Sylvie Bilodeau-Goeseels

Comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate.

The objectives of this study were to compare different methods of evaluating sperm plasmalemma and to determine their relationship with in vitro fertilization rate. A single batch of frozen semen from each of eight beef bulls was used for assessment of sperm viability and for in vitro fertilization. Conventional viability tests included sperm morphology, motility, acrosome integrity, and abnormal DNA condensation. Methods for evaluation of the sperm plasmalemma included eosin/nigrosin (EN) and trypan-blue (TB) vital stains, propidium iodide (PI) in combination with carboxyfluorescein diacetate (CFDA) or SYBR-14 (SYBR) fluorescent vital stains, and the hypoosmotic swelling test (HOST). A total of 133-150 oocytes were fertilized in vitro with sperm from each bull and cleavage rates were determined. There were high correlations between the results obtained with vital stains and good to excellent interclass correlation coefficients of agreement, indicating that these stains provide measures of the same sperm attribute, i.e. plasmalemma integrity. However, the proportions of membrane-intact sperm identified by EN or TB stains were greater (P<0.0001) than identified by CFDA/PI or SYBR/PI fluorescent stains. The results obtained with the HOST had moderate correlations but poor agreement with the results of the vital stains. The proportion of viable sperm identified by the HOST was lower (P<0.05) than the proportion identified by vital stains, indicating that response to the HOST did not depend only on the integrity of the plasmalemma. Although there were significant differences in fertilization rates and sperm viability among bulls, there was no sharp distinction for the results of sperm viability tests from bulls producing different in vitro fertilization rates. Proportions of normal, motile, acrosome-intact, and HOST-responsive sperm were identified as significant predictors of in vitro fertilizing potential; each of these endpoints explained 12-18% of the variation when evaluated separately (linear regression) and 48% when evaluated collectively (stepwise regression). In conclusion, EN and TB stains overestimated the proportion of plasmalemma-intact sperm compared to PI-based fluorescent stains. Vital stains evaluated the morphological integrity of the plasmalemma, whereas the HOST assessed plasmalemma function. In that regard, the HOST was the only plasmalemma evaluation method that significantly contributed to conventional sperm quality tests in predicting in vitro fertilization rate, indicating that the test could be incorporated to the routine of semen analysis.

Effects of oocyte quality on development and transcriptional activity in early bovine embryos.

The objective of this study was to evaluate the effects of oocyte quality on in vitro development and the level of transcriptional activity in early bovine embryos. Cumulus-oocyte complexes (COC) were divided into six classes based on their cumulus investment and on the texture of the ooplasm. Embryos originating from oocytes with more than five layers of cumulus cells and with slight expansion of the cumulus and/or granulation in the ooplasm (class II) developed to the blastocyst stage as frequently as embryos originating from oocytes of class I which showed no signs of atresia (13.9 and 13.7% for classes I and II, respectively). Oocytes with fewer than five layers of cumulus cells and homogeneous ooplasm (class III) had lower cleavage (63.1%) than oocytes with more than five layers of cumulus cells (77.2 and 83.6% for classes I and II, respectively); however, development to the blastocyst stage was similar (12.7%). More advanced atresia such as the presence of granulations in oocytes with less than five layers of cumulus cells (class IV), the absence of cumulus (class V), or the presence of expanded cumulus with dark clumps (class VI) reduced cleavage (57.4, 35.9 and 56.3% for classes IV-VI, respectively) and blastocyst formation (3.5, 0.5 and 1.9% for classes IV-VI, respectively). We examined the effects of oocyte quality on the level of transcriptional activity in in vitro produced embryos at the 2-, 4-, 8-, 16-cell stage and in embryos remaining at the 8-cell stage while the majority had progressed to the 16-cell stage (8-cell delayed embryos) by labeling with 3H-uridine followed by RNA precipitation and scintillation counting. For each developmental stage, there was no significant effect of oocyte class on uptake and incorporation of 3H-uridine into embryos, with the exception of uptake at the 8-cell stage which was higher (P<0.05) in embryos from class V-VI oocytes. Labeled uridine uptake (in embryos from classes I-II and III oocytes) and incorporation (in embryos from oocytes of all classes) increased significantly at the 16-cell stage compared to earlier stages. Eight-cell delayed embryos originating from classes I to IV oocytes incorporated significantly more 3H-uridine than normally developing 8-cell embryos. In conclusion, these results expand on previous work showing that oocytes with early signs of atresia have good development potential. No differences in transcriptional activity in embryos originating from different classes were detected. However, the results obtained with 8-cell delayed embryos indicated that transcriptional activity was determined by the interval after fertilization rather than the number of cell cycles.

