Patrick Blondin

IN VITRO PRODUCTION OF BOVINE EMBRYOS : DEVELOPMENTAL COMPETENCE IS ACQUIRED BEFORE MATURATION

Few studies have examined the importance of the time during which oocytes are left in the ovaries following animal slaughter. The objective of this study was to determine the optimal time for retrieving oocytes after slaughter and to ascertain if superovulating cows in association with this optimal time could increase the developmental competence of bovine oocytes. In Experiment 1, oocytes were left in the postmortem ovaries for 2,3,4,5,6 or 7 h and were then transported to the laboratory at approximately 30 degrees C. Recovered oocytes were processed in vitro using standard techniques. In Experiment 2, cyclic heifers (n = 18) were superovulated between Days 8 and 12 of the estrous cycle with 8 constant doses (4 mg each, twice daily) or 8 decreasing doses (2 injections of 4,3,2 and 1 mg every 12 h) of FSH-P +/- 1 mg prostaglandin 24 or 48 h before slaughter. Oocytes were left in the ovaries for 4 h and were classified according to the state of their cumulus and cytoplasm. The results indicated that oocytes aspirated from ovaries collected 4 h after slaughter produced significantly more > or =64-cell embryos after 7 d of in vitro development than those collected 2, 6 or 7 h postslaughter. Oocytes (87%) from superovulated animals had numerous layers of cumulus cells and originated from medium (2.7 to 8 mm) and large (> or =8 mm) follicles. Significantly more oocytes developed from large follicles than from medium follicles. Although individual culture of the oocytes negatively affected the percentage of embryos produced, group culture of oocytes from animals that were superovulated and left in the postmortem ovaries for 4 h resulted in exceptionally high rates of embryos after 5 d of IVD. On average, 60 to 80% of 16-cell embryos were produced, indicating that under the proper conditions, developmental competence is acquired before in vitro maturation.

THE TIME INTERVAL BETWEEN FSH ADMINISTRATION AND OVARIAN ASPIRATION INFLUENCES THE DEVELOPMENT OF CATTLE OOCYTES

Depriving the ovary of exogenous FSH for 1, 2 or 3 d following a bolus injection of FSH was shown to influence the quality of the recovered oocytes. Thus, we compared the developmental competence of oocytes from heifers which had been stimulated for 3 d with FSH (Folltropin-V) and, after an interval of 36, 48 or 60 h, underwent blind transvaginal aspiration. The ovaries of heifers with a palpable or functional corpus luteum were aspirated to remove all large follicles 2 d prior to being injected with either 6 doses of saline (S), 6 doses (20 mg/mL) of FSH (F), or in 6 decreasing doses of FSH (3, 3, 2, 2, 1, 1 mL; Fd). Follicles were counted and classified (medium: 5 to 10 mm, large: >10 mm) with ultrasonography before each aspiration. The oocytes recovered were classified, matured, fertilized, and developed in vitro. On a per animal basis, 1.5, 5.2 and 4.7 large and 1.5, 10.7 and 10.7 medium follicles were counted for S, F and Fd, respectively. A mean of 3.3, 9.1 and 7.7 oocytes was recovered for treatments S, F and Fd, respectively and 58, 94 and 82% were enclosed in a nonexpanded cumulus or a corona layer. Oocyte development rates were based on counts of embryos with 32 or more nuclei at Day 6.5. When oocytes were recovered 36 h after the last injection, an average of 1, 2.7 and 2 embryos per animal was obtained with S, F and Fd, respectively; at 48 h, 0.75, 4.25 and 1 embryo; and at 60 h, 0, 2.5 and 2.7 embryos. Variance analysis was performed, and the protected LSD test indicated that treatment F at 48 h resulted in a significantly higher embryo rate than Fd at 48 h (P<0.05) or S (all times; P<0.05). The reduced effect of the Fd regimen could be due to the decreasing FSH support during follicular growth or to the lower total amount of FSH given. In conclusion, these results indicate an advantage of using moderate (3 d) follicle stimulation followed by a period of FSH starvation to obtain optimal embryo production.

