Identification of Differentially Expressed mRNAs and miRNAs and Related Regulatory Networks in Cumulus Oophorus Complexes Associated with Fertilization.

Cumulus oophorus complexes (COCs) are the first extracellular barriers that sperm must pass through to fuse with oocytes, which have an important role in oocyte maturation and fertilization. However, little is known about the molecular mechanisms of COCs involved in fertilization. In this study, COCs were collected and then randomly divided into a test group that interacted with sperm and a control group that did not interact with sperm. Then, the total RNA was extracted; RNA transcriptome and small RNA libraries were prepared, sequenced, and analyzed. The results showed that 1283 differentially expressed genes (DEGs), including 560 upregulated and 723 downregulated genes. In addition, 57 differentially expressed miRNAs (DEMIs) with 35 upregulated and 22 downregulated were also detected. After the RNA-seq results were verified by RT-qPCR, 86 effective DEGs and 40 DEMIs were finally screened and a DEMI-DEG regulatory network was constructed. From this, the top ten hub target genes were HNF4A, SPN, WSCD1, TMEM239, SLC2A4, E2F2, SIAH3, ADORA3, PIK3R2, and GDNF, and they were all downregulated. The top ten hub DEMIs were miR-6876-5p, miR-877-3p, miR-6818-5p, miR-4690-3p, miR-6789-3p, miR-6837-5p, miR-6861-5p, miR-4421, miR-6501-5p, and miR-6875-3p, all of which were upregulated. The KEGG signaling pathway enrichment analysis showed that the effective DEGs were significantly enriched in the calcium, AMPK, and phospholipase D signaling pathways. Our study identified several DEGs and DEMIs and potential miRNA-mRNA regulatory pathways in COCs and these may contribute to fertilization. This study may provide novel insights into potential biomarkers for fertilization failure.

Bioinformatic Analysis of Human Cumulus Cells to Unravel Cellular's Processes that Could Be Used to Establish Oocyte Quality Biomarkers with Clinical Application.

Metadata analysis of public microarray datasets using bioinformatics tools has been successfully used in several biomedical fields in the search for biomarkers. In reproductive science, there is an urgent need for the establishment of oocyte quality biomarkers that could be used in the clinical environment to increase the chances of successful outcomes in treatment cycles. Adaptive cellular processes observed in cumulus oophorus cells reflect the conditions of the follicular microenvironment and may thus bring relevant information of oocyte's conditions. Here we analyzed human cumulus cells gene expression datasets in search of predictors of oocyte quality, a strategy which uncovered several cellular processes positively and negatively associated with embryo development and pregnancy potential. Secondly, the expression levels of genes that were present in the majority of processes observed were validated in house with clinical samples. Our data confirmed the association of the selected biomarkers with blastocyst formation and pregnancy potential rates, independently of patients' clinical characteristics such as diagnosis, age, BMI, and stimulation protocol applied. This study shows that bioinformatic analysis of cellular processes can be successfully used to elucidate possible oocyte quality biomarkers. Our data reinforces the need to consider clinical characteristics of patients when selecting relevant biomarkers to be used in the clinical environment and suggests a combination of positive (PTGS2) and negative (CYPB1) quality biomarkers as a robust strategy for a complementary oocyte selection tool, potentially increasing assisted reproduction success rates. Also, GPX4 expression as pregnancy potential biomarker is indicated here as a possibility for further investigations.

Cumulus cell antioxidant system is modulated by patients' clinical characteristics and correlates with embryo development.

