Corona Radiata Cells Research Articles

P–274 Undetectable viral RNA in follicular fluid (FF), cumulus cells (CC) and endometrial tissue in SARS-CoV–2 positive patients

Study questionIs there any indication for presence of viral RNA in FF, CC, immature oocytes or endometrial biopsy (EB) of SARS-CoV–2 patients undergoing ovarian stimulation?Summary answerViral RNA is undetectable in FF, CC and EB with RT-PCR. However, S-protein expression on corona radiata cells suggests susceptibility to SARS-CoV–2 infection.What is known alreadyThe effects of a SARS-CoV–2 infection on the female reproductive system are still poorly understood. Theoretically, co-localisation of the angiotensin converting enzyme (ACE2) and transmembrane serine protease 2 (TMPRSS2) on human blastocysts implies susceptibility to viral infection, mediated by the coronavirus spike (S) protein. To date, SARS-CoV–2 RNA was undetectable in mature oocytes from COVID–19 patients, despite the expression of ACE2 and TMPRSS2. The presence of viral RNA in endometrial tissue, immature oocytes, CC or FF has not yet been investigated in samples from patients with positive nasopharyngeal SARS-CoV–2 test.Study design, size, durationThis is a prospective, single-centre, observational study including ten patients with a positive nasopharyngeal swab for SARS-CoV–2, performed 48 hours before oocyte retrieval (OR), from September 2020 to January 2021. A patient was eligible if she preferred to continue treatment following adequate counselling of the unknown but presumably low risk for vertical transmission. Since a freeze-all strategy was applied, an EB was performed.Participants/materials, setting, methodsDuring OR, all protective measures were taken. Pooled FF, CC and EB from each patient were tested for viral RNA presence with RealStar® SARS-CoV–2 RT-PCR-Kit1.0 (Altona-Diagnostics). Ct values <40 were considered positive. EB was collected for pathological evaluation and cultured to obtain endometrial stromal cells (EnSC). Immature oocytes and EnSC were tested for S-protein expression by immunohistochemistry with anti-S antibody (MA5–35958, Thermo-Fisher Scientific) followed by Alexa Fluor™ 488-donkey-anti-mouse (Thermo-Fisher Scientific) and visualized with confocal microscopy.Main results and the role of chanceSARS-CoV–2 RNA was undetectable in the pooled FF, CC and EB from all patients included in the study. Histological analysis of the EB showed no pathological modifications, including inflammatory reaction, as compared to biopsies collected from swab negative patients. After staining with anti-S antibody, cultured EnSC and immature oocytes tested negative for the S-protein. However, the binding of anti-S antibody was demonstrated on the corona radiata cells remaining on the zona pellucida after oocyte denudation for intra-cytoplasmatic sperm injection, indicating presence of SARS-CoV–2. In that case, the explanation for the undetectable viral RNA in CC could be that the viral RNA concentration remained under the detection limit of the currently used RT-PCR test.Limitations, reasons for cautionThis study was conducted in a small population (ten patients included) with different viral load, with mild or without symptoms of COVID–19. Another important limitation is the absence of validation of the RT-PCR protocol for the investigation of other types of samples than nasopharyngeal swabs.Wider implications of the findings: The absence of SARS-CoV–2 RNA in all samples analysed represents a step further in reassuring a safe ART program for COVID–19 patients. However, the presence of S-protein on corona radiata cells warrants further investigation, since the theoretical possibility to infect human oocytes and/or embryos cannot be ruled out.Trial registration numberNCT04425317

Cytoplasmic and nuclear maturation of oocytes in mammals – living in the shadow of cells developmental capability

Abstract The pig is a polyestrous animal in which the ovarian cycle lasts about 21 days and results in ovulation of 10-25 oocytes. Ovum reaches 120-150 μm in diameter, with the surrounding corona radiata providing communication with the environment. The zona pellucida is composed of glycoproteins: ZP1, ZP2, ZP3. In the course of oogenesis, RNA and protein accumulation for embryonic development occurs. Maternal mRNA is the template for protein production. Nuclear, cytoplasmic and genomic maturity condition the ability of the ovum to undergo fertilization. There are several differences in protein expression profiles observed between in vitro and in vivo conditions. Oogenesis is the process of differentiating female primary sex cells into gametes. During development gonocytes migrate from the yolk sac into the primary gonads with TGF-1, fibronectin, and laminin regulating this process. Cell cycle is blocked in dictyotene. Primary oocyte maturation is resumed before each ovulation and lasts until the next block in metaphase II. At the moment of penetration of the sperm into the ovum, the metaphase block is broken. The oocytes, surrounded by a single layer of granular cells, form the ovarian follicle. The exchange of signals between the oocyte and the cumulus cells done by gap-junctions, as well as various endo and paracrine signals. The contact between the corona radiata cells provides substances necessary for growth, through the same gap junctions. Studies on follicular cells can be used to amplify the knowledge of gene expression in these cells, in order to open way for potential clinical applications.