Cows are not mice: The role of cyclic AMP, phosphodiesterases, and adenosine monophosphate‐activated protein kinase in the maintenance of meiotic arrest in bovine oocytes

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage – possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP‐elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate‐activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures. Mol. Reprod. Dev. 78:734–743, 2011.

Effects of phosphodiesterase inhibitors on spontaneous nuclear maturation and cAMP concentrations in bovine oocytes

It was previously demonstrated that inhibition of cAMP degradation with phosphodiesterase type 3 (PDE3) inhibitors resulted in the maintenance of bovine cumulus-oocyte complexes (COC) and denuded oocytes (DO) in meiotic arrest, while a PDE4 inhibitor was without effect. In this study, different inhibitors of PDE3 and PDE4 were tested for their effects on bovine oocyte nuclear maturation. Bovine COC and DO were cultured in TCM-199+10% fetal bovine serum (FBS) with or without different concentrations of the PDE inhibitors. The PDE3 inhibitor trequinsin significantly increased the percentage of COC remaining at the germinal vesicle (GV) stage after 7h of culture (19.3, 60.3, and 67.8% GV for control and trequinsin 10 and 50 nM, respectively) while Ro 20-1724 (a PDE4 inhibitor) was without effect. In DO, only trequinsin at 10 nM had a significant effect after 7h of culture (51.3 and 86.1% GV for control and trequinsin 10 nM, respectively). Trequinsin reduced the percentage of COC reaching the mature phase after 22h, but was without effect on DO. The protein kinase A (PKA) inhibitor H-89 reversed the inhibitory effect of trequinsin in COC and DO, indicating that inhibition of nuclear maturation by trequinsin involves activation of PKA. Trequinsin increased cAMP concentrations in COC but not in DO, suggesting that cumulus cells may also contain a PDE3 isoenzyme.

Selection of developmentally competent immature equine oocytes with brilliant cresyl blue stain prior to in vitro maturation with equine growth hormone.

Immature oocytes synthesize a variety of proteins that include the enzyme glucose-6-phosphate dehydrogenase (G6PDH). Brilliant cresyl blue (BCB) is a vital blue dye that assesses intracellular activity of G6PDH, an indirect measure of oocyte maturation. The objective was to evaluate the BCB test as a criterion to assess developmental competence of equine oocytes and to determine if equine growth hormone (eGH) enhanced in vitro maturation (IVM) of equine oocyte. Cumulus-oocytes complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries and were evaluated morphologically. Thereafter, COCs were exposed to BCB (26 μM) for 90 min at 39°C and selected based on the colour of their cytoplasm (BCB positive/BCB+ or BCB negative/BCB-). The COCs were allocated as follows: (a) IVM medium; (b) eGH group; (c) BCB-/IVM; (d) BCB+/IVM; (e) BCB-/eGH; and (f) BCB+/eGH. Then, COCs were cultured in vitro for 30 h, at 39°C in a 5%CO2 humidified air atmosphere. Cumulus-free oocytes were incubated in 10 μg/ml of bis-benzamide for 20 min at 39°C and nuclear maturation was evaluated with epifluorescence microscopy. Of the 39 COCs selected morphologically and subjected to BCB staining, 18/39 (46.2%) were classified as BCB+ and 21/39 (53.8%) as BCB- (P > 0.05). Maturation was not affected significantly by BCB classification, but the maturation rate was higher for oocytes that had been exposed to exogenous eGH versus controls (16/28, 57.1% versus 8/26, 30.8%, P < 0.05). In the present study, the BCB test was not useful for predicting competent equine oocytes prior to IVM. However, eGH enhanced equine oocyte maturation in vitro.