Superovulation can reduce the developmental competence of bovine embryos

This study was done to determine if different superovulatory regimens could have an effect on the percentage of embryos produced using IVM/IVF/IVC. Cyclic heifers (n = 22) were superovulated between Days 8 and 12 of the estrous cycle with 4, 6 or 8 constant doses of FSH-P (4 mg each, twice daily) +/- the addition of 1 mg prostaglandin 24 h before slaughter. Ovaries from these superovulated cows and from untreated cows were collected and the follicles dissected. Oocytes were classified according to the appearance of their cumulus and cytoplasm. Individual culture as well as group culture were performed but an individual culture reduced the percentage of oocytes developing into embryos for both untreated and superovulated animals. The results indicated that despite the superovulation regimen the developmental competence of the oocytes collected was lower (0 to 15% embryos) than that of oocytes from untreated animals (20 to 34% embryos). Small follicles ( < or = 2.7 mm) yielded mostly oocytes with an incomplete or partially expanded cumulus investment that never developed into an embryo. Differences in the morphology of the oocytes from medium (2.7 to 8 mm) and large ( > or = 8 mm) follicles were apparent, but equal developmental rates were obtained between all classes of oocytes (12 and 8% embryos, respectively). Follicular atresia was reduced significantly after superovulation (81% nonatretic follicles in treated vs 42% nonatretic follicles in untreated animals); however oocytes from atretic and slightly atretic follicles developed similarly to those from nonatretic follicles. These results suggest that although superovulation increases follicular size and decreases atresia, these conditions are not sufficient to confer developmental competence on the oocytes.

The time interval between FSH-P administration and slaughter can influence the developmental competence of beef heifer oocytes.

Superovulation alone may not be enough to result in developmentally competent oocytes. The objective of this study was to determine if a time interval between FSH administration and slaughter and between slaughter and oocyte recovery could increase the percentage of embryos. Beef heifers (n = 20) were superovulated with 1 bolus injection of 25 mg, im FSH-P diluted in saline and then slaughtered at 24, 48 or 72 h after FSH injection and the ovaries transported to the laboratory at 30 degrees C. For 6 of the heifers that received FSH-P and were then culled at 48 h post treatment, oocytes were recovered 1 to 2 h post slaughter from the first ovary and 4 to 5 h from the second ovary. Ovaries from untreated cows were collected and served as controls. The results indicated that FSH-P and culling at 48 h produced 35% >/= 32-cell embryos, significantly more than FSH-P and culling at 24 and 72 h (19 and 14%, respectively; P < 0.05). Furthermore, FSH-P and culling at 48 h produced 25% >/= 64-cell embryos, significantly more than FSH-P and culling at 24 and 72 h and the nontreatment control group (5, 7 and 15%, respectively; P < 0.05). The FSH-P group culled at 48 h produced more >/= 32-cell embryos, with an average of 84 +/- 5 cells/embryo, than the treated groups culled at 24 and 72 h and the untreated group (52 +/- 6, 60 +/- 5 and 63 +/- 3, respectively; P < 0.01). Finally, oocytes left in the postmortem ovaries for 4 to 5 h resulted in higher rates (51% and 41%) of >/= 32- and >/= 64-cell embryos, respectively, compared with that of the untreated control animals (29 and 18%; P < 0.05), but these rates were not different from oocytes left in ovaries for 1 to 2 h (33 and 24%). It is concluded that culling at 48 h after FSH treatment, as well as the conditioning effect on oocytes in warm postmortem ovaries for 4 to 5 h, increases the number of competent oocytes.