Characteristic of factors influencing the proper course of folliculogenesis in mammals

Abstract Folliculogenesis is the process of ovarian follicle formation,, taking presence during foetal period. During the follicular development, oogoniums undergo meiosis and oocytes are formed. In the ovaries of new born sows, primary and secondary follicles are present and, 90 days after birth, tertiary follicles appear. During development in the ovarian follicles growth of granulosa cells and differentiation of the thecal cells can be observed. A cavity filled with follicular fluid appears. Granulosa cells are divided into: mural cells and corona radiata, which together with the oocyte form the cumulus oophorus. Corona radiata cells, mural layers and oolemma contact each other by a network of gap junctions. Secreted from the pituitary gland, FSH and LH gonadotropin hormones act on receptors located in granular and follicular cells. In the postnatal life tertiary follicles and Graafian follicles are formed. When the follicle reaches a diameter of 1 mm, further growth depends on the secretion of gonadotropins. Mature ovarian follicles produce: progestins, androgens and oestrogens. The growth, differentiation and steroidogenic activity of ovarian follicles, in addition to FSH and LH, is also affected by prolactin, oxytocin, steroid and protein hormones, numerous proteins from the cytokine and interleukin family, metabolic hormones like insulin, glucocorticoids, leptin, thyroid hormones and growth hormones. Despite numerous studies, many processes related to folliculogenesis have not been discovered Learning the mechanisms regulating reproductive processes would allow to easily distinguish pathological processes and discover more and more genes and mechanisms of their expression in cells that build ovarian follicles.

Viability of cumulus cells is associated with basal AMH levels in assisted reproduction

Objective(s)An interesting non-invasive approach to select embryos for transfer is analyzing the health state of somatic granulosa cells surrounding the oocyte addressing their mutual dependence. This prospective study was set up to analyse whether the DNA integrity of cumulus cells correlates with preimplantation development and basal AMH levels. Study designTherefore, 56 patients who gave written consent were enrolled. Sequential denudation of the cumulus-oocyte-complexes was performed in order to separate corona radiata from outer cumulus cells. DNA integrity of both cell types was analysed using a modified chromatin dispersion test. ResultsThe percentage of viable corona radiata cells per patient showed a linear correlation to blastulation (P<0.05). These innermost cells showed significantly lower rates of strand breaks (P<0.01) as compared to outer cumulus cells. Age-corrected AMH was significantly associated with the DNA integrity of outer cumulus cells (P<0.05). Conclusion(s)For the first time it could be shown that in fact clinical embryologists deal with two different entities of cumulus cells, inner and outer ones. It seems that any protective mechanism of the female gamete follows an outward gradient, so that negative effects, e.g. apoptosis, may impair outer cumulus cells first. Age-corrected AMH reflects quality of these outer cumulus cells.Keywords: AMH; Corona radiata cells; DNA fragmentation; Outer cumulus cells; SCD test

Immunolocalization of aquaporin 5 during rat ovarian follicle development and expansion of the preovulatory cumulus oophorus

Immunofluorescent localization of aquaporin 5 (AQP5) was investigated in rat ovarian follicles during development and preovulatory cumulus oophorus expansion. Ampullary cumuli oophori complexes (COCs) were examined. Analysis revealed that AQP5 immunostaining appeared in preantral follicles and formed a characteristic ring encircling and touching the oolemma. The staining represented most likely AQP5 functioning at the ends of corona radiata cell projections, anchoring on the oocyte surface. However, several hours after the presumptive preovulatory LH surge, when the process of expansion of COCs started, the AQP5 staining appeared also on the cumulus granulosa cells and in the extracellular matrix. In the postovulatory ampullary COCs the fluorescent ring was not observed, which may be the result of the well-established preovulatory withdrawal of projections from the zona pellucida. At that time-point immunofluorescent staining of AQP5 appeared in most oocytes and was also present in the apical membrane of epithelial cells of the oviduct ampulla. The latter observation suggests that after ovulation AQP5 is involved in the transcellular movement of water in the oviduct ampulla and oocytes in rats.

