Cumulus Oophorus Cells Research Articles

Histomorphological and histochemical features of atresia of antral follicles of water buffaloes

The present study aimed to characterize the atresia of antral follicles of buffalo. Two types of atresia, cystic and obliterative, were observed in the buffalo ovary. The cystic atresia was characterized by complete degeneration of the granulosa cells with the antrum filled with fluid. In obliterative atresia, the granulosa cells degenerated, and the surrounding theca cells started invaginating into the antrum. The ingrowth of these connective tissues filled the antrum. The histological picture of tertiary follicles showed that the atresia occurred in successive stages. The atretic changes observed were pyknosis of granulosa cells, the creation of small spaces between the cells, and the loosening of the cumulus oophorus cells. Two patterns were observed based on the start of atresia, i.e. antral atresia and basal atresia. At places, call-exner bodies were observed in the granulosa cell layer. The collagen fiber-containing basal lamina started separating from the theca layer at locations and later became hypertrophied in the cystic atresia type. In contrast, in the obliterative atresia, the collagen fibers were seen as abundant in growth towards the lumen and later, it filled the whole antral cavity. Disruptions of reticular fibers as stained with Gridley’s stain were observed in the cystic type of atresia, and ingrowth of reticular fibers in the antrum and formation of corpus atreticum were contributed by reticular fibers also.

The relationship between CYP19A1 gene expression in luteinized granulosa cells and follicular estradiol output in women with endometriosis

Endometriosis was claimed to negatively affect the intrafollicular environment, hindering oocyte competence. Previous studies evaluated expression levels of cytochrome P450 aromatase (CYP19A) in granulosa and cumulus oophorus cells collected from endometriosis women, but results are controversial. To further investigate the intrafollicular environment whose alteration may potentially disturb ovarian steroidogenesis in endometriosis, gene expression of CYP19A and of its upstream enzymes, StAR and 3βHSD was assessed in luteinized granulosa cells isolated from follicular fluids (FF) collected during Assisted Reproduction Technology (ART) procedures in women with stage III-IV disease and from subjects without the condition. In a subgroup of patients, cumulus oophorus cells (COCs) were also assessed for CYP19A, StAR and 3βHSD gene expression. No difference in mRNA expression of CYP19A1, StAR and 3βHSD in both granulosa cells and COCs was observed between the two groups of patients. No significant difference was also found between estradiol FF levels detected in endometriosis patients (median=873, IQR=522–1221 ng/ml)) and control patients (median=878, IQR=609–1137 ng/ml). To gain more insight into the intrafollicular regulation of CYP19A in patients with endometriosis, associations between expression of the analyzed genes, systemic and follicular 17β-estradiol levels and ART outcomes were assessed. While in the control group, levels of CYP19A1, StAR and 3βHSD transcripts significantly correlated with follicular estradiol levels (adjusted R² of 0.60), no significant association was detected in affected women (adjusted R² of 0.23). After stratification of the populations based on the presence of the disease, CYP19A1 expression was shown to correlate with the number of oocytes retrieved [β:− 1.214;95%CI: − 2.085 - (−0.343); p = 0.007] in the control group while this association was not present in patients with endometriosis [β:− 0.003; 95%CI:− 0.468–0.461; p = 0.988)]. These results do not support data from the literature indicating a reduced aromatase expression in granulosa cells of affected women, but they highlight a potential subtle mechanism affecting the ovulation process in these women.

