Oocyte Cytoplasmic Maturation Research Articles

Oocyte zona pellucida and meiotic spindle birefringence as a biomarker of pregnancy rate outcome in IVF-ICSI treatment.

IVF-ICSI procedures are accompanied by a continuous search for predictors of ART outcome. The properties of zona pellucida (ZP) have been believed to reflect the history of oocyte cytoplasmic maturation. The meiotic spindle (MS) is crucial for chromosomal alignment and proper separation of the maternal chromosomes. There is data suggesting that birefringent ZP and MS can clinically predict the oocyte quality and developmental potential of an embryo. The aim of the study was to examine the possible effect of ZP birefringent properties and MS visualization and localization as valuable predictors of IVF-ICSI effectiveness. The prospective study was performed during a 16-month period. A total of 51 patients undergoing in vitro fertilization--embryo transfer (IVF-ET) treatment procedure with intracytoplasmic sperm injection (ICSI) were included. Controlled ovarian hyperstimulation (COH) was done using either a long n = 32 (62.75%) or an antagonist protocol n = 19. In the group of the 48 examined patients (aged 25-40), 46 ET were performed, resulting in 24 positive pregnancy tests and 19 (39.59%) clinical pregnancies. Oocytes were examined as follows: ZP birefringence autoscoring (OCTAX PolarAIDE), numeral autoscoring, thickness and clinical evaluation; MS visualization, if MS was visualized, localization of MS in relation to the polar body (PB). On day 3, 64.3% of the embryos were of good and 40.3% were of top quality. Visible differences, not statistically significant, were observed in the numeral score of ZP between oocytes selected and non-selected for ET. In cases when embryos were not of good or top quality, ZP score was higher (p = 0.005 p = 0.001). ZP manual evaluation indicated significantly stronger birefringence when pregnancy was not achieved (p = 0.022). The rate of MS positive oocytes was the highest in the group with pregnancy but it did not reach statistical significance (p = 0.471). The MS localization in relation to the PB was in most oocytes very close (< 45 degrees) in 70.9% and not different in the studied groups. Unexpected polarization microscopy imaging and rating of ZP and MS cannot be a direct predictor of the IVF outcome.

173 EFFECTS OF GLUCOSE METABOLISM DURING IN VITRO MATURATION ON CYTOPLASMIC MATURATION OF GOAT OOCYTES

It is well known that oocyte maturation consists of 2 processes: nuclear maturation and cytoplasmic maturation. Nuclear maturation refers to resumption of the first meiosis and extrusion of the first polar body (PB1), and cytoplasmic maturation is manifested as acquisition of the ability to complete pre-implantation development. Although it is recognised that energy supply is essential for oocyte maturation and there have been many reports on the effect of glucose metabolism on oocyte nuclear maturation, studies on the effect of glucose metabolism on ooplasmic maturation are limited. In the present study, goat oocytes recovered from slaughterhouse ovaries were cultured for 24 h in a simplified CR1 (sCR1) medium (NaCl, KCl, NaHCO3, CaCl2, BSA, and eCG) supplemented with glucose (10 mM) and/or lactate (3.5 mM) in the presence or absence of pentose phosphate pathway (PPP) inhibitor dehydroepiandrosterone (DHEA, 100 μM) or glycolysis inhibitor iodoacetate (1 μM). At the end of maturation culture, oocytes with PB1 were either activated by treatment with ionomycin plus 6-DMAP to observe embryo development, or assayed for total glutathione concentrations (GSX) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios. Embryos were cultured for 9 days in CR1aa medium (NaCl, KCl, NaHCO3, calcium lactate, sodium pyruvate, glutamine, EAA, NEAA, and FCS) at 38.5°C under 5% CO2 in humidified air. In the absence of inhibitors, oocyte maturation rates of 82, 65, and 76%, and blastocyst rates of 7, 0, and 7%, were obtained, respectively, after oocytes were matured in sCR1 supplemented with glucose, lactate, or both. When oocytes were matured in sCR1 containing glucose and lactate in the presence of DHEA or iodoacetate, oocyte maturation rates were 69 and 67%, respectively, with no blastocyst produced in either case. However, whereas the presence of DHEA produced 12% morulae, no morulae were observed in the presence of iodoacetate. Furthermore, GSX concentrations (pmol/oocyte) were 8.5, 6.5, and 7.2, whereas GSH/GSSG ratios were 1.8, 0.3, and 0.5, respectively, after oocyte maturation without inhibitors or with 300 μM DHEA or 3 μM iodoacetate. The difference in GSX concentration was statistically significant (P &lt; 0.05; one-way ANOVA) between DHEA and iodoacetate. In conclusion, using a culture system (sCR1 containing 3.5 mM lactate) that sustained oocyte nuclear maturation but did not support blastocyst development, we have studied the effect of PPP and glycolysis of glucose metabolism on the cytoplasmic maturation of goat oocytes. The results suggest that both PPP and glycolysis are essential for ooplasmic maturation of goat oocytes, and that both promote oocyte cytoplasmic maturation by increasing glutathione synthesis and reduction. This study was supported by grants from the National Basic Research Program of China (Nos. 2012CB944403 and 2014CB138503) and the China National Natural Science Foundation (Nos. 31272444 and 30972096).

