Claudia Lima Verde Leal - Academia.edu (original) (raw)
Papers by Claudia Lima Verde Leal
Reproduction in Domestic Animals, 2009
This study aimed to assess the effects of cyclin-dependent kinase (CDK) inhibition on factors inv... more 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.
Growth factors play an important role during early ovarian development and folliculogenesis, sinc... more Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9) and the bone morphogenetic protein 15 (BMP15), belong to the transforming growth factor beta (TGF-β) superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR). These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regu...
Semina: Ciências Agrárias, 2019
We evaluated the competence of bovine oocytes via morphological selection associated with follicu... more We evaluated the competence of bovine oocytes via morphological selection associated with follicular diameter and by brilliant cresyl blue (BCB) staining, in order to improve oocyte selection and the efficiency of in vitro embryo production. Follicles with small (2 < 4 mm), large (4–8 mm) or mixed (2–8 mm) diameters were aspirated from slaughterhouse ovaries. Next, the morphologically categorized oocytes were stained and evaluated with 26 ?M of BCB (90 min), followed by an assessment of in vitro maturation (MII) and blastocyst production. Oocytes from the large (61.1 ± 3.9%) and mixed (52.9 ± 4.6%) follicular groups showed comparable (p > 0,05) BCB+ rates, but both differed (p < 0.05) as compared to the small-diameter group (41.0 ± 4.5%). Between the BCB+ and BCB – oocytes, there was no difference (p > 0,05) in MII rate in the mixed (respectively 86.7 ± 3.4 and 75.8 ± 2.2), large (respectively 89.4 ± 2.6 and 79.7 ± 2.3) and small (respectively 76.7 ± 2.9 and 67.2 ± 2.5) ...
Zygote (Cambridge, England), Jan 27, 2017
This study aimed to examine the effects of nitric oxide (NO) and different phosphodiesterase (PDE... more This study aimed to examine the effects of nitric oxide (NO) and different phosphodiesterase (PDE) families on meiosis resumption, nucleotides levels and embryo production. Experiment I, COCs were matured in vitro with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) associated or not with the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), meiotic resumption and nucleotides levels were assessed. SNAP delayed germinal vesicle breakdown (GVBD) (53.4 ± 1.2 versus 78.4 ± 2.4% for controls, P 0.05). Cyclic GMP levels were higher in SNAP (3.94 ± 0.18, P 0.05). Embryo development did not differ from the control for SNAP and cilostamide groups (38.7 ± 5.8, 37.9 ± 6.2 and 40.5 ± 5.8%, P > 0.05), but SNAP + cilostamide decreased embryo production (25.7 ± 6.9%, P < 0.05). In conclusion, SNAP was confirmed to delay meiosis resumption by the NO/sGC/cGMP pathway, by increasing cGMP, but not cAMP. Inhibiting different PDEs to further increase ...
Theriogenology, 2017
The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction... more The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 μM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 μM: 128.5; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 μM: 173.5; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) than controls for 2.5 μM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 μM: 101.3, F 5 μM: 115.4, F 10 μM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 μM: 16.7%, F 5 μM: 11.1%, F 10 μM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 μM: 37.3%, F 5 μM: 33.2%, F 10 μM: 30.3%; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells.
Asian-Australasian Journal of Animal Sciences, 2015
Growth factors play an important role during early ovarian development and folliculogenesis, sinc... more Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9) and the bone morphogenetic protein 15 (BMP15), belong to the transforming growth factor beta (TGF-β) superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR). These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regulate absorption of amino acids, glycolysis and biosynthesis of cholesterol cumulus cells. Even though the mode of action has not been fully established, in vitro observations indicate that the factors GDF9 and BMP15 stimulate the growth of ovarian follicles and proliferation of cumulus cells through the induction of mitosis in cells and granulosa and theca expression of genes linked to follicular maturation. Thus, seeking greater understanding of the action of these growth factors on the development of oocytes, the role of GDF9 and BMP15 in ovarian function is summarized in this brief review.