PURPOSE: To study whether the cumulus cell antioxidant system varies accordingly to patients clinical characteristics' as age, infertility diagnosis, BMI, and stimulation protocol applied and if the antioxidant profile of cumulus cells could be used as a predictor of embryo development. METHODS: A prospective study including 383 human cumulus samples provided by 191 female patients undergoing intracytoplasmic sperm injection during in vitro fertilization treatments from a local in vitro fertilization center and processed in university laboratories. Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST) enzyme activity levels and reduced glutathione (GSH) levels were measured in cumulus oophorus cells individually collected from each aspirated cumulus-oocyte complex, and the results of each sample were compared considering the oocytes outcome after ICSI and patients clinical characteristics. A total of 223 other human cumulus samples from previous studies were submitted to a gene expression meta-analysis. RESULTS: The antioxidant system changes dramatically depending on patients' age, infertility diagnosis, stimulation protocol applied, and oocyte quality. SOD activity in cumulus cells revealed to be predictive of top-quality blastocysts for young patients with male factor infertility (P < 0.05), while GST levels were shown to be extremely influenced by infertility cause (P < 0.0001) and stimulation protocol applied (P < 0.05), but nonetheless, it can be used as a complementary tool for top-quality blastocyst prediction in patients submitted to intracytoplasmic sperm injection technique (ICSI) by male factor infertility (P < 0.05). CONCLUSION: Through a simple and non-invasive analysis, the evaluation of redox enzymes in cumulus cells could be used to predict embryo development, in a personalized matter in specific patient groups, indicating top-quality oocytes and improving success rates in in vitro fertilization treatments. TRIAL REGISTRATION: The trial was registered at UFRGS Research Ethics Committee and Plataforma Brasil under approval number 68081017.2.0000.5347 in June 6, 2019.

Application of ultrasound markers measured at different time points of COH cycle in the prediction of ovarian response for individualised ovulation induction.

The purpose of this study was to evaluate the predictive value of ultrasound markers measured at different time points of the controlled ovarian hyperstimulation (COH) cycle on ovarian response and outcome indicators in the IVF-ET cycle. According to the oestrogen level and the number of retrieved oocytes, patients who planned for COH treatment were separated into low-response group, normal and high-response group. The ovarian stromal artery flow parameters on the day of pituitary down-regulation, day 1, day 7, day 10, and the day of hCG injection were collected prospectively. We also have collected the data of cumulus oophorus count on the day of hCG injection by transvaginal sonography. Compared with the low-response group, on the first day of the COH cycle PI, RI, and S/D were lower in the high-response group than they were in the low-response group (p < .05). PSV and EDV were significantly higher in the high-response group than they were in the low-response group (p < .01), and the PSV on the first day of the COH cycle have statistical significance in predicting the number of high-quality embryos. The number of cumulus oophorus on the day of hCG injection has statistical significance in predicting the number of oocytes retrieved and fertilised oocytes. We conclude that the ovarian stromal artery flow parameters on the first day of the COH cycle and cumulus oophorus count on hCG injection day can serve as efficient indicators for an early assessment of ovarian response and individualised ovulation induction.IMPACT STATEMENTWhat is already known on this subject? AMH, AFC, and the age of the patient are well-known effective parameters for the evaluation of ovarian response, but these are insufficient and full of individual differences. Some researchers have investigated the value of colour Doppler ultrasound and cumulus oophorus in assessing ovarian response, but no definitive conclusion has been reached.What do the results of this study add? The hemodynamic parameters of ovarian stromal artery on the first day of the COH cycle and the number of cumulus oophorus on the day of hCG injection detected by Transvaginal Colour Doppler Sonography (TV-CDS) could be used to predict the ovarian response.What are the implications of these findings for clinical practice and/or further research? Ovarian stromal artery flow parameters and cumulus oophorus detected by TV-CDS can potentially be offered as a complementary parameter for ovarian reserve.

Sperm acrosome status before and during fertilization in the Chinese hamster (Cricetulus griseus), and observation of oviductal vesicles and globules.

The present study was conducted to determine exact location where the acrosome reaction of fertilizing spermatozoa begins in the oviduct of the Chinese hamster. Unlike spermatozoa of other rodent species, Chinese hamster spermatozoa did not spontaneously undergo the acrosome reaction in fertilization-supporting media. In naturally mated females, spermatozoa in the uterus had intact acrosomes, whereas those in the lower oviductal isthmus had visibly thin acrosomal caps. The acrosomal cap was lost when spermatozoa passed through the cumulus oophorus. Thus, Chinese hamster spermatozoa begin the acrosome reaction in the lower isthmus and complete it in the cumulus oophorus. The mucosal epithelium of the oviductal isthmus released many "transparent" vesicles into the lumen, was very fragile and readily sloughed off by rough handling or rapid flushing with medium. Globular materials that oozed out of the dissected oviduct were most likely mucosa cells destroyed by rough handling. Although the oviducts of Chinese hamsters may be exceptionally delicate, this observation nevertheless warns us to cautiously handle the oviducts of any species when studying oviduct secretions that could be involved in inducing capacitation and the acrosome reaction of spermatozoa within the female genital tract.