Embryo gene expression in response to maternal supplementation with glycogenic precursors in the rabbit

Disturbing maternal metabolism during the first pregnancy and postpartum period is associated with sub fertility in rabbit does. Nutritional strategies can be used during those periods and its effects to improve reproductive management may affect periconceptional events and early embryo development. Our goal was to elucidate if treatment with a glycogenic precursor such as propylene glycol (PG) could affect the maternal metabolic profile, follicular and oocyte quality and gene expression patterns in early embryos. Rabbit does were supplemented with 2.5% (v/v) PG from either mid-pregnancy and for 25 days of lactation (PG-GL group); only during lactation (PG-L group); or were not treated (control group). Ovarian parameters and embryos were studied at the end of treatment. At parturition serum non-esterified fatty acid concentrations increased whilst insulin decreased in all groups. Maternal feed intake was reduced in PG-supplemented does but glycaemia was maintained during the experimental period. When PG was suppressed, blood insulin immediately increased in PG-groups, but no differences in follicular population, follicular atresia, and nuclear and cytoplasmic oocyte maturation were observed compared with non-treated animals. Although embryo development was similar among groups, mRNA of SLC2A4, INSR, IGF1R, PLAC8, COX2 and IGF2R were up regulated in the blastocysts of PG-GL does. Transcripts of SOD1 were lower in PG-L embryos; but NOS3 and TP53 were similar among groups. PG did not affect the maternal metabolic profile during the postpartum period, nor the ovarian response or number of embryos developed. Nonetheless, PG supplied from mid-pregnancy modified mRNA transcripts involved in some important developmental and metabolic events in the blastocysts of those females. More experiments are needed to elucidate the physiological consequence of these results.

Oocyte and sperm factors implicated in fertilization failure after ICSI

ObjectiveTo identify and report some factors involved in fertilization failures after ICSI.DesignDescriptive.Materials and MethodsAt the present study, 2300 human oocytes with fertilization failure after ICSI were considered. Nine hundred corresponded to sub-optimal fertilization (<50%). For oocyte assessment, fixation and subsequent processing was performed. Acetylated alpha tubulin, beta tubulin and MPF (CyclinB-Cdc2) immuno-staining was performed using monoclonal antibodies. DNA detection was carried out with Hoechst-33342 and TUNEL (DNA damage). When oocyte activation failure and sperm nuclear decondensation failure were observed, Western-Blot and immunocytochemistry detection were performed over semen samples for PLCzeta (oocyte activation protein) and nuclear sperm in-vitro decondensation test was also assessed.ResultsTable 1Fertilization failures eventsICSIFert<50%TOTAL# oocytes14009002300Sperm absence8%3%6%Premature chromosome condensation (oocyte cytoplasmic immaturity)28%50%36.6%Sperm nuclear decondensation failure19%30%23.3%Pronuclear formation defects18%6%13.3%1st mitotic division arrest15%6%11.5Others12%5%9.3% Open table in a new tab Specifically, when oocyte activation failures occurred concomitantly with sperm nuclear decondensation failure (155 oocytes), 13.2% depicted a significant decrease in PLCzeta levels, 21.2% presented sperm DNA fragmentation, 39.4% showed several levels of sperm nuclear decondensation and 10.5% of the oocytes had an inability to perform an adequate chromatin remodeling, probably due to lower levels of glutation.ConclusionIn general, fertilization failure occurs in 28% of cases for an incomplete oocyte cytoplasmic maturation. This value is increased to 50% when fertilization failures take place in ≥ 50% of the oocyte cohort. The second most recurrent event was sperm DNA decondensation failure, 39.4% depicted variable decondensation levels. Oocyte features has more implications over fertilization failures after ICSI. ObjectiveTo identify and report some factors involved in fertilization failures after ICSI. To identify and report some factors involved in fertilization failures after ICSI. DesignDescriptive. Descriptive. Materials and MethodsAt the present study, 2300 human oocytes with fertilization failure after ICSI were considered. Nine hundred corresponded to sub-optimal fertilization (<50%). For oocyte assessment, fixation and subsequent processing was performed. Acetylated alpha tubulin, beta tubulin and MPF (CyclinB-Cdc2) immuno-staining was performed using monoclonal antibodies. DNA detection was carried out with Hoechst-33342 and TUNEL (DNA damage). When oocyte activation failure and sperm nuclear decondensation failure were observed, Western-Blot and immunocytochemistry detection were performed over semen samples for PLCzeta (oocyte activation protein) and nuclear sperm in-vitro decondensation test was also assessed. At the present study, 2300 human oocytes with fertilization failure after ICSI were considered. Nine hundred corresponded to sub-optimal fertilization (<50%). For oocyte assessment, fixation and subsequent processing was performed. Acetylated alpha tubulin, beta tubulin and MPF (CyclinB-Cdc2) immuno-staining was performed using monoclonal antibodies. DNA detection was carried out with Hoechst-33342 and TUNEL (DNA damage). When oocyte activation failure and sperm nuclear decondensation failure were observed, Western-Blot and immunocytochemistry detection were performed over semen samples for PLCzeta (oocyte activation protein) and nuclear sperm in-vitro decondensation test was also assessed. ResultsTable 1Fertilization failures eventsICSIFert<50%TOTAL# oocytes14009002300Sperm absence8%3%6%Premature chromosome condensation (oocyte cytoplasmic immaturity)28%50%36.6%Sperm nuclear decondensation failure19%30%23.3%Pronuclear formation defects18%6%13.3%1st mitotic division arrest15%6%11.5Others12%5%9.3% Open table in a new tab Specifically, when oocyte activation failures occurred concomitantly with sperm nuclear decondensation failure (155 oocytes), 13.2% depicted a significant decrease in PLCzeta levels, 21.2% presented sperm DNA fragmentation, 39.4% showed several levels of sperm nuclear decondensation and 10.5% of the oocytes had an inability to perform an adequate chromatin remodeling, probably due to lower levels of glutation. Specifically, when oocyte activation failures occurred concomitantly with sperm nuclear decondensation failure (155 oocytes), 13.2% depicted a significant decrease in PLCzeta levels, 21.2% presented sperm DNA fragmentation, 39.4% showed several levels of sperm nuclear decondensation and 10.5% of the oocytes had an inability to perform an adequate chromatin remodeling, probably due to lower levels of glutation. ConclusionIn general, fertilization failure occurs in 28% of cases for an incomplete oocyte cytoplasmic maturation. This value is increased to 50% when fertilization failures take place in ≥ 50% of the oocyte cohort. The second most recurrent event was sperm DNA decondensation failure, 39.4% depicted variable decondensation levels. Oocyte features has more implications over fertilization failures after ICSI. In general, fertilization failure occurs in 28% of cases for an incomplete oocyte cytoplasmic maturation. This value is increased to 50% when fertilization failures take place in ≥ 50% of the oocyte cohort. The second most recurrent event was sperm DNA decondensation failure, 39.4% depicted variable decondensation levels. Oocyte features has more implications over fertilization failures after ICSI.

Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes

BackgroundThe objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development.MethodsCumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at −50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose–response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid (SOF, n = 212); 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) tissue culture medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days.ResultsImmunoreactivity for both α and β GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control (SOF alone, P = 0.2).ConclusionsGM-CSF enhanced cumulus cell expansion of in vitro matured bovine COC. However, GM-CSF did not increase oocyte nuclear or cytoplasmic maturation rates, IGF-2 expression or subsequent embryonic development.

Absence of cumulus cells during in vitro maturation affects lipid metabolism in bovine oocytes

Cumulus cells (CC) surround the oocyte and are coupled metabolically through regulation of nutrient intake. CC removal before in vitro maturation (IVM) decreases bovine oocyte developmental competence without affecting nuclear meiotic maturation. The objective was to investigate the influence of CC on oocyte cytoplasmic maturation in relation to energy metabolism. IVM with either cumulus-enclosed (CEO) or -denuded (DO) oocytes was performed in serum-free metabolically optimized medium. Transmission electron microscopy revealed different distribution of membrane-bound vesicles and lipid droplets between metaphase II DO and CEO. By Nile Red staining, a significant reduction in total lipid level was evidenced in DO. Global transcriptomic analysis revealed differential expression of genes regulating energy metabolism, transcription, and translation between CEO and DO. By Western blot, fatty acid synthase (FAS) and hormone-sensitive phospholipase (HSL) proteins were detected in oocytes and in CC, indicating a local lipogenesis and lypolysis. FAS protein was significantly less abundant in DO that in CEO and more highly expressed in CC than in the oocytes. On the contrary, HSL protein was more abundant in oocytes than in CC. In addition, active Ser⁵⁶³-phosphorylated HSL was detected in the oocytes only after IVM, and its level was similar in CEO and DO. In conclusion, absence of CC during IVM affected lipid metabolism in the oocyte and led to suboptimal cytoplasmic maturation. Thus, CC may influence the oocyte by orienting the consumption of nutritive storage via regulation of local fatty acid synthesis and lipolysis to provide energy for maturation.

255 EXAMINING THE EFFECTS OF INSULIN-LIKE GROWTH FACTOR-I ON THE MATURATION AND DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES EXPOSED TO HEAT SHOCK