Background : A broader view of living systems complexity is bringing important contributions to b... more Background : A broader view of living systems complexity is bringing important contributions to biological sciences, since the genome expression is affected by other classes of molecules, which in their turn interact themselves in cellular metabolic pathways and biochemical networks. This level of information has been made possible by the emergence of the omic strategies, such as proteomics, metabolomics and lipidomics, that are mainly based on mass spectrometry (MS) platforms. MS has presented an incredible development over the last years, evolving to a powerful and universal analytical technique. Its ability to analyze proteins and small molecules such as lipids, sugars and metabolites at the structural level, with sensitivity and speed inconceivable a few years ago, is the major driving force in the omic fields. The development of electrospray and matrix-assisted laser desorption/ionization (MALDI) ionization techniques has decisively contributed to the many applications of this ...
Zygote, 2015
SummaryThe inhibition of nuclear maturation allows time for the oocyte to accumulate molecules th... more SummaryThe inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, ...
Systems Biology in Reproductive Medicine, 2014
The effect of different sperm washing-selection methods on sperm morphometric characteristics as ... more The effect of different sperm washing-selection methods on sperm morphometric characteristics as a study to detect differences in the subpopulational structure has been carried out in detail in a bovine model. Cryopreserved sperm samples from 5 bulls were thawed, pooled, and processed by TALP-washing centrifugation method (TWCM), selective Percoll discontinuous density-gradient centrifugation method (PDGM), and self-migration swim-up separation method (SUMM). Live-dead assay (SYBR-14/ethidium homodimer-1), chlortetracycline assay (CTC), and sperm motility were assessed, and aliquots of sperm were processed for automated sperm morphometry analysis (ASMA) simultaneously before (raw thawed sperm used as control, RTS) and after different sperm washing-selection techniques. Deleterious effects of different methods were evident, particularly on sperm membrane integrity (p50.05) and capacitation status (p50.05). Moreover, each cell was measured for four primary dimensional parameters, and three shape parameters. All sperm morphometric parameters evaluated were analyzed by principal component analysis (PCA) and multivariate clustering analyses. PCA revealed two principal components for each sperm washing or separation method explaining more than the 91% of the variance. The number of subpopulations found was the same for all methods (four) except for PDGM (three). However, irrespective of the number of subpopulations defined by PCA and clustering analyses, the sperm subpopulational structure was found to be different and strongly influenced by the sperm selection procedure due to statistical differences found regarding the sperm biophysical changes induced by each method used (p50.001). It is concluded that different sperm washing-selection methods commonly used during IVF process, may lead to alterations in sperm morphometric characteristics, which might explain the different results seen after IVF, since an important influence of these methods on sperm subpopulational structure has been demonstrated.
Reproduction, Fertility and Development, 2015
Granulosa cells (GC) are important constituents of the follicular environment for oocyte competen... more Granulosa cells (GC) are important constituents of the follicular environment for oocyte competence acquisition. However, their functionality depends on oocyte-derived factors, such as GDF9 and BMP15, which act through BMPRII receptor signalling. Gene silencing using lipofection has been used as an important tool to investigate the role of cell genes and proteins. The aim of this study was to establish the ideal conditions for lipofection in bovine GC and starting from this protocol to establish a methodology for silencing of the BMPRII gene by RNA interference, and to use this strategy to study the functions of BMPRII in GDF9 signalling. GC were obtained from slaughterhouse ovaries by aspiration of follicles (2 to 6 mm) and subsequently cultured in DMEM medium at 38.5°C and 5% CO2 in air. All data analyzes were performed by GraphPad Prism® version 5.0 software (GraphPad Software Inc., La Jolla, CA, USA), using one-way ANOVA followed by Tukey's test. For optimizing the condition...
Reproduction, Fertility and Development, 2015
Melatonin is a pineal hormone related to the control of the circadian cycle, besides the reproduc... more Melatonin is a pineal hormone related to the control of the circadian cycle, besides the reproductive seasonality of some animal species, and has shown positive effects on oocyte maturation and embryo development. The aim of this study was to assess the effects of melatonin on in vivo and in vitro maturation of mouse oocytes. Female F1 hybrids (C57BL/6 × CBA; n = 8 per group/treatment) were used in 3 different treatments (trt) groups: (I) in vivo trt: mice received 2 different doses of melatonin injections, 10 and 20 mg kg–1 per IP including a saline control dose (0 mg kg–1 per IP) for 4 days along with ovarian stimulation trt of 5 IU of eCG IP, followed by 5 IU of hCG IP 48 h later, and cumulus-oocyte complexes (COC) were collected 16 h after hCG; (II) mice received a similar in vivo melatonin trt, but ovarian stimulation trt was only 5 IU of eCG, no hCG, and COC were collected after 48 h and subsequently matured in vitro with 0.5 µg mL–1 of FSH for 16 h; (III) in vitro maturation ...