Evidence for the expression of vasorin in the human female reproductive tissues

Objective: To investigate the expression and localization of Vasorin (Vasn) in human female reproductive system. Methods: The presence of Vasorin was evaluated by RT-PCR and immunoblotting analyses in patient-derived endometrial, myometrial and granulosa cells (GCs) primary cultures. Immunostaining analyses were performed to detect Vasn localization in primary cultures and in ovarian and uterine tissues. Results: Vasn mRNA was detected in patient-derived endometrial, myometrial and GCs primary cultures without significant differences at the transcript level. Otherwise, immunoblotting analysis showed that Vasn protein levels were significantly higher in GCs than proliferative endometrial stromal cells (ESCs) and myometrial cells. Immunohistochemistry performed in ovarian tissues revealed that Vasn was expressed in the GCs of ovarian follicles at different stages of development with a higher immunostaining signal in mature ovarian follicles such as the antral follicle or on the surface of cumulus oophorus cells than in early-stage follicles. The immunostaining of uterine tissues showed that Vasn was expressed in the proliferative stroma endometrium while it was significantly less expressed in the secretory endometrium. Conversely, no protein immunoreactivity was revealed in health myometrial tissue. Conclusions: Our results revealed the presence of Vasn in the ovary and the endometrium. The pattern of Vasn expression and distribution suggests that this protein may have a role in the regulation of processes such as folliculogenesis, oocyte maturation, and endometrial proliferation.

Dinâmica da maturação nuclear e citoplasmática de oócitos bovinos cultivados in vitro em meio suplementado com fulerol

ABSTRACT The aim of this work was to evaluate, in vitro, the dynamics of nuclear and cytoplasmic maturation of bovine oocytes in traditional IVM medium (CT) and supplemented with fullerol (MF50), for 36 hours. The nuclear maturation of CT (n=300) and MF50 (n=270) every 6 hours, stained with Hoechst33342 and cytoplasmic, the mitochondrial distribution of CT (n=197) and MF50 (n=159) at every 12 hours, stained with Mitotracker Orange. At 6 hours, CT oocytes (19%) were in MI (metaphase I), while in MF50 they were in GV (germ vesicle) or GVB (GV breakeage), repeating at 12 hours. At 18 hours, 46.3% were matured in CT, and 20% in MF50. At 24 hours, 43.9% of maturation was observed in the MF50 group, and 63.8% in the CT. At 30 and 36 hours, the maturation pattern was stable, but with the onset of oocyte degeneration. There was a delay in cytoplasmic maturation with 36 hours (P<0.05) in MF50 (53.9% of mature gametes), compared to CT (69.8%). With immature cytoplasm, they were 10.4% and 31.7% for CT and MF50 (P<0.05), respectively. It was concluded that fullerol possibly interfered in the expansion of cumulus oophorus cells, as well as delayed the meiotic progression and cytoplasmic maturation.

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.

P-246 A novel non-invasive tool for oocyte selection using gene expression and artificial intelligence

Abstract Study question Is it possible to predict top quality embryos through gene expression analysis of cumulus cells and artificial intelligence before fertilization? Summary answer The artificial inteligence based tool OsteraTest is able to predict the ability of the oocyte to develop into a top quality blastocyst with 86% accuracy. What is known already Proper oocyte selection is an important bottleneck for In Vitro Fertilization (IVF) success. Nowadays, oocyte selection relies mainly in morphological analyses, which is not an unbiased method and may fail to reveal the real competence status of gametes. Cumulus oophorus cells (CC) are somatic cells that surround the oocyte at the antral follicle. It is directly involved in oocyte maturation and development, and thus is a valuable non-invasive source of biological information regarding the oocyte’s health. Artificial intelligence can be used to identify key biological processes and markers of interest through machine learning methods and could thus be applied. Study design, size, duration This is a prospective study that included data from 80 CC samples retrieved from publicly available microarray data (GSE27377) in the algorithm construction phase and 65 CC samples from each oocyte of 26 patients submitted to Intracytoplasmic Sperm Injection (ICSI) in validation phase. Samples were divided in two groups: CCs from oocytes that developed into top quality blastocysts in day 5 after ICSI and CCs from oocytes that presented arrested development. Participants/materials, setting, methods Samples were submitted to real time quantitative PCR with 25 target genes. Afterwards, gene expression levels for each gene and sample were submitted to the final algorithm, that was computed into a software, the OsteraTest, in a double-blind approach. The software indicated the development potential of each oocyte and this ranking was compared to the embryologist’s day 5 blastocyst classification according to Gardner. Main results and the role of chance The bioinformatic approach implemented resulted in the OsteraTest, composed of 8 machine learning models using a 25-gene network that altogether can predict oocyte quality, thus representing a very complex assembly. The software presented more than 86% accuracy in predicting the oocytes developmental capacity into a top-quality day 5 blastocyst. Top quality blastocysts present over 80% chance of resulting in a healthy pregnancy and live birth, and so this approach could be further used as a pregnancy potential predictor after a prospective study is conducted, analyzing CCs from oocytes that were further fertilized, developed into blastocysts and transferred in single embryo transfers. This tool can contribute greatly to improve success rates in IVF procedures and to assess egg quality in egg freezing procedures, providing information about the gametes potential even years before its use. Limitations, reasons for caution A large-scale, prospective, randomized study is necessary for further validation of these findings and to confirm the validity of the OsteraTest in the clinical environment. Such study is now being conducted in our lab. Wider implications of the findings The OsteraTest proved to be a valuable non-invasive tool to predict embryo formation and oocyte capacity even before fertilization.It can enable the clinics to anticipate successful treatments and provide a predictive report for oocyte freezing patients. Trial registration number #68081017.2.0000.5347

