Mitochondrial aggregation patterns and activity in in vitro cultured bovine oocytes recovered from early antral ovarian follicles.

The low developmental competence seen in in vitro cultured oocytes collected from early antral follicles may be related to their mitochondrial status. The aim of this study was to examine the chromatin configuration, pattern of mitochondrial aggregation and mitochondrial activity of non-cultured and in vitro-cultured bovine oocytes originating from early antral ovarian follicles. Cumulus-oocyte complexes with adjacent granulosa cells (COCGs) were recovered from early antral follicles of 0.4 to 0.8 mm diameter. Control (Day 0) oocytes were recovered from freshly collected COCGs and fixed and stained. Selected COCGs were placed in growth culture for 7 days (Day 7) or 14 days (Day 14). Following growth culture, COCs with normal appearance were placed in maturation medium (IVM) for 24 h and then fixed and stained with MitoTracker CMTM Ros Orange and Hoechst 33258. The percentage of oocytes with an immature meiotic configuration after growth culture decreased with the time of growth culture, being 96.7; 72.5 and 35.4% respectively for Day 0, Day 7 and Day 14 of culture; the remaining oocytes were degenerating or resuming meiosis. After subsequent IVM the highest proportion of oocytes in diakinesis or metaphase I was found in the D7+IVM group (59.4%). When growth culture was prolonged to day 14 and IVM, the number of degenerated oocytes increased dramatically after IVM. The mitochondrial distribution in the oocytes changed from homogeneous to heterogeneous as growth culture time increased. The respiratory activity as measured by fluorescence intensity increased over the time of growth culture, and was highest in oocytes that had resumed GVBD. In conclusion, for oocytes in isolated COCGs from early antral follicles, culture conditions longer than 7 days should be more adapted for a slow nuclear maturation accompanied by a decreased energy metabolism to prevent chromatin pycnosis.

Interactions among activity of glucose-6-phosphate dehydrogenase in immature oocytes, expression of apoptosis-related genes Bcl-2 and Bax, and developmental competence following IVP in cattle.

This study was conducted to understand whether the level of G6PDH activity assessed in immature bovine oocytes by means of BCB test was correlated with the level of expression of apoptosis-related genes such as Bcl-2 and Bax in immature and mature oocytes. This information should support previous findings suggesting that G6PDH activity is a useful marker for determining oocyte quality, thereby increasing the validity of BCB test in oocyte selection. Up to now, there are no data estimating the relation between G6PDH activity and the expression of apoptosis-related genes in oocytes. The expression of Bcl-2 and Bax genes was estimated on the mRNA and protein levels, respectively using real-time PCR and Western-blotting. To evaluate developmental competence of these oocytes, cumulus-oocyte complexes classified as BCB+ (low activity of G6PDH), BCB- (high activity of G6PDH) and Control were used for in vitro embryo production. In immature oocytes, the Bax transcript level in BCB- oocytes was significantly higher (P<0.001) in comparison to Control. In mature oocytes, the Bcl-2 transcript level was significantly lower in BCB+ oocytes (P<0.01) and in BCB- oocytes (P<0.05) in comparison to Control. However, no relation was found between the activity of G6PDH and the expression of the Bcl-2 or Bax proteins, both in immature and mature oocytes. Our results on the transcript level seem to indicate that oocytes subjected to BCB staining show tendency towards apoptosis. However, results obtained at the protein level did not confirm this conclusion. The usefulness of the BCB test as the indirect marker of apoptosis seems to be questionable. The lack of significant differences in the blastocyst rates developed from BCB+ and Control oocytes decreases the validity of BCB test in IVP technology.

Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats.

The aim of the study was to determine whether the selection of immature oocytes by a combination of cumulus-oocyte-complexes (COCs) morphology and staining with brilliant cresyl blue (BCB) would be helpful in selecting developmentally competent oocytes, and thereby increase the efficiency of blastocyst production from ovarian oocytes of FSH-primed, adult goats. In a second experiment the interaction between oocyte quality and semen donor was assessed. In a third experiment the usefulness of Vero cells for co-culture with goat embryos was investigated. In the pool of morphologically normal COCs recovered from ovaries following slicing (21.9+/-11.0), the mean rate of COCs classified as BCB+ was 85.6%, and the BCB- was approximately 11%. Oocytes classified as grade 1 and BCB+ exhibited the highest developmental competence (P<0.001) after in vitro maturation and fertilization compared with oocytes of grade 1 BCB- and grade 2 BCB+ or BCB-. There were no significant differences in developmental competence in grade 2 oocytes, regardless of BCB coloration. No significant differences in embryo cleavage and blastocyst formation rates among three bucks were observed when morphologically normal, BCB+ oocytes were used. For all tested bucks, differences in embryo production efficiency were related only to the oocyte quality. Similar blastocyst rates were developed from embryos co-cultured with goat oviduct epithelial cells (34.3%) and with Vero cells (33.3%). These results show that the most important criterion for selection of COCs before maturation is the visual assessment of morphological features. Staining with BCB of COCs recovered from adult goats does not enhance efficiency of selection of developmentally competent oocytes for IVF.

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning.

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles.

The aim of the present study was to examine the growth and survival in culture, and the subsequent meiotic competence, of bovine oocytes recovered from early antral ovarian follicles. Follicles isolated by microdissection of the ovarian slices were sorted into two size groups: (I) 0.2-0.5 mm diameter; and (II) 0.4-0.7 mm diameter. Group I follicles were cultured intact while in Group II, cumulus-oocyte complexes with pieces of parietal granulosa were dissected from the follicles and cultured. Follicles or cumulus-oocyte complexes with parietal granulose were embedded in collagen gel and cultured in TCM 199 supplemented with 3% BSA and 4 mM hypoxanthine for 14 days (Group I) or 7-10 days (Group II). After this, cumulus-oocyte complexes were recovered from the gel. Oocytes that had lost the majority of the cumulus were fixed immediately after recovery. Cumulus-oocyte complexes showing normal morphology were either fixed immediately or were subjected to IVM for an additional 24h, and then were fixed. At the end of the growth culture, 57.6% of the compact COCs in Group I follicles were preserved in the GV configuration, 16.7% had resumed meiosis, and 25.8% were degenerated or did not show detectable chromatin. After IVM, the proportion of oocytes resuming meiosis increased significantly (from 16.7% versus 42.7%; P < 0.05), and 9.1% of all oocytes had reached TI or MII. The isolated cumulus-oocyte complexes in Group II began creating follicle-like structures following 24 h of growth culture (7.1%). The proportion of these structures reached 50.8% on days 2-3, and then gradually decreased due to degeneration. On day 10 only 5.8% of cumulus-oocyte complexes were classified as intact. Of the cumulus intact oocytes recovered from the newly created follicle-like structures at 7-10 days, 54.7% were in the germinal vesicle stage, 31.0% underwent germinal vesicle breakdown, 14.3% were degenerated or the chromatin configuration was not detectable. After 24 h of IVM, 67.6% of oocytes had resumed meiosis, and 21.6% of all oocytes had reached TI and MII. These results show that isolated early follicles and cumulus-oocyte complexes from intact early antral follicles can grow in culture and can develop meiotic competence.