Insulin-like growth factor-I (IGF-I) has been suggested as a survival factor for pre-implantation bovine embryos exposed to heat shock (HS). Therefore, the aims of the study were 1) to examine the protective effects of IGF-I on the developmental competence of bovine oocytes exposed to HS, particularly the effects on oocyte cytoplasmic and nuclear maturation, and 2) to examine whether IGF-I administration contracts HS-induced apoptosis in bovine oocytes. In vitro maturation/IVF/in vitro-production procedures were performed as described previously by Gendelman and Roth (2012). Briefly, cumulus–oocyte complexes (n = 250 to 300/group; 5 replicates) were matured (TCM-199 with Earle’s salts; 22 h, 5% CO2) at 38.5°C or exposed to HS (41°C) with or without 100 µg of IGF-I (Sigma, St. Louis, MO, USA). Matured oocytes were IVF (18 h, 38.5°C, 5% CO2) and cultured in K simplex optimized medium (38.5°C, 5% CO2, 5% O2) for 8 days. Cleavage rates for 2- and 4-cell-stage embryos were assessed at 42 h post-fertilization. For each experimental group, a subgroup of matured oocytes (n = 50) was examined at the end of maturation for nuclear status (1 µg mL–1 of Hoechst 33342, Sigma), cortical granule migration (fluorescein isothiocyanate-Lens culinaris agglutinin, Sigma) and apoptotic status (TUNEL, Roche, Basel, Switzerland). Data were analysed by one-way ANOVA (JMP-6, SAS Institute Inc., Cary, NC, USA) followed by Student’s t-test. Data are presented as mean ± SE. The proportion of oocytes that cleaved to the 2- to 4-cell-stage embryos was lower in the HS group than in the control group (56.55 ± 4.49% v. 75.6 ± 4.16%, respectively; P &lt; 0.05). Although not significant, IGF-I increased the proportions of heat-stressed oocytes that cleaved to the 2- to 4-cell stage (62.32 ± 4.49% v. 56.55 ± 4.49%, for HS + IGF-I and HS, respectively). Neither maturation at 41.5°C nor IGF-I supplementation had any effect on cortical granule migration because the proportions of oocytes with a type I, type II, and type III cortical granule distribution were similar in the control and HS groups. However, the proportion of oocytes that underwent nuclear maturation (i.e. having a nucleus at the telophase-I or metaphase-II stages) was significantly lower in the HS group than in the control group (P &lt; 0.01), and IGF-I slightly increased their proportion in HS oocytes (nonsignificant). The proportion of TUNEL-positive oocytes tended to be higher in the HS group compared with the control group (47.9 ± 12.2% v. 28.0 ± 12.2%, respectively; P ≤ 0.09), and IGF-I decreased the proportion of TUNEL-positive oocytes in the HS group to a level (27.4 ± 12.2%) similar to that noted in the control group. In summary, exposing bovine oocytes to a physiologically relevant thermal stress impaired their ability to undergo first cleavages, most likely because of alteration in nuclear rather than cytoplasmic maturation. Insulin-like growth factor-I was found to slightly alleviate the deleterious effects of heat shock on bovine oocytes.

Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis

To compare nuclear and cytoplasmic RNA from a single cell type, free of cross-contamination, we studied the oocyte of the frog Xenopus tropicalis, a giant cell with an equally giant nucleus. We isolated RNA from manually dissected nuclei and cytoplasm of mature oocytes and subjected it to deep sequencing. Cytoplasmic mRNA consisted primarily of spliced exons derived from ∼6700 annotated genes. Nearly all of these genes were represented in the nucleus by intronic sequences. However, unspliced nascent transcripts were not detected. Inhibition of transcription or splicing for 1-2 d had little or no effect on the abundance of nuclear intronic sequences, demonstrating that they are unusually stable. RT-PCR analysis showed that these stable intronic sequences are transcribed from the coding strand and that a given intron can be processed into more than one molecule. Stable intronic sequence RNA (sisRNA) from the oocyte nucleus constitutes a new class of noncoding RNA. sisRNA is detectable by RT-PCR in samples of total RNA from embryos up to the mid-blastula stage, when zygotic transcription begins. Storage of sisRNA in the oocyte nucleus and its transmission to the developing embryo suggest that it may play important regulatory roles during oogenesis and/or early embryogenesis.

Ovarian response and embryo gene expression patterns after nonsuperovulatory gonadotropin stimulation in primiparous rabbits does

Ovarian stimulation with equine chorionic gonadotropin (eCG) is largely used in animal reproductive technologies to provide a larger number of oocytes and embryos and to improve the reproductive outcome. However, the consequences of maternal treatment with eCG on embryo gene expression patterns are not widely studied. The aim of this work was to assess the ovarian response (preovulatory follicular population, oocyte maturation, ovulation rate, and serum steroid concentrations), the early embryo survival and gene expression patterns of a panel of quality-genes involved in glucose intake, oxidative stress, apoptosis, proliferation, implantation, and fetal growth in embryos of lactating rabbits treated with eCG. A total of 34 primiparous rabbit does (Oryctolagus cuniculus) were randomly distributed at Day 23 postpartum into a treatment group receiving a unique nonsuperovulatory dose (25 IU) of eCG (eCG group; N = 17 does); or a control group without eCG treatment previously to artificial insemination (control group; N = 17 does). After 48 hours, 8 does of each group were euthanized and their ovarian response was studied. The rest of animals were artificially inseminated and their ovulation was induced with a GnRH analogue. Embryos were recovered 3.5 days later. The oocytes retrieved for in vitro maturation showed no differences in metaphase II rate in both experimental groups, although oocyte cytoplasmic maturation, in terms of cortical granule migration rate, was improved in eCG-treated does (P < 0.05). The mean number of preovulatory follicles was similar between groups but the ovulation rate was significantly higher in eCG-treated does compared with does not stimulated (P < 0.05). No differences were found in serum estradiol and progesterone concentrations of does the day of oocyte and embryo recovery, respectively. However, progesterone:estradiol ratio was slightly increased in eCG group on embryo recovery day (P = 0.1). The percentage of embryos recovered at the blastocyst stage was also increased in eCG-treated does (P < 0.05), nevertheless, there were no differences in the gene expression patterns of candidate genes SLC2A4, IGF1R, IGF2R, SHC1-SHC, TP53, PTGS2, and PLAC8; except for the transcripts of SOD1 mRNA which were downregulated in eCG-derived embryos (P < 0.05). In conclusion, the administration of eCG improves ovulation rate, oocyte cytoplasmic maturation, and blastocyst formation in primiparous rabbit does inseminated on Day 25 postpartum. Although it seems not to influence the gene expression patterns studied, a lower antioxidant defense of embryos developed after the maternal eCG treatment is suggested.

