Oxidative stress differentially impacts male and female bovine embryos depending on the culture medium and the stress condition.

Male and female embryos are known to differ for their metabolism and response to environmental factors very early in development. The present study aimed to evaluate the response to oxidative stress of male and female bovine embryos at the morula-blastocyst stages in terms of developmental rates, total cell number and apoptotic rates in two culture conditions. Embryos where cultured in a medium supplemented with either 5% fetal calf serum (FCS) or 4 mg/mL bovine serum albumin and a mixture of insulin, transferrin and selenium (BSA-ITS). Oxidative stress was applied at Day-5 post insemination (pi) by adding either AAPH or menadione to the culture medium, and blastocysts were analyzed at Day-7pi. The impact on development and blastocyst quality was dependent on the culture medium and the stress inducer but differed between male and female embryos. Male embryos resisted better to oxidative stress in FCS supplemented medium, no matter the stress inducer. Accordingly, the impact on blastocyst cell number tended to be higher in female blastocysts after stress induction with AAPH in FCS supplemented medium. On the other hand, in BSA-ITS supplemented medium, female embryos were more resistant to AAPH induced stress, while menadione had no impact on sex ratio. The weaker resistance of males to AAPH in this medium is in accordance with their trend to show a higher increase in apoptotic rates than females in this condition. In conclusion, this study shows that oxidative stress has differential impact on male and female bovine blastocysts depending on the culture condition and on the way oxidative stress is induced.

Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals.

BACKGROUND: We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed. RESULTS: The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells. CONCLUSIONS: Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals.

Female bovine blastocysts are more prone to apoptosis than male ones.

Female and male embryos show differences in gene expression and metabolism from the onset of their genome. Those differences are affected by environmental factors. The objective of the study was to compare the apoptotic rates of in vitro-produced female and male bovine blastocysts cultured in different conditions. Day 7 blastocysts obtained after IVF with sex-sorted semen and culture in two synthetic oviductal fluid-based media (containing fetal calf serum [FCS] or BSA, insulin, transferrin, and selenium) were simultaneously evaluated for two markers of apoptosis after 3D reconstruction from confocal images: active caspase 3 by immunofluorescence and DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling. Higher levels of apoptotic cells were observed in female embryos whatever the culture condition but with a more pronounced difference in FCS medium. This result was confirmed using the unsexed semen of two bulls. The sex effect on apoptosis was detected in both the inner cell mass and the trophectoderm but was dependent on the embryonic size. In conclusion, this study reported that female bovine blastocysts are more prone to apoptosis than male ones but that culture in FCS exacerbates the differences in apoptosis between sexes, especially in small blastocysts.

Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm.

The aim of this work was to completely replace the egg yolk a classical diluent for freezing equine semen by a cyclodextrin-cholesterol complex. At the same time, the reduction in the glycerol content used for cryopreservation and the incubation time between sperm and the freezing media were evaluated. Horse ejaculates were frozen with four different freezing extenders: a frozen reference medium (IF) containing egg yolk and 2.5% glycerol and media without egg yolk but supplemented with 1.5 mg 2-hydroxypropyl-beta-cyclodextrin cholesterol (HPßCD-C) complex and containing either 1% (G1), 2% (G2) or 3% glycerol (G3). Three incubation times (90, 120 and 180 min) at 4 °C between the fresh semen and the different media were tested before freezing. Viability and motility analyses were performed with computer assisted semen analysis (CASA). Results showed that the freezing media containing the HPßCD-C complex with 1%, 2% and 3% glycerol significantly improve the 3 in vitro parameters of post thawing semen quality (viability, progressive and total mobilities) compared to IF. The best improvement of the parameters was obtained with G1 medium and the longest contact time. The substitution of egg yolk by HPßCD-C complex allows the decrease of protein charge of the medium while favouring the cholesterol supply to membrane spermatozoa offering it a better resistance to osmotic imbalance and a better tolerance to the glycerol toxicity. Our results highlight that the egg yolk of an extender for the freezing of horse semen can be completely substituted by HPßCD-C complex.