Comparison of three sperm selection methods for ICSI-DGC, Cumulus column, and incubation with supernatant product of adipose tissue-derived adult stem cells: An experimental study.

BACKGROUND: The examination of sperm parameters and sperm DNA integrity are necessary for male fertility expression. These parameters can be affected by method of sperm separation. OBJECTIVE: To measure the damage of each sperm separation method on the sperm parameters and sperm DNA integrity. MATERIALS AND METHODS: In this experimental study, semen samples of 20 infertile men with asthenoteratozoospermia (Infertility Research Center, Qom, Iran, 2017) were processed in three ways: density gradient centrifugation (DGC), cumulus column, and incubation with supernatant products of adipose tissue-derived adult stem cells (SPAS). The results of sperm parameters and DNA fragmentation before and after the process were statistically analyzed. RESULTS: The number of separated sperms by normal morphologies during the SPAS and the cumulus column was significantly more than the corresponding population in the DGC group. In addition, although all three methods have the same ability to increase total sperm motility and the number of recovered sperms, in the field of forwarding movement and DNA fragmentation, the SPAS method performed more efficiently (p = 0.021). CONCLUSION: Sperm parameters and DNA fragmentation in the SPAS group were better than those in the DGC and cumulus column groups. Furthermore, it has been shown that the sperm capacity was increased with the SPAS method. However, the rearrangement of sperm chromatin by reducing the disulfide bridges and providing the possibility of re-histone over capacity causes a significant reduction in DNA fragmentation.

Age-related miRNome landscape of cumulus oophorus cells during controlled ovarian stimulation protocols in IVF cycles.

STUDY QUESTION: Is the microRNA (miRNA) expression pattern of cumulus oophorus cells (COCs) in women undergoing medically assisted reproduction (MAR) procedures differentially modulated according to patient age and gonadotropin treatment strategy? SUMMARY ANSWER: Maternal age is an independent factor impacting miRNA expression in COCs while gonadotropin treatment may affect follicular miRNA expression and IVF efficacy. WHAT IS KNOWN ALREADY: Epigenetic mechanisms in female infertility are complex and poorly studied. DNA methylation, histone modifications, miRNAs and nucleosome positioning influence cellular machinery through positive and negative feedback mechanisms either alone or interactively. miRNAs are important regulators during oogenesis, spermatogenesis and early embryogenesis, and are reported to play a role in regulating crosstalk between the oocyte and COCs. Although miRNome analysis has been performed in female human reproductive tissues (endometrium, myometrium, cervix and ovaries), epigenetic modifications in women with infertility have not been explored in detail. In addition, the impact of gonadotropin treatments during MAR on miRNA expression in COCs has not been fully investigated. STUDY DESIGN, SIZE, DURATION: This study was carried out in 53 COC samples obtained from mature metaphase II (MII) oocytes in 53 women undergoing MAR treatment. A total of 38 samples for assay development were pooled by maternal age and gonadotropin treatment into four predetermined subgroups: ≥36 years and recombinant human FSH (r-hFSH), n = 10; ≥36 years and r-hFSH+ recombinant human-luteinizing hormone (r-hLH), n = 10; ≤35 years and r-hFSH, n = 9; ≤35 years and r-hFSH+r-hLH, n = 9. miRNome profiles were determined and compared between subgroups. Expression of defined miRNAs was validated in the remaining fifteen samples, representative of each subgroup, by quantitative polymerase chain reaction (PCR). PARTICIPANTS/MATERIALS, SETTING, METHODS: COCs were processed for miRNA-enriched total RNA extraction and pooled in homogeneous subgroups to obtain a sufficient amount and quality of starting material to perform the analysis. Each pooled sample underwent miRNA profiling using PCR assay system to examine expression of 752 human miRNAs without pre-amplification. Data were analyzed using the delta-delta Ct method for relative quantitation and prediction of target genes (with at least four algorithms predicting the same miRNA-gene interaction pair (HIT)>4). The miRSystem database provided functional annotation enrichment (raw P-value <0.05) of co-expressed miRNAs. MAIN RESULTS AND THE ROLE OF CHANCE: We found distinctive miRNA expression profiles in each subgroup correlating with age and MAR stimulation. In addition, a number of selective and co-expressed miRNAs were revealed by comparative analysis. A cluster of 37 miRNAs were commonly but differentially expressed in all four pools. Significant differences were observed in expression regulation of 37 miRNAs between age groups (≤35 or ≥36) in women receiving r-hFSH+r-hLH compared to those receiving r-hFSH alone. Higher concentrations and increased numbers of miRNAs were recorded in younger than in older patients, regardless of treatment. Functional and expression studies performed to retrieve common miRNome profiles revealed an enrichment of biological functions in oocyte growth and maturation, embryo development, steroidogenesis, ovarian hyperstimulation, apoptosis and cell survival, glucagon and lipid metabolism, and cell trafficking. The highest scored pathways of target genes of the 37 common miRNAs were associated with mitogen-activated protein kinase (MAPK) signaling pathways, G alpha signaling, transcription regulation, tight junctions, RNA polymerase I and III, and mitochondrial transcription. We identified a potential age- and MAR stimulation-dependent signature in the miRNA landscape of COCs. LIMITATIONS, REASONS FOR CAUTION: We cannot rule out the possibility that other unknown individual genetic or clinical factors may have interfered with the reported results. Since miRNA profiling was conducted with a predefined array of target probes, other miRNA molecules, potentially modulated by age and hormonal stimulation, may have been missed in this study. WIDER IMPLICATIONS OF THE FINDINGS: miRNA expression in COCs is modulated by gonadotropin treatment and correlates strongly with age. A better understanding of the expression patterns and functions of miRNAs may lead to the development of novel therapeutics to treat ovarian dysfunction and improve fertility in older women. STUDY FUNDING/COMPETING INTEREST: This study was funded by Merck KGaA, Darmstadt, Germany. All authors declared no competing interest, except SL and TD who are fully employed by Merck KGaA. TRIAL REGISTRATION NUMBER: N/A.