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

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. 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. 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). 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. The trial was registered at UFRGS Research Ethics Committee and Plataforma Brasil under approval number 68081017.2.0000.5347 in June 6, 2019.

Diet crude protein reduction on follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows.

This study evaluated the effect of crude protein (CP) reduction in four diets (156, 139, 132, and 127 g Kg-1 DM) maintaining constant metabolizable protein (188 g/day) on the follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows. Twenty-two Girolando cows with average of 21.55 ±3.19 L daily milk yield, 105.30 ±22.62 days in lactation and 3.22 ±0.03 body condition score were selected. To reduce CP in diets and maintain constant metabolizable protein, urea and soybean meal were gradually replaced by lignosulfonate-treated soybean meal (SoyPass®, Cargill), resulting in an increase in rumen-undegradable protein and a reduction in rumen degradable protein. A linear and quadratic reduction was observed in the plasma and follicular fluid urea nitrogen concentration following CP reduction, with the most intense reduction occurring in the 127 g Kg-1 DM group (p<0.001). As CP reduced, there was a tendency for a linear increase in the follicular growth rate (P=0.0696), on the number and proportion of viable oocytes (P<0.09), and also a linear increase for the number (P=0.0397) and proportion (P<0.09) of grade I viable oocytes. Plus, there was a linear effect for the number of cumulus oophorus cells. Cows fed with the lowest amount of CP had cumulus-oocyte complexes with higher numbers of cumulus oophorus cells (P=0.0238). Also, the reduction of diet crude protein was followed by a decrease in the probability of oocytes’ DNA degradation. In conclusion, the reduction of CP in the diet of mid-lactating Girolando cows, reduces urea nitrogen concentration in both blood plasma and follicular fluid, and, as a consequence, increases the viability of oocytes and the number of cumulus oophorus cells while reducing oocytes’ DNA degradation of follicular included cumulus-oocyte complex. The reduction on dietary CP may improve in vivo oocytes’ embryo development impacting fertility of lactating dairy cows.

Expression Profile of New Gene Markers and Signaling Pathways Involved in Immunological Processes in Human Cumulus-Oophorus Cells.