Zygote (Cambridge, England), Jan 16, 2014
Summary As the standard enucleation method in mammalian nuclear transfer is invasive and damaging... more Summary As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microt...
Reproduction, Fertility and Development, 2008
The present study aimed to assess the transcripts for the proteins that embryos require, histone ... more 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 prem...
Reproduction, Fertility and Development, 2007
The present study aimed to assess the effect of meiosis block on the expression of genes involved... more 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 �M butyrolactone I (BLI) and 3 mg mL-1 BSA (B100) or with 10 �M 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 �g mL-1 LH, 0.5 �g mL-1 FSH, 0.2 mM pyruvate, and 10 �g 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 collect...
Zygote, 2005
As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three dif... more As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p <0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that st...
Theriogenology, 2005
Strontium efficiently activates mouse oocytes, however, there is limited information on its use i... more Strontium efficiently activates mouse oocytes, however, there is limited information on its use in cattle. Thus, the objective of this study was to establish a suitable protocol for activating bovine oocyte with strontium. For pronuclear development, the absence of calcium and magnesium in the activation medium (TALP) with 10 and 50 mM strontium (34.4 and 53.1%, respectively) was superior to the complete TALP (6.5 and 19.4%, respectively). In all activation media, better results were observed with 25 and 50 mM strontium (21.9-53.1 and 19.4-53.1%, respectively). Incubation for 4 h promoted similar results in all strontium concentrations. However, strontium at 15, 20, and 25 mM for 6 and 8 h (40.7, 46.7, and 48.3%, and 29.3, 48.3, and 40.7%, respectively) were superior to control (15.5 and 10%, respectively). After in vitro maturation for 26 h, strontium (S; 20 mM in Ca 2+and Mg 2+-free TALP for 6 h), ionomycin + strontium (IS), and strontium + ionomycin (SI) (60, 63.3, and 65%, respectively) were similar in pronuclear development and superior to ionomycin (I; 5 mM for 5 min; 36.7%). In treatments S and I, only 1 PN zygotes were observed. In treatment S, most of them had 1 and 2 PB (35.7 and 60.7%, respectively), and in treatment I, 0, 1, and 2 PB (14.3, 57.1, and 28.6%, respectively). Most of the zygotes in treatment IS and SI were 1 PN 2 PB (77.4 and 61.6%, respectively). The number of oocytes with clusters of cortical granules was similar in all treated groups (11-29%). Cortical granule exocytosis in treatment IS (68%) was similar to S (54%) and superior to I, SI, and control (27, 45, and 5.0%, respectively). Cleavage and blastocyst rates were www.journals.elsevierhealth.com/periodicals/the
Theriogenology, 2012
In humans and other mammals, sperm morphology has been considered one of the most important predi... more In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P Ͻ 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies.
Reproduction in Domestic Animals, 2011
The aim of this study was to determine the effect of temporary inhibition of meiosis using the cy... more The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulus-oocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 μm BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.
Reproduction, Fertility and Development, 2008
Recent progress in animal cloning by nuclear transfer (NT) has made it feasible to produce transg... more Recent progress in animal cloning by nuclear transfer (NT) has made it feasible to produce transgenic animals using genetically modified cell lines. Healthy cells and competent oocytes are needed to maximize the number of transgenic calves produced. Oocyte maturation plays a central role in oocyte competence. Prematuration inducing meiosis block is, therefore, a possible tool in transgenesis, because it allows further optimization of oocyte maturation protocols. In field conditions, it is not always possible to precisely control timing between oocyte collection and NT procedures. Therefore, temporarily and reversibly blocking maturation may also be used as a strategy to optimize cloning protocols. The aim of this study was to analyze the developmental competence of embryos reconstructed by NT using cells modified genetically as nuclei donors and oocytes submitted, or not, to meiosis block as cytoplasts. The hypothesis was that blocking meiosis does not alter the embryonic developmen...