Effect of sperm selection method by cumulus oophorus complexes and conventional sperm preparation method on sperm quality and DNA fragmentation for assisted reproduction techonology.

OBJECTIVES: To compare the effectiveness of sperm selection using cumulus oophorus complexes (COCs) and conventional sperm preparation methods on sperm quality and DNA fragmentation METHODS: Normal semen samples under the World Health Organization (WHO)'s 2010 eligibility criteria were collected and processed using conventional sperm preparation methods. The prepared sperm were divided into two groups. Spermatozoa in the study group were selected based on their ability to penetrate a layer of COCs. In the control group, spermatozoa were kept in culture medium under similar conditions. The selected-sperm were evaluated based on sperm quality and DNA fragmentation. RESULTS: Thirty normal semen samples were recruited. Spermatozoa that were able to passthrough the COCs had significantly higher sperm motility parameters than the control group (curvilinear velocity [VCL; 143.5 vs 122.2; P < 0.01], average path velocity [VAP; 83.6 vs 69.3; P < 0.01], straight-line velocity [VSL; 67.95 vs 60.45; P < 0.01]). The percentage of normal spermatozoa morphology in the COCs group was significantly higher than in the control group (21.70% vs 18.76%). In addition, there was significantly less DNA fragmentation in the COCs group than in the control group (18.83 vs 10.83). CONCLUSION: Spermatozoa selected using COCs were likely to be effective in terms of sperm quality and DNA fragmentation.

Active peristaltic movements and fluid production of the mouse oviduct: their roles in fluid and sperm transport and fertilization†.

To study how the oviduct behaves in relation to fluid secretion and sperm transport, ovary-oviduct-uterus complexes of the mouse were installed in a fluid-circulating chamber without disturbing the blood circulation or parasympathetic innervation. Injection of a bolus of Indian ink into the lower isthmus revealed very active adovarian peristalsis of the isthmus, which was most prominent during the periovulatory period. Oviduct fluid, secreted by the entire length of the isthmus, was rapidly transported to the ampulla and ovarian bursa before draining into the peritoneal cavity. The upper isthmus, in particular the isthmic-ampullary junction, was responsible for this adovarian fluid flow. Peristalsis of the oviduct, undisturbed flow of oviduct fluid from the isthmus to the peritoneal cavity, and the spermatozoon's own motility all contribute to efficient sperm ascent and to fertilization within the oviduct. Therefore, chemotaxis, rheotaxis, and thermotaxis of spermatozoa toward oocyte-cumulus complexes in the ampulla are all unlikely mechanisms for explaining sperm-oocyte contact and successful fertilization, given the rapid adovarian flow of oviduct fluid in this species.