The effect of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on in vitro maturation of equine oocytes

The objective of this study was to test the hypothesis that equine growth hormone (eGH), in combination with insulin growth factor-I (IGF-I), influences positively in vitro nuclear and cytoplasmic maturation of equine oocytes. Cumulus-oocyte complexes were recovered from follicles that were < 25 mm in diameter, characterized by morphology and were allocated randomly as follow: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 400 ng/ml anti-IGF-I antibody. Oocytes were matured for 30 h at 38.5°C in air with 5% CO2 and then stained with 10 μg/ml propidium iodide (PI) to evaluate nuclear status and 10 μg/ml Lens culinaris agglutinin-fluorescein complex (FITC-LCA) to assess cortical granule migration by confocal microscopy. The proportion of immature oocytes that developed to the metaphase II (MII) stage in the eGH + IGF-I group (15 of 45) was greater than in the groups that were treated only with IGF-I (7 of 36, p = 0.03). Oocytes that reached MII in the control group (20 of 56; 35.7%) showed a tendency to be different when compared with eGH + IGF-I group (15 of 45; 33.3%, p = 0.08). The treated group that contained anti-IGF-I (15 of 33; 45.4%) decreased the number of oocytes reaching any stage of development when compared with eGH (47 of 72; 65.3%) and eGH + IGF-I (33 of 45; 73.3%) groups (p = 0.05) when data from MI and MII were combined. We concluded that the addition of eGH to in vitro maturation (IVM) medium influenced the in vitro nuclear and cytoplasmic maturation of equine oocytes. The use of GH and IGF-I in vitro may represent a potential alternative for IVM of equine oocytes.

The involvement of growth hormone in equine oocyte maturation, receptor localization and steroid production by cumulus-oocyte complexes in vitro.

The objectives of this study were to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, and expression and subcellular localization of eGH-receptors (eGH-R) on equine ovarian follicles. Cumulus-oocyte complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries. The COCs were morphologically evaluated and randomly allocated to be cultured in either a control maturation medium or supplemented with 400 ng/mL eGH, for 30 h at 38.5°C in air with 5% CO2. The COCs were stained with 10 μg/mL propidium iodide and 10 μg/mL fluorescein isothiocyanate-labeled Lens culinaris agglutinin. Chromatin configuration and distribution of cortical granules were assessed via confocal microscopy. Compared to control, COCs incubated with eGH had: more oocytes that reached metaphase II (35/72, 48.6% vs. 60/89, 67.4%, respectively; P=0.02); greater concentrations of testosterone (0.21 ± 0.04 vs. 0.06 ± 0.01 ng/mL; P=0.01), progesterone (0.05 ± 0.01 vs. 0.02 ± 0.00 ng/mL; P=0.04), and oestradiol (76.80 ± 14.26 vs. 39.58 ± 8.87 pg/mL; P=0.05) in the culture medium, but no significant differences in concentration of androstenedione. Based on Real Time RT-PCR analyses, expression of the eGH-R gene was greater in cumulus cells and COCs at the start than at the end of in vitro maturation. Positive immunostaining for eGH-R was present in cumulus cells, the oocytes and granulosa cells. In conclusion, addition of eGH to maturation medium increased rates of cytoplasmic maturation and had an important role in equine oocyte maturation, perhaps mediated by the presence of eGH-R in ovarian follicles.

Characterization of the effects of metformin on porcine oocyte meiosis and on AMP-activated protein kinase activation in oocytes and cumulus cells.

The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.

The Control of Meiotic Arrest and Resumption in Mammalian Oocytes

In mammals, following implantation of the embryo, a small number of cells from the epiblast eventually become the precursors of primordial germ cells (reviewed by Edson et al., 2009). After mitotic proliferation of the primordial germ cells, migration to the genital ridges and rapid proliferation again, meiosis is initiated in the oogonia at about day 13 of development in mice, at day 82 in bovine and during the 3rd month in humans. A last round of DNA synthesis occurs after which the oocytes enter a long meiotic prophase which consists of different stages (leptotene, zygotene, pachytene). By the time of birth, oocytes of most mammals have reached the diplotene stage, meiosis is arrested and the nucleus is referred to as a germinal vesicle (GV). The timing of progression of oocytes through meiosis in relation to birth varies in different mammals and in a few species meiotic prophase is initiated only after birth (i.e., rabbits and cats). At about the time of birth, oocytes become surrounded by follicle cells and these primordial follicles and oocytes represent a large stockpile from which, at any given time, a few are selected to grow and develop. The gradual depletion of primordial follicles through either growth or degeneration (atresia) continues until menopause.