How to study HOX gene expression and function in mammalian oocytes and early embryos.

Mammalian oocytes and early embryos have unique characteristics and can only be obtained in small amounts. As a consequence, the techniques to be used to characterize gene expression and function have to be adapted. It is also important to keep in mind that differences exist between mammalian species. Here we describe a set of techniques useful to analyze gene expression in oocytes and early bovine embryos, including techniques to quantify maternal and embryonic transcripts by RT-qPCR, to evaluate the translation potential of maternal transcripts, to knock down HOX transcripts by injection of siRNA, and to localize HOX proteins by whole-mount immunofluorescence.

Efficient in vitro adipocyte model of long-term lipolysis: a tool to study the behavior of lipophilic compounds.

The triglycerides (TGs) stored in the white adipose tissue are mobilized during periods of negative energy balance. To date, there is no in vitro model of adipocytes imitating a long period of negative energy balance in which triglycerides are highly mobilized. Such model would allow studying the mobilization of TGs and lipophilic compounds trapped within the adipose tissue (e.g., pollutants and vitamins). The present study aims at developing a performing long-term in vitro lipolysis in adipocytes, resulting in a significant decrease of TG stores. Lipolysis was induced on differentiated rat adipocytes by a lipolytic medium with or without isoproterenol for 12 h. The condition with isoproterenol was duplicated, once with medium renewal every 3 h and once without medium renewal. Adding isoproterenol efficiently triggered lipolysis in a short time (3 h). However, a single stimulation by isoproterenol, without medium renewal, was not sufficient to reduce the TG content during a longer term (12 h). A reesterification of fatty acids occurred after a few hours of lipolysis, resulting in a novel increase of cellular lipids. Regular medium renewal combined with repeated isoproterenol stimulations led to almost emptied cells after 12 h. However, medium renewal without isoproterenol stimulation for 12 h was as efficient in terms of lipid mobilization. Our study demonstrates that, over a short-term period, isoproterenol is required to exert a significant lipolytic effect on adipocytes. During a long-term period, the presence of isoproterenol is no longer essential. Instead, medium renewal becomes the main factor involved in cell emptying. The efficiency of this protocol was demonstrated by visual tracking of the cells and by monitoring the dynamics of release of a lipophilic compound, PCB-153, from adipocytes during lipolysis.

Characterization of TALE genes expression during the first lineage segregation in mammalian embryos.

BACKGROUND: Three amino acid loop extension (TALE) homeodomain-containing transcription factors are generally recognized for their role in organogenesis and differentiation during embryogenesis. However, very little is known about the expression and function of Meis, Pbx, and Prep genes during early development. RESULTS: In order to determine whether TALE proteins could contribute to the early cell fate decisions in mammalian development, this study aimed to characterize in a systematic manner the pattern of expression of all Meis, Pbx, and Prep genes from the precompaction to blastocyst stage corresponding to the first step of cell differentiation in mammals. To reveal to what extent TALE genes expression at these early stages is a conserved feature among mammals, this study was performed in parallel in the bovine and mouse models. We demonstrated the transcription and translation of TALE genes, before gastrulation in the two species. At least one member of Meis, Pbx, and Prep subfamilies was found expressed at the RNA and protein levels but different patterns of expression were observed between genes and between species, suggesting specific gene regulations. CONCLUSIONS: Taken together, these results suggest a previously unexpected involvement of these factors during the early development in mammals.

Genetic diversity and networks of exchange: a combined approach to assess intra-breed diversity.