Profil d’expression des cellules de la corona radiata chez les patientes présentant une réserve ovarienne diminuée

ObjectivesDiminished ovarian reserve (DOR) is one of the causes of infertility. In this prospective study, gene expression profiling (GEP) of corona radiata cells (CRC) was performed to identify genes deregulated in DOR patients. Patients and methodsMicroarray-based GEP of CRC isolated from eight women undergoing IVF was performed to identify genes differentially expressed between patients with normal ovarian reserve and DOR patients. Microfluidic-based quantitative RT-PCR assay were used to validate selected transcripts on 40 independent patients. ResultsForty-eight transcripts were differentially expressed, including CXXC5 and FOXC1 down regulated in DOR, as well as CTGF, FSTL3, PTGS2 and SOCS2 up regulated in DOR. According to these transcripts, two DOR patients'subgroups (DOR Gr1 and Gr2) were identified. In DOR Gr2 patients, CITED2, CTGF, GAS-1, IRS2, PTGS2, SOCS2, VCAN were expressed at significantly higher levels, and CXXC5, FOXC1, GBP2 and ZMIZ1 at significantly lower level. Eleven of those genes are transcriptional targets of Estrogens and higher baseline oestradiol levels were observed in DOR Gr2 patients (P<0.006). Discussion and conclusionTwelve genes deregulated in CRC of DOR patients were identified, which could be involved in DOR pathogenesis. The distinction of a particular subgroup of DOR patients suggests the possibility of deregulation of estrogen response genes.

Molecular characterization of corona radiata cells from patients with diminished ovarian reserve using microarray and microfluidic-based gene expression profiling

Diminished ovarian reserve (DOR) is one of the causes of infertility in young women. In this prospective study, gene expression profiling (GEP) of corona radiata cells (CRC) was performed to identify genes deregulated in DOR patients. Microarray-based GEP of CRC isolated from eight women undergoing IVF was performed to identify genes differentially expressed between patients with normal ovarian reserve and DOR patients. Microfluidic-based quantitative RT-PCR assays were used to validate selected transcripts on 40 independent patients. A principal component analysis was used to identify more homogeneous subgroups of DOR patients. In silico analyses focusing on cis-regulation were performed to refine the interactions between patient's biological characteristics and their GEP. Forty-eight transcripts were differentially expressed, including CXXC finger protein 5 (CXXC5), forkhead box C1 (FOXC1) (down-regulated in DOR) as well as connective tissue growth factor (CTGF), follistatin-like 3 (FSTL3), prostaglandin-endoperoxide synthase 2 (PTGS2) and suppressor of cytokine signaling 2 (SOCS2) (up-regulated in DOR). According to these transcripts, two DOR patients' subgroups (DOR Gr1 and Gr2) were identified. In DOR Gr2 patients, C-terminal domain 2 (CITED2), CTGF, growth arrest-specific 1 (GAS1), insulin receptor substrate 2 (IRS2), PTGS2, SOCS2 and Versican (VCAN) were expressed at significantly higher levels and CXXC5, FOXC1, guanylate-binding protein 2 (GBP2) and zinc finger MIZ-domain containing 1 (ZMIZ1) at significantly lower levels. Higher baseline estradiol (E(2)) levels were observed in DOR Gr2 patients (P < 0.006). The in silico analyses suggested that all 11 genes differentially expressed between DOR Gr1 and DOR Gr2 subgroups could be transcriptional targets of estrogen. Despite small sample size limitations, 12 genes deregulated in the CRC of DOR patients were identified, which could be involved in DOR pathogenesis. A DOR patient's subgroup with high baseline E(2) levels and deregulated estrogen-responsive genes was also identified.