Effect of DNA damage of cumulus oophorus cells and lymphocytes analyzed by alkaline comet assay on oocyte quality and intracytoplasmic sperm injection success among patients with polycystic ovary syndrome.

AIM: Cumulus oophorus cells (COC) exhibit a close relationship with the oocytes. We aimed to determine the role of the DNA integrity of COC and lymphocytes on the oocyte and embryo quality and intracytoplasmic sperm injection (ICSI) success. METHODS: The COC obtained with the mechanical denudation of each oocyte and the lymphocytes obtained on oocyte retrieval day from 69 infertile polycystic ovary syndrome (PCOS) patients were used. The tail length, tail moment and tail DNA percentage were evaluated using an alkaline comet assay. The oocytes and embryos were graded. Clinical pregnancy was defined as the presence of a gestational sac with a beating heart. RESULTS: All of the DNA integrity parameters of the COC and lymphocytes were similar between the good and poor quality oocytes and between the good and poor quality embryos. There was no relationship between the DNA damage parameters and the fertilization and clinical pregnancy. The day 2 hormone levels, body mass index and age were negatively correlated with the DNA integrity parameters. No links were found between the embryo and oocyte scores and the DNA damage parameters. CONCLUSION: The DNA integrity of the COC and lymphocytes did not seem to be related to the oocyte and embryo quality and ICSI success.

Cumulus oophorus complexes favor physiologic selection of spermatozoa for intracytoplasmic sperm injection.

OBJECTIVE: To investigate the effectiveness of cumulus oophorus complexes (COCs) in the physiologic selection of spermatozoa for intracytoplasmic sperm injection (ICSI). DESIGN: A prospective sibling oocytes study. SETTING: Center of reproductive medicine. PATIENT(S): Couples undergoing ICSI during 2016, females aged ≤38 years, and at least six metaphase II (MII) oocytes retrieved. Sixty patients were included in the study. Of 857 MII oocytes, 429 were allocated to the study group and were injected with the sperm selected via COCs; 428 MII oocytes were allocated as controls (C) and fertilized by conventional ICSI. INTERVENTION(S): In the study group, ICSI was performed with spermatozoa that traversed the COCs in vitro. MAIN OUTCOMES MEASURE(S): Blastocyst/top blastocyst formation rate, fertilization rate, and oocyte utilization rate. RESULT(S): Oocytes injected with COC-selected spermatozoa had a significantly higher fertilization rate than the conventional ICSI group (85.31% vs. 74.77%). There were no statistically differences in cleavage and top embryo rate on day 3 between the COC-ICSI and C-ICSI groups. However, with day 5 or 6 embryos, compared with conventional ICSI, COC-ICSI significantly improved blastocyst formation rate (64.90% vs. 53.50%), blastocyst formation rate at day 5 (46.52% vs. 38.85%), top blastocyst rate (38.72% vs. 24.20%), and the usable blastocysts formation rate (62.12% vs. 46.82%). The oocyte utilization rate was improved greatly in the COC-ICSI group compared with the C-ICSI group (51.98% vs. 34.35%). Furthermore, the fertilization rate, top embryo rate on day 3, usable blastocyst rate, top blastocyst rate, and day 5 usable blastocysts rate were similar between the conventional IVF and COC-ICSI groups. Single-blastocyst transfer was performed in 82 cycles, including 44 fresh cycles and 38 frozen-thawed cycles. The cumulative embryo implantation rate in the COC-ICSI group was 64.29%, slightly higher than in the C-ICSI group (53.85%), but there was no statistical difference. CONCLUSION(S): The use of COCs to select spermatozoa for ICSI appears to be effective and led to a statistically significant improvement in blastocyst development and quality.

Effect of seasonality on the quality of bovine oocytes selected by the brilliant cresyl blue method / Efecto de la estacionalidad en la calidad de los ovocitos seleccionados por el método de azul cresil brillante / Efeito da sazonalidade na qualidade dos ovócitos bovinos selecionados pelo método azul cresil brilhante