Reproduction in Domestic Animals, 2009
This study aimed to assess the effects of cyclin-dependent kinase (CDK) inhibition on factors inv... more 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.
Growth factors play an important role during early ovarian development and folliculogenesis, sinc... more Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9) and the bone morphogenetic protein 15 (BMP15), belong to the transforming growth factor beta (TGF-β) superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR). These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regu...
Semina: Ciências Agrárias, 2019
We evaluated the competence of bovine oocytes via morphological selection associated with follicu... more We evaluated the competence of bovine oocytes via morphological selection associated with follicular diameter and by brilliant cresyl blue (BCB) staining, in order to improve oocyte selection and the efficiency of in vitro embryo production. Follicles with small (2 < 4 mm), large (4–8 mm) or mixed (2–8 mm) diameters were aspirated from slaughterhouse ovaries. Next, the morphologically categorized oocytes were stained and evaluated with 26 ?M of BCB (90 min), followed by an assessment of in vitro maturation (MII) and blastocyst production. Oocytes from the large (61.1 ± 3.9%) and mixed (52.9 ± 4.6%) follicular groups showed comparable (p > 0,05) BCB+ rates, but both differed (p < 0.05) as compared to the small-diameter group (41.0 ± 4.5%). Between the BCB+ and BCB – oocytes, there was no difference (p > 0,05) in MII rate in the mixed (respectively 86.7 ± 3.4 and 75.8 ± 2.2), large (respectively 89.4 ± 2.6 and 79.7 ± 2.3) and small (respectively 76.7 ± 2.9 and 67.2 ± 2.5) ...
Zygote (Cambridge, England), Jan 27, 2017
This study aimed to examine the effects of nitric oxide (NO) and different phosphodiesterase (PDE... more This study aimed to examine the effects of nitric oxide (NO) and different phosphodiesterase (PDE) families on meiosis resumption, nucleotides levels and embryo production. Experiment I, COCs were matured in vitro with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) associated or not with the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), meiotic resumption and nucleotides levels were assessed. SNAP delayed germinal vesicle breakdown (GVBD) (53.4 ± 1.2 versus 78.4 ± 2.4% for controls, P 0.05). Cyclic GMP levels were higher in SNAP (3.94 ± 0.18, P 0.05). Embryo development did not differ from the control for SNAP and cilostamide groups (38.7 ± 5.8, 37.9 ± 6.2 and 40.5 ± 5.8%, P > 0.05), but SNAP + cilostamide decreased embryo production (25.7 ± 6.9%, P < 0.05). In conclusion, SNAP was confirmed to delay meiosis resumption by the NO/sGC/cGMP pathway, by increasing cGMP, but not cAMP. Inhibiting different PDEs to further increase ...
Theriogenology, 2017
The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction... more The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 μM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 μM: 128.5; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 μM: 173.5; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) than controls for 2.5 μM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 μM: 101.3, F 5 μM: 115.4, F 10 μM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 μM: 16.7%, F 5 μM: 11.1%, F 10 μM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 μM: 37.3%, F 5 μM: 33.2%, F 10 μM: 30.3%; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells.
Asian-Australasian Journal of Animal Sciences, 2015
Growth factors play an important role during early ovarian development and folliculogenesis, sinc... more Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9) and the bone morphogenetic protein 15 (BMP15), belong to the transforming growth factor beta (TGF-β) superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR). These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regulate absorption of amino acids, glycolysis and biosynthesis of cholesterol cumulus cells. Even though the mode of action has not been fully established, in vitro observations indicate that the factors GDF9 and BMP15 stimulate the growth of ovarian follicles and proliferation of cumulus cells through the induction of mitosis in cells and granulosa and theca expression of genes linked to follicular maturation. Thus, seeking greater understanding of the action of these growth factors on the development of oocytes, the role of GDF9 and BMP15 in ovarian function is summarized in this brief review.