Involvement of ERK Phosphorylation in the Prevention of Ischemia-Induced Ovarian Follicular Depletion by Stem Cells

Ovarian failure is commonly caused by aging, autoimmune disease, menopause and cancer therapy. We used an ischemic model in the ovary to test the hypothesis that stem cells are helpful for ovarian regeneration after injury. Three treatment regimes were employed: sham-operated control, ligation plus PBS, and ligation plus immortalized human bone marrow stromal cells (stem cells) groups. After ligation-induced ischemia, stem cells or PBS were injected into rat ovaries. Then, pregnant mare serum gonadotropin was given intra-peritoneally to initiate folliculogenesis. The animals were then sacrificed. The ovary gland was weighed, and ovarian folliculogenesis, stem cell differentiation and vascular neogenesis were evaluated. In order to study improvement of folliculogenesis after ovarian ischemia, steroidogenic acute regulatory protein (StAR), p44/p42 MAPK (T-ERK1/2), and phospho-p44/ p42 MAPK (P-ERK1/2) expression were specifically evaluated. Results indicated that ovarian size was smaller and that the rate of folliculogenesis was lower in ovarian ischemic-reperfusion animals, but both recovered after stem cell treatment. The stem cells migrated into the ovary and differentiated into theca cells, granulosa cells, corona radiata cells and vascular endothelial cells. In addition, von Willebrand factor (vWF) expression was increased; 17beta-estradiol (E2), progesterone (P4), P-ERK1/2 and StAR protein expression was recovered by stem cells treatment in the ischemic ovaries. The serum LH was significantly increased in ovaries of ischemia-reperfusion animals, but the stem cell treatment restored the effects. These results suggest that stem cells might be helpful for ovarian regeneration after injuries by promoting vascular neogenesis and steroidogenesis through the MAPK pathway.

Metabolism of ethanol to acetaldehyde and increased susceptibility to oxidative stress could play a role in the ovarian tissue cell injury promoted by alcohol drinking

It is known that drinking alcohol can lead to reproductive problems in women. In this study, we analyzed the possibility that part of those effects were mediated through alterations of ovarian function related to ethanol oxidation to acetaldehyde occurring in situ. Biotransformation in the rat ovary cytosolic fraction was partially inhibited by allopurinol, suggesting the participation of xanthine oxidoreductase in the process. Microsomal pathway was of enzymatic nature, requiring nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), sensitive to oxygen and significantly inhibited by sodium diethyldithiocarbamate, 4-methylpyrazole and diphenyleneiodonium. Aldehyde dehydrogenase activity was detected by histochemistry in the ovarian tissue, in the strome surrounding the follicle while no alcohol dehydrogenase was detected. However, biochemical determination of alcohol dehydrogenase and aldehyde dehydrogenase activities in rat ovarian tissue revealed the presence of some activity of both enzymes but significantly lower than those found in the liver. By repetitive exposure of animals to ethanol, the microsomal metabolism to acetaldehyde was increased but not in the case of the cytosolic fraction. In these animals, t-butylhydroperoxyde-promoted chemiluminiscence was increased in comparison to control, revealing an increased susceptibility to oxidative stress due to alcohol drinking. Ultrastructure of ovarian tissue from rats exposed chronically to alcohol revealed alterations at the level of the granulosa; theca interna and pellucida zones. In the secondary follicle, alterations consisted of marked condensation of chromatin attached to the nuclear inner membrane. Intense dilatation of the outer perinuclear space could be observed. There was a marked dilatation of the rough endoplasmic reticulum accompanied of significant detachment of ribosomes from their membranes. Mitochondria appeared swollen. In the zona pellucida, most of the cell processes from oocyte and corona radiata cells were absent or broken totally or in part. Results suggest that in the rat ovary, metabolism of ethanol to acetaldehyde may play a role in alcohol effects on female reproductive function.

Reproduction of the Female Ferret (Mustela putorius furo)

The domestic ferret is a seasonally polyoestrous species. Females reach puberty at the age of 8-12 months. Females exhibit a constant oestrus between late March and early August if they are not bred. Increasing tumescence in the pink-coloured vulva is a sign of pro-oestrus. Oestrus can persist for up to 5 months, but once ovulation is induced, either pregnancy or pseudopregnancy ensues. Follicular development and atresia overlap in such a manner that there is a recent cohort of follicles available for ovulation whenever copulation might occur. Copulation may last from 15 min to 3 h, the average being 1 h. Ovulation is induced by pressure on the cervix associated with copulation. After sufficient LH release, the pre-ovulatory follicles mature and an average of 12 oocytes (5-13) per female are ovulated 30-40 h after copulation into the ovarian bursa. The ferret oocytes are most capable of being fertilized up to 12 h after ovulation, i.e. 42-52 h after copulation. Ferret oocytes ovulate at the metaphase of the second meiotic division (MII) embedded in three layers of corona radiata cells. Embryos enter the uterus over a period of several days starting on day 5 after mating. Between days 12 and 13 after mating, the embryos have become implanted in the endometrium. Implantation in the ferret is central with rapid invasion of the uterine epithelium by the trophoblast over a broad area that eventually becomes a zonary band of endotheliochorial placenta. Gestation length is 41 days (39-42 days). The domestic ferret gives birth to an average of eight kits (1-18 kits), which weigh 6-12 g at birth.

