C. L. V. Leal

Consequences of Nitric Oxide Synthase Inhibition During Bovine Oocyte Maturation on Meiosis and Embryo Development

The importance of nitric oxide synthase (NOS) in bovine oocyte maturation was investigated. Oocytes were in vitro matured with the NOS inhibitor N(w)-L-nitro-arginine methyl-ester (10(-7), 10(-5) and 10(-3) m L-NAME) and metaphase II (MII) rates and embryo development and quality were assessed. The effect of L-NAME (10(-7) m) during pre-maturation and/or maturation on embryo development and quality was also assessed. L-NAME decreased MII rates (78-82%, p < 0.05) when compared with controls without L-NAME (96%). Cleavage (77-88%, p > 0.05), Day 7 blastocyst rates (34-42%, p > 0.05) and total cell numbers in blastocysts were similar for all groups (146-171 cells, p > 0.05). Day 8 blastocyst TUNEL positive cells (3-4 cells) increased with L-NAME treatment (p < 0.05). For oocytes cultured with L-NAME during pre-maturation and/or maturation, Day 8 blastocyst development (26-34%) and Day 9 hatching rates (15-22%) were similar (p > 0.05) to controls pre-matured and matured without NOS inhibition (33 and 18%, respectively), while total cell numbers (Day 9 hatched blastocysts) increased (264-324 cells, p < 0.05) when compared with the controls (191 cells). TUNEL positive cells increased when NOS was inhibited only during the maturation period (8 cells, p < 0.05) when compared with the other groups (3-4 cells). NO may be involved in meiosis progression to MII and its deficiency during maturation increases apoptosis in embryos produced in vitro. Nitric oxide synthase inhibition during pre-maturation and/or maturation affects embryo quality.

Endothelial and inducible nitric oxide synthases in oocytes of cattle

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (IVM) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM, but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity.

Identification of sperm head subpopulations with defined pleiomorphic characteristics in ejaculates of captive Goeldi's monkeys (Callimico goeldii).

The aim of this study was to evaluate the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates from the vulnerable Goeldis monkey (Callimico goeldii) by using a combination of computerized analysis system and Principal Component Analysis (PCA) methods. Each sperm head was measured for four primary spermatozoal head dimensional parameters (area [A (μm(2))], perimeter [P (μm)], length [L (μm)] and width [W (μm)]) and three head shape derived parameters (ellipticity [(L/W)], elongation [(L-W)/(L+W)] and rugosity [(4πA/P(2))]). Six separate subpopulations (SPs) were identified: SP1, constituted by very large, narrow and very elliptical spermatozoa (A=16.85±1.56μm(2), W=2.75±0.42μm and ellipticity=2.16±0.24); SP2, characterized by average sized, short, wide and round spermatozoa (A=15.00±1.92μm(2), L=5.06±0.49μm, W=3.51±0.31μm and ellipticity=1.44±0.15); SP3, represented by small, wide and slightly round spermatozoa (A=14.95±1.75μm(2), W=3.47±0.29μm and ellipticity=1.48±0.14); SP4 included very small, short and very round spermatozoa (A=14.15±2.38μm(2), L=4.90±0.57μm and elongation=0.18±0.05); SP5 consisted of average sized and slightly elliptical spermatozoa (A=15.14±1.72μm(2) and ellipticity=1.49±0.14); and SP6 included large and round spermatozoa (A=16.30±1.62μm(2) and elongation=0.19±0.04). There were differences in the sperm subpopulation distribution (P<0.001) among the five donors analyzed. In conclusion, the results of the current study confirmed that the use of computer sperm analysis methods combined with PCA cluster analyses are useful methods to identify, classify, and characterize different sperm head morphometric subpopulations in neotropical primates. Broadening our knowledge of C. goeldii sperm morphometric abnormalities as well as developing reliable techniques for sperm evaluation may be essential for ex situ conservation of this threatened species.