BACKGROUND: Cryopreservation of three endangered Belgian sheep breeds required to characterize their intra-breed genetic diversity. It is assumed that the genetic structure of a livestock breed depends mostly on gene flow due to exchanges between herds. To quantify this relation, molecular data and analyses of the exchanges were combined for three endangered Belgian breeds. METHODS: For each breed, between 91 and 225 sheep were genotyped with 19 microsatellites. Genetic differentiations between breeds and among herds within a breed were evaluated and the genetic structure of the breeds was described using Bayesian clustering (Structure). Exchanges of animals between 20, 46 and 95 herds according to breed were identified via semi-directed interviews and were analyzed using the concepts of the network theory to calculate average degrees and shortest path lengths between herds. Correlation between the Reynolds' genetic distances and the shortest path lengths between each pair of herds was assessed by a Mantel test approach. RESULTS: Genetic differentiation between breeds was high (0.16). Overall Fst values among herds were high in each breed (0.17, 0.11 and 0.10). Use of the Bayesian approach made it possible to identify genetic groups of herds within a breed. Significant correlations between the shortest path lengths and the Reynolds' genetic distances were found in each breed (0.87, 0.33 and 0.41), which demonstrate the influence of exchanges between herds on the genetic diversity. Correlation differences between breeds could be explained by differences in the average degree of the animal exchange networks, which is a measure of the number of exchanges per herd. The two breeds with the highest average degree showed the lowest correlation. Information from the exchange networks was used to assign individuals to the genetic groups when molecular information was incomplete or missing to identify donors for a cryobank. CONCLUSIONS: A fine-scale picture of the population genetic structure at the herd level was obtained for the three breeds. Network analysis made it possible to highlight the influence of exchanges on genetic structure and to complete or replace molecular information in establishing a conservation program.

HOX genes are expressed in bovine and mouse oocytes and early embryos.

HOX proteins are transcription factors that play a major role in patterning the body axis of vertebrates from the gastrulation stage. While nothing has been reported so far about their roles at earlier stages, there is evidence that some HOX genes are expressed before gastrulation. The objective of this work was to study the pattern of expression of several HOX genes during oocyte maturation and early embryonic development up to the blastocyst stage. Using nested PCR, HOXD1, HOXA3, HOXD4, HOXB7, HOXB9, and HOXC9 transcripts were detected in bovine oocytes and early embryos at various frequencies depending on the stage of development. Quantitative PCR was performed on bovine oocytes and early embryos: relative expression of HOXD1, HOXA3, and HOXC9 decreased sharply after the 5-8 cell stage. HOXB9 relative expression increased between the oocyte and the morula stage. All transcripts seemed to be of maternal origin before the maternal to embryonic transition, as demonstrated by blocking transcription with α-amanitin. Reverse transcription was performed with either hexamers or oligo-dT, allowing for the determination that HOXC9 transcripts were slightly deadenylated during oocyte maturation; HOXD1, HOXA3, and HOXB9 transcripts were not, indicating that they could be translated. Hoxd1, Hoxa3, Hoxb9, and Hoxc9 expression was also detected in mouse oocytes and early embryos. A similar pattern of expression was found in the two species. In conclusion, mammalian HOX genes might be implicated in the control of oocyte maturation, the maternal-to-embryonic transition or the first steps of embryo differentiation.

Peroxiredoxins in gametogenesis and embryo development.

Reactive oxygen species have been implicated in gametogenesis and embryo development in animals. As peroxiredoxins are now recognized as important protective antioxidant enzymes as well as modulators of hydrogen peroxide-mediated signaling, we addressed here the putative role of this novel family of peroxidases in gamete maturation and during embryogenesis in mammals and insects.

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development.

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.

Impact of pro-oxidant agents on the morula-blastocyst transition in bovine embryos.

Exposing day 5 bovine morulae to reactive oxygen species induces a delayed degeneration of some blastocysts on day 8 post-insemination (pi) but without affecting the blastocyst rates. The aim of this study was to characterize the resisting and the degenerating population of blastocysts. The kinetics of degeneration of the embryos exposed to the two pro-oxidant agents: 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and buthionine sulfoximine (BSO) was evaluated using time-lapse cinematography. With both agents the first signs of degeneration appeared at day 7.5 pi but the duration of the degeneration process was shorter in presence of AAPH than BSO (4.2 vs. 12.5 hr, ANOVA, P < 0.05). The resisting blastocysts derived from morulae with a larger diameter (mean diameter: 161 vs. 154 microm, ANOVA, P < 0.05) and showed an earlier cavitation (135 vs. 142 hpi, P < 0.05) than the degenerating ones. The profile of protein neosynthesis at day 7 was not affected by the treatment. The proportion of male embryos was more important in the resisting than in the degenerating population (70 vs. 55%, chi2, P < 0.05) especially when the stress was induced by AAPH. The quality of the resisting embryos, measured by the total cell number and the rate of apoptosis, did not seem to be affected when compared to control embryos. In conclusion, resistance to oxidative stress seems related to the kinetics of development and/or the sex of the embryos. Resisting embryos apparently display a quality similar to untreated embryos.