Ultrastructural Alterations and Occurrence of Apoptosis in Developing Follicles Exposed to Low Frequency Electromagnetic Field in Rat Ovary

Humans are continuously exposed to harmful environmental factors, including Electromagnetic Fields (EMF) produced by home appliances, diagnostic tools and industrial instruments. The present study investigates morphologically the effects of EMF on ovarian follicles. Female Wistar rats were exposed to 3 mili Tesla EMF for 4 h day(-1) for 4 months, the ovaries from both experimental and control groups were processed for Transmission Electron Microscopy (TEM) and TUNEL reaction technique. TEM revealed that in the oocytes from experimental group the nuclei were shrunken and zona pellucida appeared narrower than those in the control group. The number of microvilli were decreased significantly and in their cytoplasm there were several lipid droplets and organels were dispersed. Signs of apoptosis such as condensed nuclei, chromatin margination and nuclear membrane dilatation were common in granulosa and corona radiata cells from the EMF-exposed rats than those from the control group. Primary morphological changes in the granulosa cells from this group were retraction of granulosa cells, loss of microvilli and condensation or loss of mitochondrial cristae. The morphological changes in oocytes seem to indicate the cytotoxic effect of EMF and the changes occurred in granulosa cells coincide with initiation of apoptosis in granulosa cells. Present results suggest that EMF exposure may interfere with normal process of folliculogenesis.

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5–8 mm) and small (2–3 mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.

229CHARACTERIZATION OF CONNEXIN 43 IN THE CANINE OVARIAN FOLLICLE

Connexin 43 (Cx43) is the most abundant gap-junction protein in ovarian follicle cell interactions and has been well characterized for several mammalian species. However, the involvement of Cx43 in canine follicular development has not been extensively investigated. The objective of this study was to 1) characterize the spatial patterns of Cx43 localization within the canine ovarian follicle at various physiological states and during the estrous cycle (prepubertal, anestrus, proestrus, estrus and diestrus), and 2) to characterize the canine Cx43 mRNA transcript. The spatial expression pattern of Cx43 protein was evaluated by immunofluorescence microscopy in canine ovaries. Cx43 was not detected in primordial follicles, but was detected in primary and secondary follicles at each physiological state at all granulosa cell borders. In secondary follicles, definitive, punctate staining patterns were localized along mural granulosa cells and also in the surrounding granulosa-cumulus cell borders. Notably, more intense staining was observed in the corona radiata cells immediately surrounding the oocyte, as well as in trans-zonal projections and at the perivitelline membrane. Patterns of localization were most similar between proestrus and diestrus, and between prepubertal and anestrus in secondary follicles. Estrus-stage follicles showed a decrease in localization at the corona-oocyte cell borders as compared to proestrus and diestrus. In large, healthy antral follicles from proestrus and estrus stages, Cx43 was present in the stroma, theca, and granulosa layers. However, antral follicles from estrus-stage ovaries showed more intense staining in the mural granulosa and theca layers, and less intense in the stroma as compared to those of proestrus stage. The most intense pattern of punctate staining was observed in the corpora lutea of diestrus-stage ovaries. Additionally, gene-specific primers were designed from highly conserved regions of Cx43 mRNA among bovine, human and mouse. RNA was isolated from canine uterus and used as a template for RT-PCR. The PCR products were then sequenced and verified in GenBank to assess homology. The sequenced coding region for canine Cx43 mRNA shares 93% sequence homology with bovine vascular smooth muscle. Canine specific primers for this sequence have been designed, and expression analysis studies in canine ovarian follicles are currently underway. These results indicate that the pattern of localization of Cx43 is similar to that reported for the cow and the pig, except that in canine ovarian preantral follicles, Cx43 is also localized to the peri vitelline membrane. Additionally, these results suggest that the localization of Cx43 is dependent on the physiological state of the ovary, and is likely necessary for folliculogenesis and subsequent oocyte development in canines.