The Influence of Morphology, Follicle Size and Bcl‐2 and Bax Transcripts on the Developmental Competence of Bovine Oocytes

This study analysed two non-invasive oocyte selection methods in relation to in vitro embryo development capacity and expression of apoptosis-related genes. Selection was based on morphological quality of oocytes or follicle diameter. Oocytes were classified as grade I (GI ≥3 layers compact cumulus cells and homogeneous cytoplasm; grade II (GII ≤3 layers compact cells and homogeneous cytoplasm;, and grade III (GIII ≥3 layers, but cells with slight expansion and slightly granulated cytoplasm). Blastocyst development was lower for GII (28.5%) than for GIII (47.7%, p < 0.05), and GI was similar to both (36.9%, p > 0.05). Relative expression of Bcl-2 gene was lower in the GI (1.0, p < 0.05) than in the GII (1.8) and GIII (2.2), which were not different (p > 0.05). There was no difference (p > 0.05) between GI (1.0), GII (0.92) and GIII (0.93) regarding the Bax transcript. However, the Bax and Bcl-2 transcript ratios in GII (Bax; 0.92 and Bcl-2; 1.8) and GIII (Bax; 0.93 and Bcl-2; 2.2) were different (p < 0.05). Regarding oocytes from follicles of different sizes, cleavage and blastocyst rates for 1-3 mm (82.5; 23.7%) were lower (p < 0.05) than for 6-9 mm (95.6; 41.1%), but similar (p > 0.05) to 3-6 mm (93.7; 35.4%), which were not different (p > 0.05). Regarding Bax and Bcl-2 expression, the oocytes were similar (p > 0.05) for 1-3 mm (Bax; 1.0 and Bcl-2; 1.0), 3-6 mm (Bax; 1.0 and Bcl-2; 0.93) and 6-9 mm (Bax; 0.92 and Bcl-2; 0.91). In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs taken from follicles >3 mm in diameter are better suited to in vitro embryo development.

Effect of cyclin-dependent kinase (CDK) inhibition on expression, localization and activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) in bovine oocytes.

This study aimed to assess the effects of cyclin-dependent kinase (CDK) inhibition on factors involved in the control of meiosis in bovine oocytes: maturation promoting factor (MPF) (p34(cdc2) and cyclin B1) and mitogen activated protein kinase (MAPK). Oocytes were maintained at germinal vesicle (GV) stage in vitro with 10 μM of the CDK inhibitor butyrolactone I (BLI) for 24 h (inhibited). After this period, some of the oocytes were transferred to in vitro maturation (IVM) culture for 24 h (inhibited and matured). Control oocytes were assessed immediately after follicle aspiration (immature) or after in vitro maturation for 24 h (matured). Real-time PCR analyses showed that transcripts for p34(cdc2) and MAPK were detected in immature and inhibited oocytes and decreased after maturation, irrespective of CDK inhibition with BLI. Cyclin B1 was detected at similar levels in all oocyte groups. The p34(cdc2) and MAPK proteins were detected by Western blotting at similar levels in all oocyte groups, and cyclin B1 protein was detected only after maturation. Immunofluorescence detection showed that p34(cdc2) was localized in the cytoplasm and GV of immature oocytes, and then throughout the cytoplasm after maturation. Cyclin B1 and MAPK were detected in the cytoplasm in all oocyte groups. Maturation promoting factor and MAPK activities were similar throughout most of maturation for oocytes treated with or without BLI. In conclusion, CDK inhibition did not affect the expression (mRNA and protein levels) and localization of MPF and MAPK, and had nearly no effect on kinase activities during maturation.

259 PRELIMINARY INVESTIGATIONS OF THE EFFECT OF NITRIC OXIDE ON THE EXPRESSION OF GENES INVOLVED IN THE NITRIC OXIDE/CYCLIC GUANOSINE MONOPHOSPHATE/CYCLIC ADENOSINE MONOPHOSPHATE PATHWAY DURING MEIOSIS RESUMPTION IN BOVINE OOCYTES