Background : A broader view of living systems complexity is bringing important contributions to b... more Background : A broader view of living systems complexity is bringing important contributions to biological sciences, since the genome expression is affected by other classes of molecules, which in their turn interact themselves in cellular metabolic pathways and biochemical networks. This level of information has been made possible by the emergence of the omic strategies, such as proteomics, metabolomics and lipidomics, that are mainly based on mass spectrometry (MS) platforms. MS has presented an incredible development over the last years, evolving to a powerful and universal analytical technique. Its ability to analyze proteins and small molecules such as lipids, sugars and metabolites at the structural level, with sensitivity and speed inconceivable a few years ago, is the major driving force in the omic fields. The development of electrospray and matrix-assisted laser desorption/ionization (MALDI) ionization techniques has decisively contributed to the many applications of this ...
Zygote, 2015
SummaryThe inhibition of nuclear maturation allows time for the oocyte to accumulate molecules th... more SummaryThe inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, ...
Systems Biology in Reproductive Medicine, 2014
The effect of different sperm washing-selection methods on sperm morphometric characteristics as ... more The effect of different sperm washing-selection methods on sperm morphometric characteristics as a study to detect differences in the subpopulational structure has been carried out in detail in a bovine model. Cryopreserved sperm samples from 5 bulls were thawed, pooled, and processed by TALP-washing centrifugation method (TWCM), selective Percoll discontinuous density-gradient centrifugation method (PDGM), and self-migration swim-up separation method (SUMM). Live-dead assay (SYBR-14/ethidium homodimer-1), chlortetracycline assay (CTC), and sperm motility were assessed, and aliquots of sperm were processed for automated sperm morphometry analysis (ASMA) simultaneously before (raw thawed sperm used as control, RTS) and after different sperm washing-selection techniques. Deleterious effects of different methods were evident, particularly on sperm membrane integrity (p50.05) and capacitation status (p50.05). Moreover, each cell was measured for four primary dimensional parameters, and three shape parameters. All sperm morphometric parameters evaluated were analyzed by principal component analysis (PCA) and multivariate clustering analyses. PCA revealed two principal components for each sperm washing or separation method explaining more than the 91% of the variance. The number of subpopulations found was the same for all methods (four) except for PDGM (three). However, irrespective of the number of subpopulations defined by PCA and clustering analyses, the sperm subpopulational structure was found to be different and strongly influenced by the sperm selection procedure due to statistical differences found regarding the sperm biophysical changes induced by each method used (p50.001). It is concluded that different sperm washing-selection methods commonly used during IVF process, may lead to alterations in sperm morphometric characteristics, which might explain the different results seen after IVF, since an important influence of these methods on sperm subpopulational structure has been demonstrated.
Reproduction, Fertility and Development, 2015
Granulosa cells (GC) are important constituents of the follicular environment for oocyte competen... more Granulosa cells (GC) are important constituents of the follicular environment for oocyte competence acquisition. However, their functionality depends on oocyte-derived factors, such as GDF9 and BMP15, which act through BMPRII receptor signalling. Gene silencing using lipofection has been used as an important tool to investigate the role of cell genes and proteins. The aim of this study was to establish the ideal conditions for lipofection in bovine GC and starting from this protocol to establish a methodology for silencing of the BMPRII gene by RNA interference, and to use this strategy to study the functions of BMPRII in GDF9 signalling. GC were obtained from slaughterhouse ovaries by aspiration of follicles (2 to 6 mm) and subsequently cultured in DMEM medium at 38.5°C and 5% CO2 in air. All data analyzes were performed by GraphPad Prism® version 5.0 software (GraphPad Software Inc., La Jolla, CA, USA), using one-way ANOVA followed by Tukey's test. For optimizing the condition...
Reproduction, Fertility and Development, 2015
Melatonin is a pineal hormone related to the control of the circadian cycle, besides the reproduc... more Melatonin is a pineal hormone related to the control of the circadian cycle, besides the reproductive seasonality of some animal species, and has shown positive effects on oocyte maturation and embryo development. The aim of this study was to assess the effects of melatonin on in vivo and in vitro maturation of mouse oocytes. Female F1 hybrids (C57BL/6 × CBA; n = 8 per group/treatment) were used in 3 different treatments (trt) groups: (I) in vivo trt: mice received 2 different doses of melatonin injections, 10 and 20 mg kg–1 per IP including a saline control dose (0 mg kg–1 per IP) for 4 days along with ovarian stimulation trt of 5 IU of eCG IP, followed by 5 IU of hCG IP 48 h later, and cumulus-oocyte complexes (COC) were collected 16 h after hCG; (II) mice received a similar in vivo melatonin trt, but ovarian stimulation trt was only 5 IU of eCG, no hCG, and COC were collected after 48 h and subsequently matured in vitro with 0.5 µg mL–1 of FSH for 16 h; (III) in vitro maturation ...