Abstract Background: The brilliant cresyl blue (BCB) staining is a non-invasive test to select the best-suited oocytes for embryonic development. This makes it a useful tool to select best-quality oocytes at the times of the year when there is forage restriction. Objective: To evaluate the effect of seasonality on the nuclear maturation and quality of oocytes selected by the BCB test. Methods: The cumulus-oophorus complexes (COCs) were obtained in summer and winter of 2010 and 2011. Selected COCs were maintained for 90 min at 38.5 °C in a CO2 incubator, in TCM 199 medium containing 10% fetal bovine serum and antibiotics, and supplemented with 26 µM brilliant cresyl blue. Afterwards, they were divided according to the ooplasm staining (BCB+ -blue; BCB− -unstained). Subsequently, COCs were matured for 22 h. Nuclear maturation was evaluated at 22 h of culture. Results: The proportion of BCB− oocytes was higher in the winter of 2010, but there was no increase in this group in the winter of 2011. The percentage of oocytes that reached metaphase II was higher in control and BCB+ groups in relation to oocytes from BCB− group. Conclusion: The season of the year influences the percentage of oocytes best suited for embryonic production in situations in which oocyte donors receive pasture-based feeding, since the method was effective in determining the effect of seasonality on the competence of bovine oocytes to reach nuclear maturation.

Resumen Antecedentes: La tinción con azul cresil brillante (BCB) es un método no invasivo para seleccionar ovocitos aptos para el desarrollo embrionario. Por tanto, es una herramienta útil para selecionar los ovocitos de mejor calidad en temporadas de restricción de forraje. Objetivo: Evaluar el efecto de la estacionalidad sobre la maduración nuclear y calidad de los ovocitos seleccionados por el test BCB. Métodos: Los complejos cumulus-oophorus (CCOs) fueron obtenidos durante el verano y el invierno de 2010 y 2011. Los CCOs seleccionados se mantuvieron durante 90 min a 38,5 oC en una incubadora de CO2 en un medio TCM 199 con 10% de suero fetal bovino y antibióticos, suplementado con 26 µM de azul cresil brillante. Luego se separaron según el color del citoplasma (BCB+ -azul y BCB− -incoloro). Posteriormente, los CCOs se maduraron durante 22 h. La evaluación de la maduración nuclear se realizó a las 22 h de cultivo. Resultados: La proporción de ovocitos BCB− fue mayor en el invierno de 2010, pero no hubo un aumento de ese grupo en el invierno de 2011. El porcentaje de ovocitos que alcanzaron la etapa de metafase II fue mayor en el grupo control y BCB+ con respecto al grupo BCB−. Conclusión: La estación del año influye en el porcentaje de ovocitos más aptos para la producción de embriones en situaciones donde las donadoras de ovocitos reciben alimentación a base de pastos, ya que este método fue eficaz para determinar el efecto de la estacionalidad en la competencia de ovocitos bovinos en alcanzar la maduración nuclear.

Resumo Antecedentes: O método do azul cresil brilhante (BCB) não é invasivo e seleciona ovócitos mais aptos ao desenvolvimento embrionário. Portanto é ferramenta útil para selecionar ovócitos de melhor qualidade em épocas do ano que ocorre restrição de pastagem. Objetivo: Avaliar o efeito da sazonalidade sobre a maturação nuclear e a qualidade dos ovócitos selecionados pelo teste BCB. Métodos: Os complexos cumulus oophorus (CCOs) foram obtidos no verão e inverno de 2010 e 2011. Os CCOs selecionados foram mantidos por 90 min, a 38,5 °C, em incubadora de CO2, em meio TCM 199 contendo 10% de soro fetal bovino e antibióticos, e suplementado com 26 µM de azul cresil brilhante. Em seguida, estes foram divididos de acordo com a coloração do citoplasma (BCB+ -azuis e BCB− -incolores). Então os CCOs foram maturados durante 22 h. A avaliação da maturação nuclear foi realizada às 22 h de cultivo. Resultados: A proporção dos ovócitos BCB− foi maior no inverno de 2010, mas não houve aumento desse grupo no inverno de 2011. O percentual de ovócitos que atingiu o estágio de metáfase II foi maior no controle e no grupo BCB+ em relação ao grupo BCB−. Conclusão: A estação do ano influencia o percentual de ovócitos mais aptos a produção de embriões, em situações onde as doadoras de ovócitos recebem alimentação baseada em pastagens, já que o método se mostrou efetivo para determinação do efeito da sazonalidade sobre a competência de ovócitos bovinos em atingirem a maturação nuclear.

Follicular Fluid redox involvement for ovarian follicle growth.