The function of the immune system extends from defense against external pathogens to the recognition and elimination of mutated or dying cells, aiding elimination of malignant potential and/or maintaining homeostasis. The many cell types of the immune system secrete a broad range of factors to enable cellular signaling that is vital to physiological processes. Additionally, in the ovary, follicular selection and maturation, as well as ovulation, are directly regulated by the nearby immune cells. Additionally, ovulation and rupture of the follicle have been observed to resemble a local inflammatory response. Cells of the cumulus–oocyte complex (COC) show evolving gene expression profiles throughout the oocytes’ lifespan, including genes associated with immunological processes. Analysis of these genes allows the identification of useful molecular markers, as well as highlighting gene functions and interactions in these cells. Cumulus cells were obtained from hormonally stimulated patients undergoing an in vitro fertilization procedure and studied under long-term culture conditions. The microarray technique made it possible to compare the level of CCs’ gene expression on the 1st, 7th, 15th and 30th day of cultivation. Additionally, RNA microarray analysis was performed to map gene expression in these cells, associated with immunological processes and associated cytokine signaling. Subsequently, the use of DAVID software allowed us to identify the “defense response to other organism”, “defense response”, “defense response to virus”, “cytokine secretion”, “cytokine production” and “cytokine-mediated signaling pathway” GO BP terms, as well as allowing further analysis of the most differentially expressed genes associated with these processes. Of the 122 genes involved, 121 were upregulated and only one was downregulated. The seven most upregulated genes related to the abovementioned terms were ANXA3, IFIT1, HLA-DPA1, MX1, KRT8, HLA-DRA and KRT18. Therefore, genes involved in immunological defense processes are upregulated in CC cultures and could serve as useful molecular markers of growth and development in the COC, as well as the proliferation of granulosa and cumulus cells.

Double HCG trigger improves recovery of oocytes in women with a paucifollicular response to ovarian stimulation: A pilot study.

To examine whether adding a second HCG trigger, 12.5h after the first (36.5h before ovarian puncture), can facilitate recovery of oocytes in women with a paucifollicular response to ovarian stimulation. A total of 85 women aged 35-42years, with a paucifollicular response to ovarian stimulation and who had experienced a total failure of oocyte recovery after the standard HCG ovulation trigger 36.5h before ovarian puncture, were subsequently treated by the same protocol but with the addition of a second HCG trigger 12.5h later. The recovered oocytes were inseminated by intracytoplasmic sperm injection (ICSI) and all available embryos were transferred 3days later. The double trigger enabled recovery of cumulus oophorus cells from most of the follicles in the women who experienced failure of total recovery of oocytes after a single trigger. Fifteen patients became pregnant, and no signs of ovarian hyperstimulation syndrome were observed. Nine women delivered a healthy child. In women aged 35-42years with a paucifollicular response to ovarian stimulation, a double HCG trigger appears to improve the rate of oocyte recovery. The conclusion of this pilot study needs to be confirmed by larger prospective trials.

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

STUDY QUESTIONIs 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 ANSWERMaternal age is an independent factor impacting miRNA expression in COCs while gonadotropin treatment may affect follicular miRNA expression and IVF efficacy.WHAT IS KNOWN ALREADYEpigenetic 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, DURATIONThis 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, METHODSCOCs 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 CHANCEWe 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 CAUTIONWe 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 FINDINGSmiRNA 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 INTERESTThis 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 NUMBERN/A

Sperm-Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus-Oocyte Complex.

We have recently described a new model to study gamete interaction in mammalian species. The model recreates the spherical surface of the oocyte by using magnetic Sepharose beads coated with a layer of a recombinant protein involved in gamete interaction (such as ZP2, or the IZUMO1 receptor JUNO) and an external layer of cumulus oophorus cells, thus mimicking, to some extent, a native cumulus-oocyte complex. Once generated, this 3D model can be used in a sperm-binding assay to obtain valuable information about the molecular basis of gamete interaction, since different recombinant proteins can be used to coat the bead surface, thus generating a variety of models to be used for several species. Furthermore, thanks to the ability of the model to decoy sperm, the physiological status of the bound sperm can be studied, making this a powerful tool to select sperm with high fertilizing capacity, to unmask subfertile animals in livestock breeding centers, or for toxicological studies. Here, we describe how to generate and use this model for sperm-binding assays, using porcine sperm as an example, and ZP2, a protein from zona pellucida, as the recombinant protein of interest. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of the in vitro 3D model Alternate Protocol 1: Binding cumulus oophorus cells to the model Basic Protocol 2: Quality control of the model by SDS-PAGE electrophoresis and western blot Support Protocol 1: Immunochemistry to confirm proper protein distribution on surface of beads Support Protocol 2: Elution of recombinant conjugated proteins Basic Protocol 3: Sperm-binding assay Alternate Protocol 2: Sperm preparation by the swim-up method.