Zygote (Cambridge, England), Jan 16, 2014
Summary As the standard enucleation method in mammalian nuclear transfer is invasive and damaging... more Summary As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microt...
Reproduction, Fertility and Development, 2008
The present study aimed to assess the transcripts for the proteins that embryos require, histone ... more 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 prem...
Reproduction, Fertility and Development, 2007
The present study aimed to assess the effect of meiosis block on the expression of genes involved... more 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 �M butyrolactone I (BLI) and 3 mg mL-1 BSA (B100) or with 10 �M 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 �g mL-1 LH, 0.5 �g mL-1 FSH, 0.2 mM pyruvate, and 10 �g 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 collect...
Zygote, 2005
As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three dif... more As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p <0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that st...
Theriogenology, 2005
Strontium efficiently activates mouse oocytes, however, there is limited information on its use i... more Strontium efficiently activates mouse oocytes, however, there is limited information on its use in cattle. Thus, the objective of this study was to establish a suitable protocol for activating bovine oocyte with strontium. For pronuclear development, the absence of calcium and magnesium in the activation medium (TALP) with 10 and 50 mM strontium (34.4 and 53.1%, respectively) was superior to the complete TALP (6.5 and 19.4%, respectively). In all activation media, better results were observed with 25 and 50 mM strontium (21.9-53.1 and 19.4-53.1%, respectively). Incubation for 4 h promoted similar results in all strontium concentrations. However, strontium at 15, 20, and 25 mM for 6 and 8 h (40.7, 46.7, and 48.3%, and 29.3, 48.3, and 40.7%, respectively) were superior to control (15.5 and 10%, respectively). After in vitro maturation for 26 h, strontium (S; 20 mM in Ca 2+and Mg 2+-free TALP for 6 h), ionomycin + strontium (IS), and strontium + ionomycin (SI) (60, 63.3, and 65%, respectively) were similar in pronuclear development and superior to ionomycin (I; 5 mM for 5 min; 36.7%). In treatments S and I, only 1 PN zygotes were observed. In treatment S, most of them had 1 and 2 PB (35.7 and 60.7%, respectively), and in treatment I, 0, 1, and 2 PB (14.3, 57.1, and 28.6%, respectively). Most of the zygotes in treatment IS and SI were 1 PN 2 PB (77.4 and 61.6%, respectively). The number of oocytes with clusters of cortical granules was similar in all treated groups (11-29%). Cortical granule exocytosis in treatment IS (68%) was similar to S (54%) and superior to I, SI, and control (27, 45, and 5.0%, respectively). Cleavage and blastocyst rates were www.journals.elsevierhealth.com/periodicals/the
Theriogenology, 2012
In humans and other mammals, sperm morphology has been considered one of the most important predi... more In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P Ͻ 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies.
Reproduction in Domestic Animals, 2011
The aim of this study was to determine the effect of temporary inhibition of meiosis using the cy... more The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulus-oocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 μm BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.
Reproduction, Fertility and Development, 2008
Recent progress in animal cloning by nuclear transfer (NT) has made it feasible to produce transg... more Recent progress in animal cloning by nuclear transfer (NT) has made it feasible to produce transgenic animals using genetically modified cell lines. Healthy cells and competent oocytes are needed to maximize the number of transgenic calves produced. Oocyte maturation plays a central role in oocyte competence. Prematuration inducing meiosis block is, therefore, a possible tool in transgenesis, because it allows further optimization of oocyte maturation protocols. In field conditions, it is not always possible to precisely control timing between oocyte collection and NT procedures. Therefore, temporarily and reversibly blocking maturation may also be used as a strategy to optimize cloning protocols. The aim of this study was to analyze the developmental competence of embryos reconstructed by NT using cells modified genetically as nuclei donors and oocytes submitted, or not, to meiosis block as cytoplasts. The hypothesis was that blocking meiosis does not alter the embryonic developmen...