Improvement of bovine in vitro embryo production by vitamin K2 supplementation

Mitochondria play an important role during early development in mammalian embryos. It has been shown that properly controlled follicular preparation increases the likelihood of in-vitro-produced bovine embryos reaching the blastocyst stage and that competent embryos exhibit heightened expression of genes associated with mitochondrial function. We hypothesized that apparently incompetent embryos could be rescued by restoring mitochondrial function. It has been shown that vitamin K2 (a membrane-bound electron carrier similar to ubiquinone) can restore mitochondrial dysfunction in eukaryotic cells. The aim of this study was therefore to investigate the effects of vitamin K2 on bovine embryonic development in vitro. The vitamin was found most effective when added 72u200ah after fertilization. It produced a significant (P<0.05) increase in the percentage of blastocysts (+8.6%), more expanded blastocysts (+7.8%), and embryos of better morphological quality. It improved the mitochondrial activity significantly and had a measurable impact on gene expression. This is the first demonstration that current standard conditions of in vitro production of bovine embryos may be inadequate due to the lack of support for mitochondrial function and may be improved significantly by supplementing the culture medium with vitamin K2.

Papaverine-sensitive phosphodiesterase activity is measured in bovine spermatozoa

Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well‐known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP‐PDE activity in bovine spermatozoa. Total cAMP‐PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2 ± 49.5 and 1252.6 ± 86.5 fmoles/min/106 spermatozoa, respectively. Using different family‐specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP‐PDE activity was papaverine‐sensitive (44.5% and 57.5%, respectively, at 400 nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post‐acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.

Cyclic nucleotide phosphodiesterases in human spermatozoa and seminal fluid: Presence of an active PDE10A in human spermatozoa

BACKGROUNDnCyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for sperm functions such as capacitation, motility and acrosome reaction. It is well known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. The present study was undertaken to characterize cAMP-PDE activity in human semen.nnnMETHODSncAMP-PDE activity was measured in human sperm and seminal plasma using family specific PDE inhibitors. Three sperm fractionation methods were applied to assess cAMP-PDE activity in spermatozoa. Western blots were used to validate the presence of specific family in sperm and seminal plasma.nnnRESULTSnUsing three sperm fractionation methods, we demonstrated that in human sperm, the major cAMP-PDE activity is papaverine-sensitive and thus ascribed to PDE10. In seminal plasma, total cAMP-PDE activity was 1.14±0.39fmol of cAMP hydrolyzed per minute per μg of protein. Using specific inhibitors, we showed that the major cAMP-PDE activity found in human seminal plasma is ascribed to PDE4 and PDE11. Western blot analysis, immunoprecipitation with a specific monoclonal antibody, and mass spectrometry confirmed the presence of PDE10 in human spermatozoa.nnnCONCLUSIONnThis study provides the first demonstration of the presence of functional PDE10 in human spermatozoa and functional PDE4 and PDE11 in human seminal plasma.nnnGENERAL SIGNIFICANCEnSince the contribution of cyclic nucleotides in several sperm functions is well known, the finding that PDE10 is an active enzyme in human spermatozoa is novel and may lead to new insight into fertility.

Characterization of cAMP-phosphodiesterase activity in bovine seminal plasma

The second messenger cyclic adenosine monophosphate (cAMP) has a central role in sperm physiology. Extracellular cAMP can be sequentially degraded into 5′AMP and adenosine by ecto‐phosphodiesterases (ecto‐PDE) and ecto‐nucleotidases, a phenomenon called extracellular cAMP‐adenosine pathway. As cAMP‐adenosine pathway is involved in sperm capacitation, we hypothesize that extracellular PDEs are functionally present in seminal plasma. Exclusively measuring cAMP‐PDE activity, total activity in bovine seminal plasma was 10.1 ± 1.5 fmoles/min/μg. Using different family‐specific PDE inhibitors, we showed that in seminal plasma, the major cAMP‐PDE activity was papaverine sensitive (47.5%). These data support the presence of PDE10 in bovine seminal plasma and was further confirmed by western blot. In epididymal fluid, total cAMP‐PDE activity was 48.2 ± 14.8 fmoles/min/μg and we showed that the major cAMP‐PDE activity was 3‐isobutyl‐methylxanthine insensitive and thus ascribed to PDE8 family. PDE10A mRNAs were found in the testis, epididymis, and seminal vesicles. cAMP‐PDE activity is present in bovine seminal plasma and epididymal fluid. The results suggest a role for ecto‐PDEs present in those fluids in the signaling pathways involved in sperm functions.