No single way to explain cytoplasmic maturation of oocytes from prepubertal and cyclic gilts

The objective of this study was to evaluate selected aspects of cytoplasmic maturation in oocytes from prepubertal and cyclic crossbred gilts before and after in vitro maturation. For this purpose, cortical granule redistribution, mitochondrial DNA content and mitochondria translocation were analyzed. Moreover, for the first time the fatty acid profiles in follicular fluid (FF) of both gilt categories was evaluated. The nuclear maturation (the percentage of metaphase II oocytes was 83% in prepubertal gilts compared with 87% in cyclic gilts), cortical granule relocation from the cortex to peripheral ooplasm (98.7% vs. 98.8% of oocytes, respectively) and mitochondrial DNA content (227 543 vs. 206 660, respectively) was not affected by sexual maturity of the donor gilt. However, the redistribution of active mitochondria during in vitro maturation was observed only in the oocytes of cyclic gilts. With regard to FF analysis, saturated, unsaturated, and monounsaturated fatty acids were significantly more abundant in the FF of prepubertal females. In particular, stearic (C18:0) and palmitic (C16:0) fatty acids had significantly higher concentrations in the FF of prepubertal gilts. In conclusion, although the oocytes of prepubertal gilts matured in vitro at a rate similar to those of cyclic gilts, they differed with respect to the selected factors attributed to cytoplasmic maturation. We suggest that the higher content of particular fatty acids, which is known to have a negative influence on oocyte maturation, as well as impaired mitochondria redistribution are factors limiting the maturation potential of oocytes from prepubertal gilts.

Translation of incenp During Oocyte Maturation Is Required for Embryonic Development in Xenopus laevis1

The chromosome passenger complex (CPC) consists of Aurora-B kinase and several other subunits. One of these, incenp, binds Aurora-B and regulates its kinase activity. During Xenopus oocyte maturation, incenp accumulates through translation, contributing to aurora-b activation. A previous study has demonstrated that inhibition of incenp translation during oocyte maturation diminishes aurora-b activation but does not interfere with oocyte maturation, characterized by normal maturation-specific cyclin-b phosphorylation, degradation, and resynthesis. Here we have extended these findings, showing that inhibition of incenp translation during oocyte maturation did not interfere with meiosis I or II, as indicated by the normal emission of the first polar body and metaphase II arrest, followed by the successful emission of the second polar body upon parthenogenetic egg activation. Most importantly, however, when transferred to host frogs and subsequently ovulated, the incenp-deficient eggs were fertilized but failed to undergo mitotic cleavage. Thus, translation of incenp during oocyte maturation appears to be part of oocyte cytoplasmic maturation, preparing the egg for the rapid mitosis following fertilization.

The influence of 9-cis-retinoic acid on nuclear and cytoplasmic maturation and gene expression in canine oocytes during in vitro maturation

Retinoids have important roles in regulation of oocyte nuclear and cytoplasmic maturation. The present study investigated the effects of a retinoid metabolite on nuclear maturation, cytoplasmic maturation, and gene expression in canine oocytes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were harvested from ovaries by slicing. Only oocytes that were >120 μm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. Varying concentrations of 9-cis retinoic acid (9-cis-RA; 0, 5, 50, and 500 nm) were included in the maturation medium, and the following were measured: (i) oocyte nuclear maturation after culture for 48 h; (ii) cytoplasmic granular migration by labeling of oocytes with fluorescein isothiocyanate labeled lectins; and (iii) relative expression of genes related to apoptosis (BAX and BclII) in cumulus cells detached from oocytes, by semiquantitative reverse transcriptase-polymerase chain reaction. After 48 h culture with IVM, the highest percentage of oocytes that had developed to the metaphase II (MII) stage were in the 5 nm 9-cis-RA treatment group (18.3 ± 2.5%; P < 0.05). Complete granular migration was observed in oocytes matured with 5 nm 9-cis-RA, consistent with a commensurate gain in developmental competence. Treatment with 5 nm 9-cis-RA had no effect on BclII gene expression, but downregulated BAX expression. In conclusion, since 5 nm 9-cis-RA was beneficial to nuclear and cytoplasmic maturation of canine oocytes, we inferred an important role for 9-cis-RA during IVM.

Biomolecules of Human Female Fertility - Potential Therapeutic Targets for Pharmaceutical Design

Scientists are able to advance the frontiers of human reproduction by employing a variety of molecular biological techniques to understand the biological processes intricately linked to oocyte and ovarian follicle development. Despite an abundance of knowledge concerning essential pathways which may have critical roles in oogenesis and folliculogenesis, the repertoire of medications to treat female fertility problems remains limited to a few classes of drugs involved in the induction or suppression of folliculogenesis and ovulation; many of which are not specific in their drug actions and can give rise to complications during clinical application. This paper aims to review biomolecules and pathways (e.g. PI3K, WNT, MAPK) pertinent to ovarian follicular development and active in human oocytes, including those involved in communication between somatic cells within the ovary (cumulus cells, granulosa cells and thecal cells) and the oocyte itself. These biomolecules which are involved in the nuclear and cytoplasmic maturation of oocytes and the control of ovarian folliculogenesis have potential as targets for improved ovarian stimulation, optimisation of oocyte maturation, and as biomarkers of oocyte viability assessment.