Effects of polyadenylation inhibition on meiosis progression in relation to the polyadenylation status of cyclins A2 and B1 during in vitro maturation of bovine oocytes.

The control of protein synthesis during maturation in oocytes is mainly exerted through cytoplasmic polyadenylation of stored mRNAs. We first analyzed the polyadenylation status of cyclins A2 and B1 during in vitro maturation (IVM) of bovine oocytes, using Rapid Amplification of cDNA Ends-Polyadenylation Technique (RACE-PAT). An inconstant elongation of the poly(A) tail was observed for cyclin A2 transcripts after maturation, while a constant lengthening was observed for cyclin B1, occurring during the first 12 hr of incubation. We then evaluated the effects of the polyadenylation inhibitor 3'-deoxyadenosine (3'-dA), on polyadenylation and nuclear maturation. The presence of 0.02 mM 3'-dA during the whole incubation period or from 6 hr after its beginning completely prevented meiosis progression in 100% of the oocytes. Polyadenylation of cyclin B1 was also completely prevented when 3'-dA was added at 0 hr, and greatly reduced when added at 6 hr. When 3'-dA was added at 12 hr, around metaphase I (MI), 46.9% of the oocytes have reached metaphase II (MII, vs. 78.8% in the control group) at 24 hr. The use of the same concentration of 3'-deoxyguanosine (3'-dG), that impairs transcription but not polyadenylation, did not affect cyclins polyadenylation, nor nuclear maturation, whatever was the timing of addition. These results suggest that the polyadenylation of cyclin B1 could be related to the first peak of activity of MPF, occurring around MI (10-12 hr after the onset of the maturation period). They also show that, in our culture conditions, inhibition of polyadenylation prevents meiosis progression, especially up to the MI stage, while inhibition of transcription does not.

Porcine embryo development and fragmentation and their relation to apoptotic markers: a cinematographic and confocal laser scanning microscopic study.

Porcine embryo selection prior to transfer is mainly influenced by morphological criteria. However, the relationship between embryonic morphology, developmental potential and cell death by apoptosis in porcine embryos is still unclear. The aim of this study was to establish embryo quality parameters for in vivo fertilised porcine embryos based on timing of development in vitro, embryo morphology and the presence of apoptosis. The kinetics of development and morphological parameters were investigated in a time-lapse cinematographic experiment. Possible links between embryo morphology and apoptosis were examined via a confocal laser scanning experiment, analysing nuclear changes, annexin V and terminal dUTP nick-end labelling. The timing of early cleavages was firmly linked to embryo developmental competence in vitro. Attainment of at least the 5-cell stage before 77 h post insemination and attainment of the morula stage before 102 h post insemination significantly increased the odds for reaching the early blastocyst stage. Overall, a negative effect of fragmentation percentage and fragmentation pattern on subsequent embryonic development was observed, but the developmental potential of embryos experiencing slight fragmentation (0-5%) was not different from embryos without fragmentation. Correlations detected between developmental arrest and fragmentation, and fragmentation and apoptosis were 0.60 and 0.87 (P < 0.05) respectively. Only a minority of the embryos arrested between the 1- and 4-cell stage displayed biochemical characteristics of apoptosis. Consequently, a significant correlation (0.57) between developmental arrest and apoptosis could only be established for embryos arrested after embryonic genome activation.

Influence of antral follicle size on oocyte characteristics and embryo development in the bovine.