New markers of human cumulus oophorus cells cultured in vitro – transcriptomic profile

Abstract The presence of CCs around the oocyte after ovulation is one of the key elements contributing to oocyte developmental competence. In the presented study, we used CCs from 12 patients aged 18-40 diagnosed with infertility. After harvesting cells on day 1, 7, 15 and 30 of culture, total RNA was isolated and transcriptomic analysis was performed. The DAVID software indicated the following GO BP terms: “cell junction organization”, “cell migration”, “cell morphogenesis involved in differentiation”, “cell morphogenesis” and “cell motility”. Of the genes belonging to all ontological groups, the most downregulated were: SLC7A8, DFNB31, COL1A1, CDC42SE1, TGFBR3, HMGB1, with the most upregulated genes being: ANXA3, KIAA1199, HTR2B, VCAM1, DKK1. While many studies focus on attempts to obtain fully competent oocytes, scientists still have difficulty attaining adequate results in vitro. Lack of adequate knowledge often results in low in vitro fertilization efficiency. Therefore, our research focuses on CCs cells, thanks to which the oocyte most likely acquires developmental competence. The main purpose of the study was to identify the potential molecular markers responsible for cell junction organization, migration, differentiation, morphogenesis and motility. Running title: New markers of human cumulus oophorus cells cultured in vitro

The Role of Taste Receptor mTAS1R3 in Chemical Communication of Gametes.

Fertilization is a multiple step process leading to the fusion of female and male gametes and the formation of a zygote. Besides direct gamete membrane interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves gamete communication via chemical molecules triggering various signaling pathways. This work focuses on a mouse taste receptor, mTAS1R3, encoded by the Tas1r3 gene, as a potential receptor mediating chemical communication between gametes using the C57BL/6J lab mouse strain. In order to specify the role of mTAS1R3, we aimed to characterize its precise localization in testis and sperm using super resolution microscopy. The testis cryo-section, acrosome-intact sperm released from cauda epididymis and sperm which underwent the acrosome reaction (AR) were evaluated. The mTAS1R3 receptor was detected in late spermatids where the acrosome was being formed and in the acrosomal cap of acrosome intact sperm. AR is triggered in mice during sperm maturation in the female reproductive tract and by passing through the egg surroundings such as cumulus oophorus cells. This AR onset is independent of the extracellular matrix of the oocyte called zona pellucida. After AR, the relocation of mTAS1R3 to the equatorial segment was observed and the receptor remained exposed to the outer surroundings of the female reproductive tract, where its physiological ligand, the amino acid L-glutamate, naturally occurs. Therefore, we targeted the possible interaction in vitro between the mTAS1R3 and L-glutamate as a part of chemical communication between sperm and egg and used an anti-mTAS1R3-specific antibody to block it. We detected that the acrosome reacted spermatozoa showed a chemotactic response in the presence of L-glutamate during and after the AR, and it is likely that mTAS1R3 acted as its mediator.

Effects of Breed, Age, Body Condition Score, and Nutritional Status on Follicular Population, Oocyte Yield, and Quality in Three Cameroonian Zebus CattleBos indicus