200 SYNCHRONIZATION OF OOCYTE MEIOTIC MATURATION IN SUPEROVULATED MICE IMPROVES IN VITRO FERTILIZATION RATE

In vitro synchronization of oocyte nuclear and cytoplasmic maturation has been found to improve the IVF rate of ovarian oocytes in several species, including humans, in comparison with nonsynchronized in vitro-matured oocytes. Here, we tested the hypothesis that synchronization of oocyte meiotic maturation by an in vivo system in superovulated mice would increase the oocyte fertilization rate when compared to that of conventional superovulated oocytes. Recently, we observed that cilostazol (CZL), a PDE3-I, was able to inhibit mouse oocyte meiotic maturation in both in vitro and in vivo systems. Administering CZL at 7.5 mg, 4 or 7 h pre-hCG allowed retrieval of ovulated oocytes of which &gt;95% were at MI stage, scored by Nikon stereo microscope (SMZ 1500). A conventional superovulation program was adapted in all treated and their control groups, in which mice were injected with eCG and after 48 h with hCG (7.5 IU for each hormone). On the second morning, 13 to 14 h post-hCG, mice were killed and oocytes were collected from oviducts and in vitro fertilized (control). For the treated groups, CZL was administered in a single 7.5 mg oral dose (gavage) 4 or 7 h before the hCG injection. On the second morning, CZL-treated animals were killed at the same timing as control animals and oocytes were retrieved from the oviduct and in vitro matured for 6 h (for those gavaged with CZL, 4 h pre-hCG) or 3 h (for those gavaged with CZL, 7 h pre-hCG) to MII oocytes before IVF. These groups were designated as in vivo-in vitro synchronized/matured oocytes. In other groups treated with CZL, 4 or 7 h pre-hCG, the ovulated oocytes were allowed to mature in the oviduct (full in vivo synchronization and maturation) and oocytes were retrieved and fertilized with the same fertilization timings as the in vivo-in vitro synchronized/matured oocytes. Oocytes were cultured for 1 day after IVF and examined for cleavage. Statistical differences were analyzed by cross-tabulated chi-square test. The full in vivo synchronization and maturation (for both CZL dose timings of 4 and 7 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [89% (n = 219) and 92.2% (n = 374) vs 81.8% (n = 198); P = 0.034 and P &lt; 0.0001, respectively]. The in vivo-in vitro synchronized/matured oocytes (CZL dose timing at 7 h, but not 4 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [88.5% (n = 339) vs 83.4% (n = 458), respectively; P = 0.043]. However, the increase of the IVF rate of the oocytes from mice treated with CZL, 4 h pre-hCG, in the in vivo-in vitro synchronized/matured group was not significantly different from the control group [88.5% (n = 399) vs 83.4% (n = 458), respectively; P = 0.43]. It is concluded from the present study that synchronization of oocyte meiotic maturation by the in vivo and in vivo-in-vitro protocols can increase the IVF rate of oocytes in superovulated mice.

199 PREMATURATION OF BOVINE OOCYTES WITH BUTYROLACTONE I AND/OR CILOSTAMIDE: EFFECTS ON MEIOSIS PROGRESSION, CYTOPLASMIC MATURATION AND GENE EXPRESSION