The developmental competence of bovine oocytes isolated from antral follicles of different sizes was assessed in three European laboratories (Belgium, UCL; Denmark, DIAS; France, INRA). Using the same protocol for in vitro production of embryos, the oocytes isolated from follicles with a diameter > or = 6 mm always gave a higher blastocyst rate than oocytes from follicles < 4 mm (UCL: 42% versus 14%, DIAS: 50% versus 35%, INRA: 39% versus 22%; P < 0.05). Blastocyst cell number was not affected by follicle size. Several parameters were investigated for these oocytes. The energy metabolism of cumulus-oocyte-complexes and of denuded oocytes was assessed by the oxygen and pyruvate uptake and by lactate release both at the beginning and the end of the maturation. No effect of follicle size could be detected but lactate release increased after maturation. The global profile of transcripts, the pattern of protein neosynthesis and the kinetics of meiosis resumption were not affected by follicle size. The developmental kinetics of derived embryos was also analysed. Whatever the follicle size, viable embryos had a shorter first and third embryonic cell cycle. Among the viable embryos, the size of the follicle interfered with the fourth cell cycle duration. A higher percentage of blastocysts issued from large follicle presented a short fourth cell cycle (9h) (35% versus 6%; P < 0.05). Beside, blastocysts derived from small follicles had a delayed cavitation and expansion. Thereby, a higher developmental competence for oocytes from follicle > or = 6 mm versus < 4 mm was demonstrated in three laboratories although no differences could be displayed directly at the oocyte level.

Expression of peroxiredoxins in bovine oocytes and embryos produced in vitro.

Peroxiredoxins (PRDXs) form a family of peroxidases involved in antioxidant protection and cell signaling. Due to their peroxide reductase activity, these enzymes might be involved in fine-tuning peroxide levels in embryos during in vitro production. In this study, RT-PCR was used to examine the expression of the six PRDX isoforms (PRDX1 to PRDX6) in bovine oocytes and embryos. PRDXs were detected in oocytes both before and after in vitro maturation. Besides, PRDX6 was up-regulated after maturation. Single embryos were analyzed from the two-cell to the blastocyst stages. PRDX1 and PRDX5 transcripts were detected throughout development. PRDX2, PRDX3, and PRDX6 were not expressed around the 9- to 16-cell stage. PRDX4 transcripts were weakly detected in pools of embryos from the 9- to 16-cell stage onwards. In situ immunodetection of PRDX5, which was previously reported to exhibit the widest subcellular distribution among PRDXs in adult mammalian cells, showed a mitochondrial distribution pattern in the bovine embryo. Finally, the potential modulation by oxidative stress of PRDX expression around the major embryonic genome activation was evaluated by culturing embryos under 20% O2 instead of 5%. No significant difference in the pattern of PRDX expression was observed under 20% O2. In conclusion, our data show for the first time that PRDXs are expressed in mammalian oocytes and early embryos. Moreover, the bovine transcripts exhibit various patterns of expression that might be related to the potential role of PRDXs in oocyte maturation and embryo development.

Effect of prematuration, meiosis activating sterol and enriched maturation medium on the nuclear maturation and competence to development of calf oocytes.

New strategies were proposed to improve the developmental competence of calf oocytes through in vitro technologies. Cumulus-oocyte complexes were first prematured for 24 h in the presence of meiosis inhibitors. Both Roscovitine alone (50 microM) or in combination with Butyrolactone-I (12.5 microM Rosco+6.25 microM BL-I) prevented the progression of meiosis. Their effect on nuclear maturation was reversible after a further 17 or 24 h maturation step. However, a dramatic decrease in embryo development was observed after fertilization (abattoir oocytes: 4-9% blastocyst rate versus 14-17% for control embryos). Similar results were obtained with oocytes collected by Ovum Pick Up from living donors. No pregnancy was obtained after single transfer of two blastocysts obtained from prematured oocytes (0/2 versus 4/12 for control embryos). Adding low concentrations (1, 3 or 10 microM) of follicular fluid-meiosis activating sterol (FF-MAS) during the maturation step had a beneficial effect on nuclear maturation (73-86% metaphase II versus 58% for control oocytes). However, subsequent embryo development was not improved. Enriching the maturation medium, namely with hormones, growth factors and precursors of glutathione, induced a sixfold increase in glutathione in the oocyte and had a beneficial effect on embryo development (38% increase in blastocyst rate). In conclusion, in opposition to the results reported with adult oocytes, prematuring calf oocytes had a negative impact on their developmental potential. Although FF-MAS improved nuclear maturation, its addition in the maturation medium did not increase embryo development. However, enriching the maturation medium had a positive effect on embryo development, indicating that cytoplasmic maturation was improved.