This study was aimed at investigating the effects of breed, age, body condition score, and nutritional status on follicular dynamics, quality, and oocyte yield in Cameroonian zebus cattleBos indicus(Gudali and Red and White Fulani). A total of 95 cows were investigated for breed, age, body condition score, and nutritional status. Blood samples and ovaries of these cows were collected during slaughtering. Follicles on each ovary were counted and measured using electronic digital calipers and then classified into three categories: small (&lt;3 mm), medium (3 – 8 mm), and large (&gt; 8 mm) diameter. The oocytes were retrieved by slicing each ovary into a Petri dish containing collected medium. The oocytes recovered were examined under a stereoscope (x 10) and classified into four groups based on the morphology of cumulus oophorus cells and cytoplasmic changes. From these ovaries, 3888 follicles were counted with an average population of 27.81 ± 6.88 follicles per cow. The average numbers of small, medium, and large follicles per cow were 17.26 ± 1.36 (62.06%), 9.64 ± 1.00 (34.66%), and 0.52 ± 0.32 (1.86%), respectively. A total of 1822 oocytes were harvested with an average of 19.11 ± 0.89 oocytes per cow. Oocytes graded I, II, III, and IV were 7.03 ± 2.30 (36.65%), 4.53± 2.01 (23.61%), 3.19 ± 0.85 (16.63%), and 4.43 ±1.62 (23.09%), respectively. This study also showed that ovarian weight, number of follicles, and oocytes’ yield of right ovary were higher than left ones (P &lt; 0.05). Younger and normal cows (BCS = 3) presented higher (P &lt; 0.05) number of follicles and oocytes. Urea and cholesterol levels were higher in Gudali and White Fulani cows (P &lt; 0.05). This study indicated that ovaries of zebus harvested in slaughterhouse are an important source of follicular population, oocytes yield, and quality. Right ovaries are heavy and offer more numbers of follicles and oocytes than the left ones. BCS is the best indicator of nutritional status of cows. Important follicle populations are obtained from cows aging 6-9 years with BCS = 3.

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.

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. 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. 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. The DNA integrity of the COC and lymphocytes did not seem to be related to the oocyte and embryo quality and ICSI success.

Age-related expression of TGF beta family receptors in human cumulus oophorus cells.

During ovarian follicle growth, local cellular interactions are essential for oocyte quality acquisition and successful fertilization. While cumulus cells (CCs) nurture oocytes, they also deliver oocyte-secreted factors (OSFs) that activate receptors on CCs. We hypothesized that disturbance of those interactions contributes to age-related lower reproductive success in women submitted to assisted reproductive technology treatments. Women aged 27-48, without recognized personal reproductive disorder, were enrolled in the study and divided in <35- and ≥35-year-old groups. CCs collected upon follicle aspiration were processed for immunocytochemistry and RNA extraction. The expression patterns of OSF receptors BMPR2, ALK 4, ALK5, and activin receptor-like kinase (ALK6) were studied. Independently of age, receptors were found mostly in the cell periphery. The quantitative assay revealed that in older women, BMPR2, ALK 4, and ALK6 were all significantly decreased, whereas ALK5 was slightly increased. Femaleage imparts an effect on the expression of OSF receptors inCCs. The findingsindicate that reproductive aging affects the local regulation of signaling pathways mediated by BMPR2, ALK6, and ALK4 receptor activation, suggesting their joint involvement.

The opioid peptide beta-endorphin stimulates acrosome reaction in human spermatozoa.

The acrosome reaction occurs in vivo following sperm capacitation and is essential for the acquisition of sperm fertilization ability. However, little is known about the molecular identity of the physiological acrosome reaction regulators. In addition to progesterone, which is produced by cumulus oophorus cells and known to regulate acrosome reaction by activating the specific calcium channel CatSper, endogenous opioid peptides such as beta-endorphin and met-enkephalin are present at high concentrations in the follicular fluid suggesting that the opioid system may be involved in the mechanisms regulating the acrosome reaction in humans. By using Reverse Transcription-PCR, western blot and immunofluorescence approaches, we described the presence and localization of the beta-endorphin precursor, pro-opiomelanocortinin the middle section and in flagellum of human spermatozoa, and inside the seminiferous tubules of human testis. Flow cytometry and intracellular calcium analyses showed that beta-endorphin causes an inversely dose-dependent increase in the percentage of acrosome-reacted sperm cells by a calcium-independent protein kinase C pathway. These findings are important for future studies of sperm physiology and provide new insight into the function of the opioid system as a target of fertility management.