As the human ovarian follicle enlarges in the course of a regular cycle or following controlled ovarian stimulation, the changes in its structure reveal the oocyte environment composed of cumulus oophorus cells and the follicular fluid (FF).In contrast to the dynamic nature of cells, the fluid compartment appears as a reservoir rich in biomolecules. In some aspects, it is similar to the plasma, but it also exhibits differences that likely relate to its specific localization around the oocyte. The chemical composition indicates that the follicular fluid is able to detect and buffer excessive amounts of reactive oxygen species, employing a variety of antioxidants, some of them components of the intracellular milieu.An important part is played by albumin through specific cysteine residues. But the fluid contains other molecules whose cysteine residues may be involved in sensing and buffering the local oxidative conditions. How these molecules are recruited and regulated to intervene such process is unknown but it is a critical issue in reproduction.In fact, important proteins in the FF, that regulate follicle growth and oocyte quality, exhibit cysteine residues at specific points, whose untoward oxidation would result in functional loss. Therefore, preservation of controlled oxidative conditions in the FF is a requirement for the fine-tuned oocyte maturation process. In contrast, its disturbance enhances the susceptibility to the establishment of reproductive disorders that would require the intervention of reproductive medicine technology.

The Behavior and Acrosomal Status of Mouse Spermatozoa In Vitro, and Within the Oviduct During Fertilization after Natural Mating.

Although 90%-100% of mouse oocytes can be fertilized in vitro with capacitated spermatozoa within 1 h after insemination, oocytes within the oviduct are fertilized one by one over a period of several hours. In vitro experiments showed that both acrosome-intact and acrosome-reacted spermatozoa entered the cumulus oophorus, but that acrosome-reacted spermatozoa reached the surface of oocytes more readily than acrosome-intact spermatozoa. During the period of fertilization within the oviduct, acrosome-reacted spermatozoa were seen throughout the isthmus, but with higher incidence in the upper than in the mid- and lower segments of the isthmus. Very few spermatozoa were present in the ampulla, and almost all were acrosome reacted. Although the cumulus oophorus and zona pellucida are known to be able to induce or facilitate the acrosome reaction of spermatozoa, this picture makes it likely that almost all fertilizing mouse spermatozoa within the oviduct begin to react before ascending from the isthmus to the ampulla. We witnessed a reacted spermatozoon that stayed on the zona pellucida of a fertilized oocyte for a while; it then moved out of the cumulus before reaching the zona pellucida of the nearby unfertilized oocyte. We noted that only a few spermatozoa migrate from the isthmus to the ampulla during the progression of fertilization, and this must be one of the reasons why we do not see many spermatozoa swarming around a single oocyte during in vivo fertilization.

Expressão gênica em ovócitos suínos de diferentes classificações morfológicas / Gene expression in swine oocytes in different morphological classifications

A produção in vitro de embriões suínos tem alcançado resultados insatisfatórios: ovócitos maturados in vivo produzem uma porcentagem maior de embriões em relação aos maturados in vitro. O sucesso da maturação in vitro está diretamente relacionado com a competência ovocitária. Somente ovócitos competentes são capazes de serem fecundados e terem desenvolvimento embrionário normal. A competência ovocitária pode ser avaliada por vários parâmetros. Recentemente têm sido utilizados como parâmetro os estudos da expressão de genes associados com a competência. O presente trabalho teve por objetivo avaliar diferenças na expressão dos genes BMP15, RYBP, MATER e ZAR1 em ovócitos imaturos de diferentes classes morfológicas, sendo elas: 1, 2, 3 e 4, com a finalidade de proporcionar importantes marcadores moleculares relacionados com a capacidade ovocitária. O RNA total dos ovócitos foi extraído e utilizado como molde para a síntese da primeira fita de cDNA. Os resultados da expressão gênica foram analisados utilizando-se modelo misto, considerando os dados de expressão gênica variável dependente e as classes ovocitárias variáveis independentes. Os genes BMP15, ZAR1 e RYBP apresentaram expressão semelhante nas classes ovocitárias 1, 2 e 3; somente a categoria 4 diferiu na expressão desses genes (P<0,05). O gene MATER foi expresso de forma semelhante em todas as classes ovocitárias estudadas (P>0,05). A técnica de RT-qPCR foi eficiente para detecção desses transcritos em ovócitos de diferentes classes. No entanto, para melhor entendimento do envolvimento desses transcritos na aquisição da competência ovocitária, são necessários mais estudos avaliando ovócitos de diferentes classes morfológicas, em diferentes fases de desenvolvimento, e implicação de outros genes envolvidos com a competência ovocitária.