Meiotic block during a prematuration culture (pre-IVM) before in vitro maturation (IVM) is suggested as a way to provide additional time to synchronize oocyte-somatic cell communication, leading to improved cytoplasmic maturation and nuclear meiotic competence and the acquisition of critical cellular functions necessary for developmental competence. This study was designed to evaluate the effects of the inhibitors butyrolactone I (Bl-I) and cilostamide on oocyte nuclear and cytoplasmic maturation. For that, 2 well-established methods of pre-IVM and IVM for cilostamide (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011) or Bl-I (Hashimoto et al. 2002 Biol Reprod. 66, 1696–1701) were used. Abattoir-collected oocytes were IVM in maturation medium (MM: mSOF with 0.8% BSA and hormones) for 24 h, without a previous pre-IVM culture (control). Cilostamide-group oocytes were treated for the first 2 h in vitro (pre-IVM) with 100 μM of the adenylate cyclase activator forskolin and 500 μM IBMX (nonspecific phosphodiesterase inhibitor) and then oocytes underwent extended IVM for 30 h in the presence of 20 μM cilostamide (type 3 phosphodiesterase inhibitor; pre-IVM 2 h + IVM 30 h). The Bl-I-group oocytes were pre-IVM for 24 h with 100 μM Bl-I diluted in TCM-199 supplemented with 0.2 mM pyruvate and then were IVM in MM for 20 h (pre-IVM 24 h + IVM 20 h). The Bl-I + Cilost group was a combination of both procedures: oocytes were first pre-IVM with Bl-I for 24 h and then cultured as described for the cilostamide group (pre-IVM 24 h + IVM 30 h). Cultures were carried out at 38.5°C in 5% CO2 in humidified air. After IVM, oocytes were stained with 500 nM mitotracker red to assess the mitochondrial membrane potential (Δψm) and with 10 μg mL–1 of Hoescht 33342 to evaluate the nuclear maturation (n = 207). Images were captured by an Olympus confocal microscope and analyzed with Fluoview software. The TUNEL assay was used to detect oocyte DNA fragmentation (n = 74). Relative amounts of mRNA for apoptotic-related genes were quantified after IVM in individual oocytes using real-time PCR (Kameyama et al. 2007 Reproduction 133, 423–32). Means were compared by ANOVA and Tukey's test or by chi-square (P ≤ 0.05). The percentage of oocytes reaching metaphase II after IVM did not differ among groups (73.8 to 90.4%; P ≥ 0.05), indicating that in vitro meiotic resumption was normal. The Δψm, expressed in arbitrary units of fluorescence, was 1.0 ± 0.1a (control), 2.7 ± 0.4b (Bl-I), 3.2 ± 0.5b (Cilost) and 2.1 ± 0.3ab (Bl-I + Cilost). The percentage of TUNEL-positive oocytes (18.8–41.3%) did not differ among groups (P ≥ 0.05). The relative abundance of BAX (1.0 ± 0.4 to 2.3 ± 0.4) and BCL-XL (1.0 ± 0.3 to 0.3 ± 0.1) transcripts was unaffected by pre-IVM and IVM (P ≥ 0.05). In conclusion, except for an increase in mitochondrial activity, pre-IVM with cilostamide and/or Bl-I did not affect cytoplasmic and nuclear oocyte maturation. However, oocyte developmental potential needs to be better evaluated in a future study through assessment of embryonic development. We acknowledge FAPESP and NSERC.

Detection of the expression of aldehyde deshydrogenase 1A2 (ALDH1A2) in cumulus cells (CC) By differential gene expression analysis: Implications for maturation

ALDH1A2 catalyzes the formation of retinoic acid (RA). RA is important during the formation of primordial follicles in humans and oocyte cytoplasmic maturation in the bovine. Expressions of RA receptors have been found in CC and higher RA concentration are found in follicular fluids of dominants follicles which was correlated with estradiol (E2) concentration. Objective

Vitrification of Bovine Oocytes: Effect of the Meiotic Stage on Nuclear and Cytoplasmic Maturation.

Cryopreservation of the female gamete is an important tool for the assisted reproductive technologies. However, because of their large size and marked sensitivity to cooling, cryopreservation of oocytes is very difficult. Although vitrification has been indicated as the election method no suitable results have yet been reported. The aim of this study was to evaluate nuclear and cytoplasmic maturation of oocytes vitrified at different moments of in vitro maturation using cryotop method. Bovine oocytes obtained from slaughterhouse ovaries, were select and allocated to four groups: non vitrified, used as control (CG), vitrified immediately after selection (V0), vitrified at 8 hours (V8) and vitrified at 22 hours (V22) of in vitro maturation. After warming, oocytes were placed in the incubator to complete 24 h of maturation. At the end of maturation period oocytes were denuded, fixed and stained with lacmoid or were in vitro fertilized and cultured. Oocytes of all treatments that showed metaphase II plate were classified as mature, those with any chromosomal aberrations as abnormal, and those with diffusely stained cytoplasm or undefined chromatin, as degenerate. Cleavage rate was evaluated on D2 after in vitro insemination (pi) and blastocyst rates on D7 and D8 pi. Data were compared using Chi-square analysis (P<0.05). Vitrified treatments, V0 (n = 51), V8 (n = 50) and V22 (n = 39) presented lower (P < 0.05) percentage of oocytes reaching metaphase II compared to CG (n = 36), with maturation rates of 47%, 50%, 56% and 83%, respectively. All cryopreserved groups showed a higher (P<0.05) percentage of abnormal (V0: 33%; V8: 30% and V22: 23%) and degenerated chromatin (V0:19%; V8:18% and V22:17%) than CG (11%; 5%). Cleavage and blastocyst rate on D7 and D8 were similar (P<0.05) among vitrified groups V0 (19%, 2.6% and 2.6%; n = 112), V8 (18%, 2.6%, and 2.6%; n = 115) and V22 (21%, 3.6% and 3.6%; n = 109). However, CG (n = 119) showed a higher (P<0.05) cleavage (93%) and a higher (P < 0.05) percentage of oocytes that developed to blastocyst on D7 (34%) and D8 (37%). The results suggest that, stage of meiosis in which oocytes are vitrified did not minimize the deleterious effect of cryopreservation on nuclear and cytoplasmic maturation of bovine oocytes. However, even with low rates, they were capable of been fertilized and develop to blastocyst stage. Research supported by CNPq and Embrapa-Cenargen. (poster)

Developmental, but Not Meiotic, Competence of Cat Oocytes Is Affected by Seasonal Anestrus.