Oocyte–sperm interaction in the course of IVF: a scanning electron microscopy analysis

Interaction between the spermatozoon and the zona pellucida during the first steps of fertilization was analysed on approximately 500 polyploid and unfertilized IVF oocytes by scanning electron microscopy (SEM). Oocytes demonstrate high inter- and intra-individual variations in size and morphology which do not correlate either with the maturity of the oocytes or with the age of the women. During gamete interaction, corona radiata cells are widely dispersed around the zona but are still in contact with it through cytoplasmic filaments. Channels between granulosa cells guide spermatozoa towards the zona. In the course of fertilization, different types of attachment of the spermatozoon to the oocyte occur. Most commonly, a flat, tangential attachment of the sperm head to the surface of the zona appears, which is then followed by intrusion into the zona in precisely this horizontal position. However, vertical binding with penetration by the tip of the head first also occurs. In oocytes where large, cluster-like numbers of bound spermatozoa are visible, vertical binding and penetration is the most usual position. In the process of gamete interaction, both spermatozoa and zona pellucida are actively involved. Spermatozoa, including their tails, which are attached to the zona, are overgrown by filaments of zona material. These filaments of the zona are made of granules, which are the basic components of zona material. After the removal of the zona pellucida by laser, the oolemma becomes visible. It is covered by microvilli of highly variable numbers. Between these microvilli, cortical granules are evident, and appear even before sperm penetration.

Development of in vitro embryo production systems for red deer ( Cervus elaphus) : Part 2. The timing of in vitro nuclear oocyte maturation

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2 mm in diameter. Oocytes with compact cumulus cells were matured in 50 μl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 μg LH and FSH, 1 μg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 °C and 5% CO 2 in air. At 3 h intervals (0–27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6 h of incubation. The nuclear membrane began to disperse after 6 h and by 10.6±0.6 h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7±0.4 and 24.8±0.9 h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12 h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24 h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.

In vitro parthenogenesis of mouse oocytes under several experimental conditions.

Although the in vitro fertilisation index is a parameter commonly employed to investigate sperm functional activity, little attention has been given to the occurrence of parthenogenesis. The purpose of this study was to study at 6 h or 22 h incubation: (a) the cleavage-related events that occur in in vitro incubated mouse oocytes, in the absence (parthenogenesis) or presence of homologous spermatozoa; (b) the effect of mineral oil, commonly used in in vitro fertilisation assays; (c) the effect of piroxicam, a prostaglandin synthesis inhibitor, on the parthenogenetic rate; and (d) the influence on parthenogenesis of spontaneous loss of the cumulus oophorus coat during incubation. Under the experimental conditions employed, there was parthenogenetic activation and activation due to fertilisation. Both increased in a time-dependent manner. The mineral oil enhanced the parthenogenetic rate at 22 h incubation. However, it did not have any effect when the oocytes were inseminated. Since we can not discriminate how much of this activation was due to fertilisation and how much to parthenogenesis we must be very careful with this comparison. Piroxicam 10(-8) M did not show any effect on the mouse oocyte parthenogenetic rate at neither 6 h or 22 h incubation. Our results suggest that oocyte susceptibility to spontaneous parthenogenetic activation may be modified by the presence of the cumulus and corona radiata cells. In conclusion, we consider that further rigorous studies on these influences are necessary in order to confer more reliability on the results.

Morphology of the oocyte-follicular connection in the mare.

The present study characterised the oocyte-follicular connection (i.e., oocyte fixation site) in Graafian follicles of the mare morphologically. Antral follicles were dissected in toto from ovaries obtained from oestrous, dioestrous and transitional mares after slaughter. The location of the cumulus oophorus complex in relation to the ovulation fossa, the width and density of the blood vessels surrounding the cumulus oophorus complex, the relative dimensions and histological aspects of the cumulus oophorus were investigated. For ultrastructural analysis of the junctional regions, cumulus-oocyte complexes were recovered in vivo by transvaginal ultrasound-guided follicle aspiration. The location of the oocyte fixation site was independent of mare, follicular size and stage of the oestrous cycle. In 82% of follicles, the oocytes were embedded in a broad based cell mount. The width and density of the blood vessels surrounding the oocyte fixation site were correlated to each other, but independent of follicular size and cyclic stage. The histological appearance of the cumulus oophorus varied, especially in respect to the compactness, and loosening of the cumulus cell population was observed in several medium-sized follicles from dioestrous mares. Loosening of the cumulus cell population was apparently associated with decreased interdigitation between adjacent corona radiata cells. It can be concluded that the fixation site of the equine cumulus oophorus complex represents a firm cellular anchorage between follicular wall and oocyte. Furthermore, the location of the cumulus oophorus complex in relation to the ovulation fossa and characteristics of the surrounding blood vessels is independent of follicular size and cyclic stage.