Peroxiredoxin 6 is upregulated in bovine oocytes and cumulus cells during in vitro maturation: role of intercellular communication.

Peroxiredoxins are peroxidases involved in antioxidant defense and intracellular signaling. Expression of transcripts coding for peroxiredoxin 6 (PRDX6) has been previously described to be upregulated in oocytes after in vitro maturation, a period during which general transcription decreases dramatically in oocytes. The aim of the present work was to evaluate PRDX6 regulation in bovine cumulus-oocyte complexes in relation to maturation and intercellular communication. PRDX6 expression was analyzed by reverse transcription-PCR and Western blotting in oocytes and cumulus cells before and after in vitro maturation. PRDX6 was found to be upregulated at the mRNA and protein levels in both cell types after maturation. The effect of paracrine and gap junctional communication on PRDX6 expression was then assessed by culturing cumulus clusters in the presence or absence of denuded oocytes. While PRDX6 upregulation in oocytes required intact cumulus-oocyte junctions, the presence of denuded oocytes was necessary but sufficient for the upregulation to occur in cumulus cells. Finally, the influence of recombinant mouse growth differentiation factor-9 (GDF-9) on PRDX6 expression in cumulus cells was studied. GDF-9 induced cumulus expansion and PRDX6 upregulation in bovine cumulus clusters. Altogether, our data suggest that PRDX6 upregulation in cumulus-oocyte complexes during in vitro maturation is mutually regulated by both cell types: PRDX6 upregulation in oocytes would require gap junctions with cumulus cells, while upregulation in cumulus would depend on secretion of oocyte paracrine factor(s) with GDF-9 being a likely candidate.

Poly(A) RNA is reduced by half during bovine oocyte maturation but increases when meiotic arrest is maintained with CDK inhibitors.

Variations in the amount of different RNA species were investigated during in vitro maturation of bovine oocytes. Total RNA content was estimated to be 2 ng before meiosis, and after meiosis resumption, no decrease was observed. Ribosomal RNA did not appear to be degraded either, whereas poly(A) RNA was reduced by half after meiosis resumption, from 53 pg to 25 pg per oocyte. Real-time polymerase chain reaction was performed on growth and differentiation factor-9 (GDF-9), on cyclin B1, and on two genes implicated in the resistance to oxidative stress, glucose-6-phosphate-dehydrogenase (G6PD) and peroxiredoxin-6 (PRDX6). When these transcripts were reverse-transcribed with hexamers, the amplification results were not different before or after in vitro maturation. But when reverse transcription was performed with oligo(dT), amplification was dramatically reduced after maturation, except for cyclin B1 mRNA, implying deadenylation without degradation of three transcripts. Although calf oocytes have a lower developmental competence, their poly(A) RNA contents were not different from that of cow oocytes, nor were they differently affected during maturation. When bovine oocytes were maintained in vitro under meiotic arrest with CDK inhibitors, their poly(A) RNA amount increased, but this rise did not change the poly(A) RNA level once maturation was achieved. The increase could not be observed under transcription inhibition and, when impeding transcription and adenylation, the poly(A) RNA decreased to a level normally observed after maturation, in spite of the maintenance of meiotic arrest. These results demonstrate the importance of adenylation and deadenylation processes during in vitro maturation of bovine oocytes.