Immature cat oocytes collected during seasonal anestrus (August-January) require high FSH concentrations in vitro to achieve nuclear and cytoplasmic maturation. The normally compromised meiotic and developmental competence of these oocytes is not due to reduced FSH-receptor (FSHR) abundance. To better understand the physiological mechanisms of this seasonal difference, we assessed the impact of the seasonal anestrus on oocyte nuclear and cytoplasmic maturation in vivo as well as the expression of maturation-related genes. Twelve adult cats were exposed for 3 to 5 months to a natural breeding season (14 hours of light and 10 hours of dark) and then to a similar interval simulating anestrus (8 hours of light and 16 hours of dark). After ovarian stimulation with exogenous eCG/hCG, oocytes were aspirated from preovulatory follicles by laparoscopy. Grade 1 cumulus-oocyte complexes (COCs; dark oocyte cytoplasm and several layers of expanded cumulus cells) were selected and inseminated (n = 30 during the breeding season; n = 45 during anestrus) with frozen-thawed sperm. Presumptive embryos then were cultured for 8 days, fixed in 70% v/v ethanol and stained with Hoechst to determine the number of blastomeric nuclei. During each interval, a proportion of Grade 1 COCs (n = 8 during the breeding season; n = 11 during anestrus) were collected for relative quantification of maturation-related genes (ESR2, PTGS2, EGFR). Total RNA was extracted using a Picopure RNA isolation kit and cDNA generated using random primers and Superscript III reverse transcriptase. cDNA samples were assessed by real-time qPCR to compare relative expression levels of FSHR, ESR2, PTGS2 and EGFR (normalized to ACTB, RPS7 and YWHAZ expression). Proportions of Grade 1 COCs at the metaphase II stage were similar between the two intervals. Relative expression levels of developmental markers ESR2, PTGS2, EGFR also were comparable. A trend for decreased EGFR expression was associated with a lower proportion of oocytes fertilized and reaching an advanced embryo stage (P<0.05) during anestrus compared to the breeding season. Therefore, although oocyte meiotic competence is unimpaired in vivo, oocyte developmental competence is compromised during the anestrual compared to the breeding season. Generally, this effect on developmental capacity was not reflected in all maturation-related genes. Collective results infer that alternate functions of FSH unrelated to downstream gene regulation (such as EGFR) may be responsible for seasonal decreases in oocyte developmental capacity in vivo. Further investigation of FSHR functionality will identify alternate mechanisms to manipulate FSH responsiveness in vivo. (Supported by the Kelly Fellowship and Lalor Foundation) (poster)

Structural and functional changes linked to, and factors promoting, cytoplasmic maturation in mammalian oocytes.

In most mammals, oocyte maturation is the final process of oogenesis, from the prophase of the first meiosis (germinal vesicle stage) to the metaphase of the second meiosis (MII), during which the oocyte acquires fertilizable competence as well as post-fertilization development competence. The nuclear and cytoplasmic maturation processes occur in synchrony but independently. Cytoplasmic maturation entails biochemical and structural changes in the cytoplasm, which give rise to oocytes capable of being fertilized and developing into embryos. Herein we review the literature and results from our own experiments on the structural and molecular events regulating cytoplasmic maturation in oocytes, concentrating on (1) the appropriate reorganization of active mitochondria and the endoplasmic reticulum, a structural and functional feature of cytoplasmic maturation, and (2) factors involved in regulatory mechanisms such as cumulus cell-oocyte gap junctional signaling, cumulus cell-oocyte bidirectional paracrine signaling, and the complex interactions of these signaling processes and follicular fluid constituents in the follicle environment.

Evidence of melatonin synthesis in the cumulus oocyte complexes and its role in enhancing oocyte maturation in vitro in cattle

Melatonin is a multifunctional molecule that mediates several circadian and seasonal reproductive processes. The exact role of melatonin in modulating reproduction, however, is not fully understood-especially its effects on the ovarian follicles and oocytes. This study was conducted to investigate the expressions of the ASMT and melatonin-receptor MTNR1A and MTNR1B genes in bovine oocytes and their cumulus cells, as well as the effects of melatonin on oocyte nuclear and cytoplasmic maturation in vitro. Cumulus-oocyte complexes (COCs) from abattoir ovaries were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10, 50, and 100 ng/ml. The expression of ASMT, MTNR1A, and MTNR1B genes was evaluated by RT-PCR. Moreover, the effects of melatonin on cumulus cell expansion, nuclear maturation, mitochondrial characteristics and COCs steroidogenesis were investigated. Furthermore, the level of reactive oxygen species (ROS) was evaluated in denuded oocytes. Our study revealed that ASMT and MTNR1A genes were expressed in COCs, while the MTNR1B gene was expressed only in oocytes. Additionally, melatonin supplementation at 10 and 50 ng/ml to in vitro maturation medium significantly enhanced oocyte nuclear maturation, cumulus cell expansion and altered the mitochondrial distribution patterns, but had no effects on oocyte mitochondrial activity and COCs steroidogenesis. Melatonin-treated oocytes had a significantly lower level of ROS than controls. The presence of melatonin receptors in COCs and its promoting effects on oocyte nuclear and cytoplasmic events, indicate the potentially important roles of this hormone in regulating bovine oocyte maturation. Moreover, the presence of ASMT transcript in COCs suggests the possible involvement of these cells in melatonin biosynthesis.