Nitric oxide is a chemical messenger generated by the activity of the enzyme NO synthase (NOS) and has been implicated in the control of oocyte maturation. Nitric oxide stimulates guanylate cyclase (GC) to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases (PDE) that may interfere with cyclic adenosine monophosphate (cAMP) levels, a nucleotide also involved in meiosis resumption. In a previous study, we found that increasing NO levels in the presence of a NO donor (S-nitroso-N-acetylpenicillamine, SNAP) resulted in a delayed resumption of meiosis and a lower rate of germinal vesicle breakdown after 9 h of in vitro maturation. A temporary increase in cGMP levels was also observed with the same treatment, which was reversed by inhibiting GC activity with oxadiazolo-quinoxaline-one (ODQ; unpublished data). These results suggest that NO acted via GC/cGMP and that even a temporary increase in the cGMP level led to a delay of meiosis resumption. The aim of the present study was to determine the role played by NO on the expression of genes encoding for enzymes of the NO/GC/cGMP and cAMP pathways during the first 9 h of oocyte maturation. Cumulus–oocyte complexes were in vitro matured for 9 h in a semi-defined medium (TCM-199 + 3 mg mL–1 of BSA) with 10 to 7 M SNAP associated or not associated with 100 µM ODQ, a GC inhibitor. A group of oocytes incubated in the absence of inhibitors was considered the control. Total RNA was extracted from pools of 20 denuded oocytes with TRIzol (Life Technologies, Grand Island, NY, USA) and reverse transcribed into complementary DNA using a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed by real-time PCR using SYBR Green (Applied Biosystems). The genes that had their expression measured pertained to one of the following groups: 1) genes encoding for enzymes that synthesise NO (NOS2 and NOS3); 2) genes involved in the control of cGMP levels (GUCY1B3 and PDE5A) or the enzymes activated by it (PKG1 and PKG2); or 3) genes involved in the control of cAMP levels (ADCY3, ADCY6, ADCY9, PDE3A, and PDE8A) or one of the enzymes activated by it (PKA1). GAPDH and PPIA were selected as housekeeping genes using qbasePLUS version 2.3 (Biogazelle, Zwijnaarde, Belgium). Data from 5 replicates were analysed using LinRegPCR version 11.1 and SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). All genes were found to be expressed in the three experimental groups; however, a significant difference in gene expression levels was not found among groups. Results suggest that NO does not act on oocyte maturation by affecting the expression of the investigated genes in oocytes. To our knowledge, this is the first report to demonstrate the expression of the ADCY3, ADCY6, and ADCY9 genes in bovine oocytes. Further research is in progress to study the effect of the SNAP treatment on the expression of these genes in cumulus cells. Financial support from FAPESP 2010/18023-9.

237 EXPRESSION OF THE GENES HSP 70.1, ZAR-1, AND MATER IN BOVINE OOCYTES SUBMITTED TO PREMATURATION AND/OR IN VITRO MATURATION

The present study aimed to assess the transcripts for the proteins that embryos require, histone 2a (H2a-FZ), heat shock 70 kDa protein 1A (HSP 70.1), zygote arrest 1 (ZAR-1), and maternal antigen (MATER), in bovine oocytes submitted to prematuration (PM) culture and/or in vitro maturation (IVM). Follicles (2–6 mm diameter) were aspirated from slaughterhouse-derived ovaries. Oocytes were selected and randomly distributed among treatments. For PM, oocytes were cultured 24 h in TCM-199 medium supplemented with 10 µm butyrolactone I, 0.2 mm pyruvate, and 10 µg mL–1 gentamicin (BGV group). Part of the prematured oocytes were washed and transferred to IVM culture (BMII group). For IVM, oocytes were cultured in TCM-199 supplemented with 10% FCS, 5.0 µg mL–1 LH, 0.5 µg mL–1 FSH, 0.2 mm pyruvate, and 10 µg mL–1 gentamicin for 22 h. As controls one group of oocytes was collected immediately after aspiration (GV group) and another group was matured in vitro (MII group) without undergoing prematuration. All cultures were carried out in 100-µL droplets of culture medium under mineral oil at 38.5°C and 5% CO2 in air. Oocytes from each treatment were denuded and frozen at 80°C (3 pools of 200 oocytes) in PBS with 0.1% polyvinyl alcohol (PVA) and 1 UI µL–1 RNase inhibitor. RNA was extracted using the RNeasy Protect Kit (Qiagen, Sao Paulo, Brazil) according to the manufacturers instructions. The extracted RNA was used for reverse transcription by the enzyme Improm-II Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturers instructions. cDNA was produced in a thermocycler for 60 min at 42°C, followed by warming to 70°C for 15 min and cooling to 4°C for freezing at –20°C. Relative quantification of the transcripts for the genes H2a-FZ, HSP 70.1, ZAR-1, and MATER was performed by real-time PCR using the SYBR GREEN Kit (Applied Biosystems do Brasil, Sao Paulo, Brazil) according to manufacturers instructions. Data were analyzed by the REST© software (Relative Expression Software Tool; Pfaffl et al. 2002 Nucl. Acids Res. 30, e36) 2005 BETA V1.9.9; a level of significance of 5% was considered to show differences among transcripts using H2a-FZ to normalize data. No differences were observed (P 0.05) MII (1.0, 1.0, and 1.0) and BMII (0.52 ± 0.25, 0.5 ± 0.1, and 0.6 ± 0.25) for transcripts HSP 70.1, ZAR-1, and MATER, respectively. A reduction in transcripts in group BMII may influence oocyte competence, reducing embryo development and/or quality.