Effects of follicle-stimulating hormone, bovine somatotrophin and okadaic acid on cumulus expansion and nuclear maturation of blue fox (Alopex lagopus) oocytes in vitro.

The meiotic competence and meiosis resumption of Blue fox (Alopex lagopus) oocytes from anoestrous animals were followed. Oocyte-cumulus complexes (OCC) were cultured in modified TC 199 medium with or without FSH, recombinant bovine somatotrophin (bST) and okadaic acid (OA). The results showed that oocytes less than 100 microns in diameter did not achieve germinal vesicle breakdown (GFBD) by 72 h of culture, which indicates their meiotic incompetence. Oocytes larger than 100 microns in diameter underwent GVBD after 48 h of culture (27%) and reached metaphase II (MII) after 72 and 96 h (20% and 27%) in control medium. Both bST and OA accelerated resumption of meiosis (bST: 55% GVBD and 42% MII after 48 h; OA: 66% GVBD after 18 h). In contrast, FSH significantly reduced meiosis resumption (only 3% GVBD and MII after 72 h) and induced changes in the shape of cumulus granulosa (CG) cells and F-actin assembly typical for cumulus expansion. However, the innermost layers of CG cells (corona radiata) remained connected with the oocyte via gap junctions until the end of culture. Cumuli of oocytes cultured in control, bST-supplemented or OA-supplemented medium did not expand (changes in cell shape and F-actin redistribution did not occur). Moreover, especially in media with bST and OA an increased detachment and rapid disconnection of their gap junctions with the oocyte were observed. These results suggest that under in vitro conditions FSH stimulates expansion of the CG cells and the attached membrana granulosa cells but in contrast it secures heterologous gap junctions between cytoplasmic processes of the corona radiata cells and oolemma during 3 days of culture. Thus, in agreement with the in vivo situation in which Canidae oocytes are ovulated in the GV stage, the cumulus, mainly corona radiata cells, controls resumption of meiosis in Blue fox oocytes under in vitro conditions also.

Calcium elevation in sheep cumulus-oocyte complexes after luteinising hormone stimulation.

We investigated Ca2+ levels in intact cumulus-oocyte complexes (COCs) on exposure to peak levels of luteinising hormone (LH). Specific preparations were used where cumulus corona cells were loaded with a membrane-permeant Ca(2+)-sensitive dye (FLUO-3AM), whereas the oocyte was injected directly with the nonpermeant form of the dye (FLUO-3). After exposure to LH, cumulus and corona radiata cells showed distinct rises in intracellular Ca2+ in 50-200 sec. The pattern of Ca2+ response varied in the different cells both for the duration of the transients and for their persistence. Interestingly, Ca2+ elevations were recorded in all the layers of the cumulus mass, including the innermost layer of corona cells, demonstrating the wide diffusion of LH receptors. Following the Ca2+ raise in somatic cells, an intracellular Ca2+ elevation also was recorded within the oocyte with a delay of 100-300 sec. The elevation started at the cortex of the oocyte and then spread all over the ooplasm. The addition of verapamil or manganese chloride did not prevent LH-induced Ca2+ elevation in the COC, whereas mechanical uncoupling of cumulus cells from the oocyte prevented any Ca2+ response within the oocyte. The results indicate that cumulus corona cells are capable of transducing LH message by rising intracellular Ca2+ and show that this signal is rapidly transferred into the oocyte through gap junctions. This may result from the direct diffusion of Ca2+ or its putative releaser IP3 from cumulus cells to the oocyte.

Progesterone accelerates spermatozoa penetration time in homologous in vitro fertilization in the pig

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2 mm in diameter. Oocytes with compact cumulus cells were matured in 50 μl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 μg LH and FSH, 1 μg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 °C and 5% CO2 in air. At 3 h intervals (0–27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6 h of incubation. The nuclear membrane began to disperse after 6 h and by 10.6±0.6 h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7±0.4 and 24.8±0.9 h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12 h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24 h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.