Production and quality of bovine oocytes and embryos.

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.

Bovine embryo technologies.

Embryo technologies are a combination of assisted reproduction, cellular and molecular biology and genomic techniques. Their classical use in animal breeding has been to increase the number of superior genotypes but with advancement in biotechnology and genomics they have become a tool for transgenesis and genotyping. Multiple ovulation and embryo transfer (MOET) has been well established for many years and still accounts for the majority of the embryos produced worldwide. However, no progress has been made in the last 20 years to increase the number of transferable embryos and to reduce the side effects on the reproductive performance of the donors. In vitro embryo production (IVP) is a newer and more flexible approach, although it is technically more demanding and requires specific laboratory expertise and equipment that are most important for the quality of the embryos produced. Somatic cell cloning is a rapidly developing area and a very valuable technique to copy superior genotypes and to produce or copy transgenic animals. More knowledge in oocyte and embryo biology is expected to shed new light on the early developmental events, including epigenetic changes and their long lasting effect on the newborn.Embryo technologies are here to stay and their use will increase as advances in the understanding of the mechanisms governing basic biological processes are made.

Bovine oocytes treated prior to in vitro maturation with a combination of butyrolactone I and roscovitine at low doses maintain a normal developmental capacity.

Butyrolactone I (BL-I) and Roscovitine (ROS), two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, were used to block germinal vesicle breakdown (GVBD) of cattle oocytes. A concentration 6.25 microM BL-I and 12.5 microM ROS blocked over 93.3 +/- 2.5% of oocytes in germinal vesicle (GV) stage during a 24-hr culture period. Following a second 24-hr culture step in maturation medium (IVM) almost all (91.5 +/- 3.0%) inhibited oocytes resumed meiosis and reached the metaphase II (MII) stage. The MII kinetics was different for inhibited and control oocytes. Fifty percent MII was reached at 13-14 hr in BL-I + ROS treated oocytes, compared to 18 hr in control oocytes. Therefore, control oocytes were fertilised (IVF) after 22 hr IVM and inhibited oocytes after 16 or 22 hr IVM. After IVF, percentage of grade 1 freezable embryos on day 7 (D + 7) as well as percentage of blastocyst formation on D + 8 in the group of BL-I + ROS treated oocytes fertilised after 16 hr IVM were higher (P < 0.05) compared with the other experimental group fertilised after 22 hr IVM but not different in comparison with the control. Survival to freezing and thawing of grade 1 embryos frozen on D + 7 was employed as viability criteria and was similar in all groups. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes determines a reversible meiotic block, without compromising their subsequent developmental competence.

Nitric oxide synthase expression and nitric oxide/cyclic GMP pathway in swine granulosa cells.

The present investigation was undertaken to verify if the two nitric oxide synthase isoforms, eNOS and iNOS, are present in swine granulosa cells and whether the enzyme soluble guanylate cyclase is functionally active in the same cells and can account for NO effects. Using western blotting, the presence of endothelial NO synthase was demonstrated in freshly collected cells; on the contrary, iNOS expression was not observed in the same cells either before or after culture with the inflammatory cytokine hTNF-alpha. The treatment with a strong NO donor (S-Nitroso-L-acetyl penicillamine, SNAP) determined an increase of cGMP levels in culture media, which was attenuated by the combined treatment with an inhibitor of NO-sensitive soluble guanylate cyclase, 1H-[1,2,3]oxadiaziolo [4,3a]quinoxaline -1-one (ODQ). The cGMP analog, 8 bromo-cGMP, mimicked the strong inhibitory effect exerted by SNAP on estradiol 17 beta and progesterone production, while ODQ did not modify steroids concentrations in culture media. These observations demonstrate the presence of a follicular NO-generating system, which in swine granulosa cells seems to include only the endothelial NOS isoform. Furthermore, the nitric oxide/cyclic GMP system seems to be functionally active in these cells, since cGMP appears to mediate NO action, even if it cannot account completely for NO inhibitory effect on steroidogenesis.

Lipid hydroperoxide and cGMP are not involved in nitric oxide inhibition of steroidogenesis in bovine granulosa cells.

The present study was performed to explore two of the possible signalling mechanisms through which nitric oxide (NO) inhibits steroidogenesis in bovine granulosa cells. Because cGMP is generally known to play a pivotal role in NO signal transduction, the first aim of the present study was to verify the presence of a functional NO-cGMP signalling pathway. Because non-cGMP-dependent pathways could be involved in the inhibition of steroidogenesis by NO, we examined the formation of lipid hydroperoxides (LPOs), possibly induced by NO. Using bovine granulosa cells collected from small (< 5 mm) and large (> 8 mm) follicles, the effectiveness of the NO donor s-nitroso-N-acetylpenicillamine (SNAP; 10(-3), 10(-4) and 10(-5) M) in stimulating cGMP production and the formation of LPOs was examined. The second aim of the present study was to determine whether the effects of NO on steroidogenesis could be mimicked by treatment of cells with a cGMP analogue (8-bromo-cGMP (8-Br-cGMP); 10(-3), 10(-4) and 10(-5) M) and whether these effects could be reversed by [1H]-[1,2,3]oxadiaziolo[4,3a]quinoxaline-1-one (ODQ; 10(-5) and 10(-4) M) an inhibitor of NO-sensitive soluble guanylate cyclase. The highest dose of SNAP used induced a significant (P<0.01) increase in cGMP levels, while other concentrations tested were ineffective. Neither concentration of ODQ used significantly inhibited basal cGMP output, while both concentrations counteracted the stimulatory effect of SNAP. Treatment of cells with 8-Br-cGMP and ODQ was ineffective in modifying steroidogenesis. Treatment with SNAP, at the three concentrations tested, had no significant effect on the level of LPOs. The present results suggest that NO inhibits steroidogenesis in bovine granulosa cells without involving cGMP and LPOs.

Is nitric oxide an autocrine modulator of bovine granulosa cell function?

Nitric oxide (NO) is an important intra- and intercellular messenger controlling many biological processes. It is synthesized by NO synthases, which have been found also in granulosa cells. The present study examined whether NO is present in bovine follicular fluid and is produced by granulosa cells in culture. Secondly, it aimed to determine if NO affects the main parameters of granulosa cell function. The NO donor S-nitroso-L-acetyl-penicillamine (10(-3), 10(-4), 10(-5) M) was used to evaluate whether NO might influence steroidogenesis, proliferation and apoptosis in bovine granulosa cells collected from follicles divided according to their size in small (<5 mm) and large (>8 mm). The data demonstrate the presence of NO in follicular fluid and its production by granulosa cells in culture: the most active cells in producing NO are those from the small follicles, as confirmed by the NO levels in follicular fluid. This study also shows that NO donor significantly (P<0.001) inhibits progesterone (P4) and oestradiol 17beta (E2) production by the granulosa cells from both kinds of follicle; moreover, the highest concentration of NO donor significantly (P<0.001) inhibits DNA fragmentation in all the cells whereas the lowest concentration stimulates (P<0.001) cellular apoptosis only in granulosa cells from large follicles. NO donor does not seem to modify cell proliferation. Taken together these data lead point to NO as a local modulator of granulosa cell function.