337 GENE EXPRESSION IN BOVINE OOCYTES AFTER MEIOSIS BLOCK WITH BUTYROLACTONE I

The present study aimed to assess the effect of meiosis block on the expression of genes involved in apoptosis. Slaughterhouse bovine ovaries were collected soon after slaughter and transported to the laboratory in saline. Follicles with a diameter of 2–6 mm were aspirated, and oocytes were selected and distributed to the different treatments. For meiosis block (MB), oocytes were cultured for 24 h in TCM-199 supplemented with 100 µM butyrolactone I (BLI) and 3 mg mL-1 BSA (B100) or with 10 µM BLI, but without BSA (B10). After the 24-h MB culture, the oocytes were subjected to IVM in TCM-199 supplemented with 10% fetal calf serum (FCS), 5.0 µg mL-1 LH, 0.5 µg mL-1 FSH, 0.2 mM pyruvate, and 10 µg mL-1 gentamicin for 22 h. All cultures were at 38.5°C under an atmosphere of 5% CO2 in air. As controls (C), a group of oocytes was collected immediately after aspiration (immature oocytes) and after IVM without prior meiosis block (mature oocytes). In the treated groups, oocytes were collected at the end of MB culture (immature oocytes) and at the end of IVM post-MB (mature oocytes). After removal of cumulus cells and zonae pelucidae, oocytes were frozen at -80°C in PBS with 0.1% PVA and 1 IU µL-1 RNase inhibitor. Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen Brasil, Ltda., Sao Paulo, Brazil) according to the manufacturers recommendations in an Applied Biosystems 7500 fast real-time PCR system (Applied Biosystems, Sao Paulo, Brazil). Efficiency of the reactions was assessed by the software LinRegPCR (Ramakers et al. 2003 Neurosci. Lett. 339, 62–66) and submitted to REST Beta 2005 V1.9.9 analysis (Relative Expression Software Tool). A level of significance of 5p was used. Relative expression of BAX and BCL2 genes did not differ (P > 0.05) when the expression of the constitutive gene GAPDH was used to normalize the data. However, without normalization, a significant reduction in expression was observed after IVM (P < 0.05). For immature oocytes, GAPDH, BAX, and BCL2 expression was 1.0p (for the three genes) for control; 0.26, 0.36, and 0.56p for B100; and 0.6, 0.44, and 0.26p for B10, respectively. After IVM, expressions were 0.06, 0.33, and 0.26p for control; 0.06, 0.19, and 0.25p for B100; and 0.02, 0.15, and 0.3p for B10, respectively. The results show a reduction in transcripts (GAPDH, BAX, and BCL2) after meiosis block, and this reduction was more pronounced in matured oocytes, irrespective of treatment. The reduction in transcripts during MB could influence oocyte competence, but the reduction of these transcripts during maturation seems to be inherent to the oocytes. This work was supported by Fapesp, Brazil, grant n 03/01479-6.

