paralleled their nuclear developmental changes (formation of the 2nd metaphase spindle of meiosis, pronuclear formation and nuclear division, respectively). In Experiment 2, embryos were maintained in vitro until they ceased to undergo cell division. A total of 39 aspiration procedures was carried out on 14 red deer hinds. Forty-four cumulus-oocyte complexes (COC) were aspirated from 95 large Graafian follicles; of these, 27 were classed as mature/nondegenerated on the basis of cumulus/cytoplasmic morphology. Seventeen oocytes cleaved following in vitro fertilization, yielding six 2-cell embryos, six 4-cell embryos, four 8-cell embryos and one 16-cell embryo. The results indicate that laparoscopic aspiration of mature oocytes from hormone-treated females offers a valuable source of genetic material for assisted deer breeding programs.     

Ovulation and early embryogenesis in swine

Thirty gilts were used to examine if the sequence in which oocytes were released at ovulation contributed to differences in embryonic development and uterine secretions by Day 12 (Day 0 = onset of estrus). Oocytes of follicles destined to ovulate last were recovered 42 h after injecting proestrous gilts with hCG, incubated with a fluorescent stain, and returned to the donor's oviduct. These later-maturing oocytes subsequently became the lesser-developed (p less than 0.01) embryos on Day 4. In a second experiment, lesser- vs. more-developed Day 4 embryos from additional gilts were transferred to ligated uterine horns of nonpregnant gilts. Subsequently, the lesser-developed Day 4 embryos became the smaller (p less than 0.01) blastocysts within a litter on Day 12. Uterine flushings associated with lesser-developed embryos on Day 12 contained less estradiol (p less than 0.01), less total protein (p less than 0.10), and less acid phosphatase activity (p less than 0.05), but total content of calcium was not different compared to flushings that contained more-developed embryos. Analysis of uterine flushings with two-dimensional PAGE procedures indicated advanced uteroferrin-associated glycoprotein secretion from the horn that contained more-developed embryos. Results of these experiments suggested that oocytes of later-ovulating follicles were progenitors of smaller embryos, which probably stimulated uterine secretion later than more advanced littermates on Day 12.     

Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes

To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.     

Chromatin and microtubule organization in the first cell cycle in rabbit parthenotes and nuclear transplant embryos

Artificial activation and nuclear transfer in rabbit oocytes have been used in past years in an attempt to develop viable techniques for cloning in cattle. The procedures established in our laboratory, using the rabbit as a model, consistently lead to high rates of development to the blastocyst stage. However, the rate of embryos developing to term is considerably lower. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of electrical pulse-activated oocytes and of nuclear transfer embryos. Our goal was to investigate the responses of the cell to the different stimuli applied and to establish the sequence of events leading to first cleavage in the absence of normal fertilization. Our results show that, in both electrically activated oocytes and nuclear transfer embryos, although the initial development patterns are rather unusual, embryos become synchronized at the time of the formation of a pronuclear-like structure, and then organize metaphase spindles and cleave. These spindles consistently present small defects, suggesting that problems in the formation of the mitotic apparatus during the first cell cycle may have a long-term effect leading to embryo mortality. © 1993 Wiley-Liss, Inc.     

Ovum pick-up in sheep: efficiency of in vitro embryo production, vitrification and birth of offspring

The production of offspring involving available technologies like ovum pick-up, in vitro embryo production and cryopreservation has not been fully described in the sheep. We tested the overall efficiency of these procedures on 20 Sarda dairy ewes that were twice stimulated for recovery of follicular oocytes. In total, 415 oocytes were aspirated from 522 follicles (11.5 oocytes/ewe), and 328 of them (9.1 oocytes/ewe) were selected for in vitro embryo production procedure. Development into blastocysts occurred in 98 embryos (2.7 blastocysts/ewe), of which 64 were vitrified and 34 were transferred, in pairs, directly to recipients. The pregnancy rate, diagnosed at 80 d for fresh and vitrified embryos, did not differ significantly (47.1 vs 42.8%, respectively), but there were significant differences in lambing rates between the 2 groups (41.2 vs 23.8%, respectively). Overall, 24 lambs were born; all weighed within the range for the breed, but head deformities were observed in 2 cases. The results of this study show that with application of the above techniques, it is possible to obtain repeatedly embryos and viable offspring.     

Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development

Actin is one of the major proteins in mammalian oocytes. Most developmental events are dependent on the normal distribution of filamentous (F-) actin. Polymerization of nonfilamentous (G-) actin into F-actin is important for both meiosis and mitosis. This study examined G- and F-actin distribution in pig oocytes and embryos by immunocytochemical staining and confocal microscopy. Actin protein was quantified by electrophoresis and immunoblotting. G-Actin was distributed in the whole cytoplasm of oocytes and embryos irrespective of their stages. F-Actin was distributed at the cortex of oocytes and embryos at all stages, at the joint of blastomeres in the embryos, in the cytoplasm around the germinal vesicle (GV), and in the perinuclear area of 2- to 4-cell-stage embryos. No differences in the amount of actin protein were found among oocytes and embryos. Oocytes cultured in medium with cytochalasin D (CD), an inhibitor of microfilament polymerization, underwent GV breakdown and reached metaphase I but did not proceed to metaphase II. Two- to 4-cell-stage embryos cultured in medium with CD did not develop to blastocysts. When GV-stage oocytes or 2- to 4-cell-stage embryos treated with CD for 6 h were re-cultured in media without CD, oocytes or embryos re-assembled actin filaments and underwent a meiotic maturation or blastocyst formation similar to that of controls. These results indicate that it is the polymerization of G-actin into F-actin, not actin protein synthesis, that is important for both meiosis and mitosis in pig oocytes and embryos.     

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。