The in vitro production of pig embryos has achieved unsatisfactory results; in vivo matured oocytes produce a higher percentage of embryos compared to in vitro maturation. The success of in vitro maturation is directly related to oocyte competence. Only competent oocytes are capable of being fertilized and have normal embryonic development. The oocyte competence can be assessed using several parameters. Recently these parameters have been used for gene expression studies associated with competence. This work aimed to evaluate differences in gene expression BMP15, RYBP, MATER, ZAR1 as endogenous control and the constitutive gene GAPDH in immature oocytes of different morphological classes which are: 1, 2, 3 and 4, in order to provide significant molecular markers linked to the ability of development. Oocytes Total RNA was extracted and used as a template for synthesis of the first cDNA strand. The results of gene expression were analyzed using a mixed model, considering the dependent gene expression data and independent ovocitary variable classes. The genes BMP15, RYBP ZAR1 and showed similar ovocitary expression in classes 1, 2 and 3 differ only in category 4 in their expression (P<0.05). The MATER gene was similarly expressed in all ovocitary classes studied (P>0.05). The RTQ-PCR technique was effective for detection of these transcripts in oocytes from different classes. However, for better understanding of the involvement of these transcripts in the acquisition of oocyte competence more studies are needed to evaluate different morphological classes of oocytes at different stages of development and the implication of other genes involved in oocyte competence.

Notable Aspects of Glycan-Protein Interactions.

This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host's immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses).

Obesity accelerates ovarian follicle development and follicle loss in rats.

OBJECTIVE: Studies have shown that excess body fat negatively affects reproductive functions in females. However, whether obesity affects the ovarian follicle development and ovarian lifespan and the underlying mechanism has not been well elucidated. The aim of the present study was to investigate the association between obesity and ovarian follicle development. METHODS: Adult female Sprague-Dawley rats (n = 36) were randomly divided into three groups: the normal control (NC) group, the caloric restriction (CR) group (fed 70% food of the NC group) and the high-fat diet (HF) group. They were maintained on these regimens for 18 weeks. RESULTS: The body weight, ovary weight and visceral fat in the HF group were significantly higher than those in the NC group and the CR group at the end of treatment. Histological analysis showed that the HF rats had significantly less number and percentage of primordial follicles, but greater number and percentage of developing and atretic follicles than the NC rats and CR rats. Western blot analysis demonstrated that the level of mTORC1 and p-S6K1 proteins significantly increased in the ovaries of HF rats, whereas that of SIRT1, SIRT6, FOXO3a and NRF-1 decreased compared to the NC rats. In contrast, the expression of mTORC1 and p-S6K1 dramatically declined, while that of SIRT1, SIRT6, FOXO3a and NRF1 increased in the ovaries of CR rats. CONCLUSIONS: Our study suggests that the HF diet induced obesity may accelerate the ovarian follicle development and rate of follicle loss through activating mTOR and suppressing SIRT1 signaling, thus leading to POF, and that CR may inhibit the activation of primordial follicles, follicular development and loss, thus extending the ovarian lifespan through suppressing mTOR and activating SIRT1 signaling.

Sperm competition risk generates phenotypic plasticity in ovum fertilizability.

Theory predicts that sperm competition will generate sexual conflict that favours increased ovum defences against polyspermy. A recent study on house mice has shown that ovum resistance to fertilization coevolves in response to increased sperm fertilizing capacity. However, the capacity for the female gamete to adjust its fertilizability as a strategic response to sperm competition risk has never, to our knowledge, been studied. We sourced house mice (Mus domesticus) from natural populations that differ in the level of sperm competition and sperm fertilizing capacity, and manipulated the social experience of females during their sexual development to simulate conditions of either a future 'risk' or 'no risk' of sperm competition. Consistent with coevolutionary predictions, we found lower fertilization rates in ova produced by females from a high sperm competition population compared with ova from a low sperm competition population, indicating that these populations are divergent in the fertilizability of their ova. More importantly, females exposed to a 'risk' of sperm competition produced ova that had greater resistance to fertilization than ova produced by females reared in an environment with 'no risk'. Consequently, we show that variation in sperm competition risk during development generates phenotypic plasticity in ova fertilizability, which allows females to prepare for prevailing conditions during their reproductive life.