292 OOCYTES FROM FOLLICLES OF DIFFERENT DIAMETERS: EMBRYO IN VITRO DEVELOPMENTAL POTENTIAL AND QUALITY

The present study aimed to assess the developmental potential and quality of embryos produced from oocytes originating from follicles of different sizes. Ovaries were collected at a slaughterhouse and follicles of 6 mm were aspirated. Follicle diameters were estimated based on the size of their exposed surface on the ovarian cortex using a ruler as reference for the first aspirations and were then based on visual evaluation. Aspirated oocytes, separated by their follicular origin, were matured in vitro in TCM-199 supplemented with 10% FCS, 5.0 µg mL-1 of LH, 0.5 µg mL-1 of FSH, 0.2 mM pyruvate, and 10 µg mL-1 of gentamicin for 22 h. After in vitro maturation, oocytes were in vitro-fertilized (IVF) using frozen–thawed semen prepared by Percoll gradient. Sperm cells were co-cultured with the oocytes at a final concentration of 2 × 106 sperm cells mL-1 in TALP-IVF medium supplemented with 2 µM penicillamine, 1 µM hypotaurine, 250 µM epinephrine, and 20 µg mL-1 of heparin. After 20 h, presumptive zygotes were partially denuded and transferred to in vitro culture (IVC) medium (TCM-199 supplemented with 10% FCS, 2.0 mM pyruvate, and 10 mg mL-1 of gentamicin). All cultures were incubated at 38.5°C under 5% CO2 in air and maximum humidity. The cleavage rate was assessed after 48 h of IVC, and blastocyst development was assessed on Day 7 (D7). On Day 9 (D9), the hatching rate was assessed and the hatched embryos were fixed for 1 h (3% paraformaldehyde in PBS), permeabilized for another h (0.5% Triton-X, 0.1% sodium citrate, and 0.1% plyvinyl alcohol in PBS), and evaluated by the TUNEL technique (in situ cell death detection kit). Total cell number and TUNEL-positive cells in embryos were counted under an epifluorescence microcope. Data of 4 replicates were analyzed by ANOVA and comparisons among groups were made by the Tukey test. The level of significance used was 5%. From the oocytes used ( 6 mm = 88), cleavage rates increased with increasing follicular diameter. Oocytes originating from 6-mm follicles resulted in 75, 93.6, and 95.5% cleavage rates, respectively (P > 0.05). For blastocyst rates on D7, 6 mm = 41.1%; P > 0.05). Regarding the hatching rate, total cell numbers, and TUNEL-positive cells on D9, 6 mm (32.3%, 237, and 0.23%, respectively) were not different (P > 0.05). The results suggest that oocytes from larger follicles are more competent for cleavage and blastocyst development on D7; however, when oocytes reach the blastocyst stage, hatching, total cell numbers, and apoptotic cell numbers are not influenced by follicle diameter. Financial suport for this work was provided by the FAPESP, Brazil.

354 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS AFTER NITRIC OXIDE INHIBITION

Nitric oxide (NO) is a chemical messenger detected in several cell types such as endothelial cells, neurons, and macrophages, exerting varied functions including vasodilatation, neurotransmission, and cell death induction. NO is generated by the activity of the enzyme nitric oxide synthase (NOS), which has been detected in several organs and tissues including the reproductive system. The aim of the present study was to assess the dose-response effect of N-omega-nitro-l-arginine-methyl ester (l-NAME), an NOS inhibitor, on in vitro nuclear and cytoplasmic maturation of bovine oocytes. Slaughterhouse ovaries were collected and their follicles (2–6 mm) were aspirated to obtain cumulus–oocyte complexes (COCs). Increasing l-NAME concentrations (0, 10-7, 10-5, 10-4, and 10-3 M) were added to IVM medium (TCM-199, supplemented with 10% fetal calf serum, 0.5 µg mL-1 FSH, 5.0 µg mL-1 LH, 0.2 mM pyruvate, and 10 mg mL-1 gentamicin); oocytes were cultured for 22 h. Nuclear maturation was assessed by propidium iodide staining (10 µg mL-1). For IVF, frozen–thawed semen prepared by Percoll gradient was used. Sperm cells were co-cultured with the oocytes at a final concentration of 2 × 106 sperm cells mL-1 in TALP-IVF medium supplemented with 2 µM penicillamine, 1 µM hypotaurine, 250 µM epinephrine, and 20 µg mL-1 heparin. After 20 h, presumptive zygotes were partially denuded and transferred to IVC medium (TCM-199 supplemented with 10% fetal calf serum, 2.0 mM pyruvate, and 10 mg mL-1 gentamicin). All cultures were at 38.5°C under 5% CO2 in air and maximum humidity. Cytoplasmic maturation was assessed by blastocyst development rates on Day 7. DNA fragmentation was assessed on Day 8 embryos by TUNEL (In Situ–Cell Death Detection kit, fluorescein; Roche Diagnostica Brasil, Sao Paulo, Brazil). Data were analyzed by ANOVA using the GLM procedure (SAS Institute, Inc., Cary, NC, USA), and means were compared by Duncan test at a 5% level. After IVM, the control group (0 M l-NAME) showed a greater number of oocytes in metaphase II (MII: 95.8 ± 3.7%; P 0.05). Many oocytes remained in metaphase I (MI: 18–22%). Cleavage rates at 48 h IVC was not affected (77–88%; P > 0.05). Blastocyst rates (34.0 ± 7.2% to 41.5 ± 4.8%; P > 0.05) and total cell numbers (151 to 174) were also unaffected by NO inhibition by l-NAME. However, the number of TUNEL-positive cells was lower in the control group (1.4 ± 4.7; P 0.05). In conclusion, NO synthesis inhibition in oocytes during IVM reduces nuclear maturation, particularly during MI–MII transition, and increases apoptosis in blastocysts, suggesting that NO may be involved in oocyte maturation and apoptosis protection.