Evaluation of bovine (Bos indicus) ovarian potential for in vitro embryo production in the Adamawa plateau (Cameroon)

An abattoir study was conducted to evaluate the ovarian potential of 201 local zebu cattle from Ngaoundere, Adamawa region (Cameroon) for in vitro embryo production (IVEP). The ovaries were excised, submerged in normal saline solution (0.9%) and transported to the laboratory for a detailed evaluation. Follicles on each ovary were counted, their diameters (F) measured and were grouped into 3 categories: small (F &lt; 3 mm), medium (3 = F = 8 mm) and large (F &gt; 8 mm). Each ovary was then sliced into a petri dish; the oocytes were recovered in Dulbecco’s phosphate buffered saline, examined under a stereoscope (x10) and graded into four groups based on the morphology of cumulus oophorus cells and cytoplasmic changes of the oocytes. Grade I (GI): oocytes with more than 4 layers of bunch of compact cumulus cells mass with evenly granulated cytoplasm; grade II (GII): oocyte with at least 2–4 layers of compact cumulus cell mass with evenly granulated cytoplasm; grade III (GIII): oocyte with at least one layer of compact cumulus cell mass with evenly granulated cytoplasm; grade IV (GIV): denuded oocyte with no cumulus cells or incomplete layer of cumulus cell or expanded cells and having dark or unevenly granulated cytoplasm. The effects of both ovarian (ovarian localization, corpus luteum, size and weight of ovary) and non-ovarian factors (breed, age, body condition score (BCS) and pregnancy status of cow) on the follicular population and oocyte recovery rate were determined. There were an average of 16.75±0.83 follicles per ovary. The small, medium and large follicles were 8.39±0.60, 8.14±0.43 and 0.21±0.02 respectively. Oocyte recovery was 10.97±0.43 per ovary (65%). Oocytes graded I, II, III and IV were 3.53±0.19 (32.21%), 2.72±0.15 (24.82%), 2.24±0.15 (20.43%) and 2.47±0.20 (22.54%) respectively. The oocyte quality index was 2.26. Younger non pregnant cows having BCS of 3 and large ovaries presented higher number of follicles and oocyte quality (P &lt; 0.05) compared with other animals. Oocytes with quality (grade I and II) acceptable for IVEP constituted 57.15% of the harvest. This study indicated that factors such as age, pregnancy status, BCS and ovarian size must be taken into account to increase the potential of the ovary for IVEP.

Contribution of CBX4 to cumulus oophorus cell phenotype in mice and attendant effects in cumulus cell cloned embryos

Cumulus oophorus cells play an essential role in oocyte development. They are also widely employed as donor cells for cloning by somatic cell nuclear transfer. Our previous studies revealed that Cbx4 mRNA was overexpressed in cloned two-cell embryos. These data indicated that CBX4 may regulate normal cumulus cell differentiation and that its overexpression in clones could contribute to aberrant gene regulation. We used siRNA-mediated knockdown of Cbx4 to assess its role in determining cumulus cell phenotype and compared the effects of this knockdown to published data for aberrant gene regulation in cloned embryos. We observed widespread effects on the expression of genes related to diverse processes in cultured cumulus cells, including cell assembly/proliferation and DNA replication/repair, endocrine function, carbohydrate and lipid metabolism, inflammation, and cell morphology, with apparent effects of CBX4 in promoting cumulus cell proliferation and survival and inhibiting differentiation. Overall, the data implicate CBX4 as a key component in the pathway integrating endocrine signals, intraovarian paracrine factors, and oocyte-derived factors in the control of cumulus cell functions. We also observed altered expression of 25 cumulus cell markers of oocyte quality, indicating an important role of CBX4 in production of high quality oocytes. Finally, we found that about one-quarter of the genes showing aberrant transcription in cloned embryos are sensitive to Cbx4 knockdown in cumulus cells, consistent with a role for aberrant Cbx4 regulation in elaborating abnormal cloned embryo characteristics.