Cumulus-corona Cells Research Articles

Design and implementation of a lab-on-a-chip for assisted reproductive technologies.

The microfluidic device is highly optimized to remove oocytes from the cumulus-corona cell mass surrounding them. Additionally, it effectively captures and immobilizes the oocytes, aiding in assessing their quality and facilitating the injection of sperm into the oocyte. In this study, a novel microfluidic chip was designed and manufactured using conventional soft lithography methods. This research proposes the utilization of a microfluidic chip as a substitute for the conventional manual procedures involved in oocyte denudation, trapping, and immobilization. The microfluidic chip was modeled and simulated using COMSOL Multiphysics® 5.2 software to optimize and enhance its design and performance. The microfluidic chip was fabricated using conventional injection molding techniques on a polydimethylsiloxane substrate by employing soft lithography methods. A hydrostatic force was applied to guide the oocyte through predetermined pathways to eliminate the cumulus cells surrounding the oocyte. The oocyte was subsequently confined within the designated trap region by utilizing hydraulic resistance along the paths and immobilized by applying vacuum force. The application of this chip necessitates a lower level of operator expertise compared to enzymatic and mechanical techniques. Moreover, it is feasible to continuously monitor the oocyte's state throughout the procedure. There is a reduced need for cultural media compared to more standard approaches.

P–171 Automated oocyte and zygote denudation using a novel microfluidic device supervised by a computer vision algorithm

Abstract Study question Is it possible to remove cumulus cells using a 16-well microfluidic device with automated flows to facilitate vitrification, ICSI, NI-PGT or non-invasive metabolomics analysis? Summary answer The designed automated system and protocol efficiently denude 16 samples simultaneously with a x10 lower shear stress than the manual process and without human intervention. What is known already Most processes involved in IVF such as insemination, washing, denudation, embryo culture and selection are still manually performed, labor-intensive and require highly skilled professionals. This leads to a significant variability in the clinical outcomes achieved by different embryologists and labs. The automation of these processes is a promising approach to reduce costs and improve the accessibility to assisted reproductive therapies. Although a simple procedure, standardization of cumulus oocyte complex (COCs) and zygotes denudation is key to facilitate ICSI, vitrification and to avoid DNA contamination for NI-embryo testing (PGT or metabolomics), while avoiding damage to the oocyte by excessive shear stress. Study design, size, duration A total of 160 cow COCs were used due to their size similarity with human COCs. Half were denuded 16–20 hours post-insemination and half pre-insemination for 5–10 minutes. COCs were classified as partially denuded if fertilization assessment, ICSI or vitrification was possible, and completely denuded if no cumulus cells remained. COCs controls were manually denuded (Stripper® capillary 145μm ID) to compare shear stress between procedures. This study was conducted during 2020 – 2021. Participants/materials, setting, methods We developed a customized microfluidic biochip that exerts a particular fluid motion while avoiding egg entrapment within microfluidic channels. The denudation efficacy was established by subjectively scoring images of bovine oocytes after generating a continuous “Push & Pull” fluid motion inside the biochip wells. A Computer Vision model was developed in parallel in order to optically assess denudation completion. The model used was a Pytorch implementation of Faster-RCNN with ImageNet pretrained weights Main results and the role of chance 96 bovine COCs were microfuidically handled post insemination achieving complete (56/96) or partial (40/96) removal of the cumulus cells on day 1, while for day 3 double denudation group, 89/96 (92.7%) were completely denuded while the rest remained partially denuded. In comparison, 80/80 (100%) of manually denuded cow COCs, achieved complete denudation (50% post-insemination group and 50% pre-insemination group). In addition, 48/64 (75%) cow COCs treated pre-insemination were partially denuded, enough to carry out ICSI after 5–10 min of treatment. The results here obtained indicate that media needs to flow through the device at a rate that can generate enough shear to strip off the cumulus-corona cells while avoiding emptying of the reservoirs containing the fertilization or culture medium. The shear stress of our design was calculated to be smaller than 4.4 Pa, about ten times lower than the one applied by the manual process (∼44Pa). The deep learning algorithm was tested on 20 unseen human oocytes on day 1, with 10 true positives 9 true negatives, and 1 false negative (95% accuracy). Limitations, reasons for caution The success of the denudation procedure was dependent on the design of the biochip wells and the microfluidic protocol used. The accuracy of our findings is still limited because of the difficulty in manufacturing prototype biochips. Wider implications of the findings: Complete denudation is key to avoid DNA contamination for NI-PGT or metabolomics analysis, while avoiding damage to the oocyte by excessive shear stress. Our device, which has the potential of scaling up and treat each oocyte individually, can improve automation and increase efficiency of current ART procedures Trial registration number NA

On-chip oocyte denudation from cumulus-oocyte complexes for assisted reproductive therapy.

Human infertility can be treated using assisted reproductive technology (ART) such as intracytoplasmic sperm injection (ICSI). But current ART techniques suffer from multiple cumbersome processes requiring technically skilled personnel. Microfluidics technologies offer unique opportunities to streamline ART procedures, reduce stress imposed upon gametes and embryos, and minimize the operator-to-operator variability. However, there have been no automated and continuous processing systems that can reduce the dependence on well-trained embryologists to obtain ICSI-ready oocytes from patients. In this study, using mouse models, we developed a microfluidic device to denude oocytes from the surrounding cumulus-corona cell mass, facilitating the evaluation of oocyte quality and the injection of sperm. Enzyme-treated cumulus-oocyte complexes pass through a series of jagged-surface constriction microchannels of optimized geometries. The jagged inner wall of constriction channels facilitates stripping off of the cumulus-corona cell mass. Oocytes that were denuded by the device showed comparable fertilization and developmental competence compared with mechanical pipetting. The device developed in this study achieves the automation of a manual process for oocyte denudation in a continuous flow, as well as improving standardization and ease-of-use. Our denudation-on-a-chip approach requires inexpensive and simple equipment, which represents one step forward towards improving the accessibility and affordability of assisted reproductive therapy.

Differences in collagen expression in cumulus cells induced by LH-activity of highly purified menotrophin in comparison to recombinant FSH in a follicle culture model

Differences in collagen expression in cumulus cells induced by LH-activity of highly purified menotrophin in comparison to recombinant FSH in a follicle culture model

The Fallopian tubes in domestic mammals: how vital is their physiological activity?

Set in an historical perspective, this essay examines diverse physiological aspects of Fallopian tube function in domestic animals and man. Microsurgical experiments are described that established the role of the isthmus in imposing a sperm gradient up to the site of fertilisation. Resection of the isthmus followed by reanastomosis of the remaining portions of the tube generated a high incidence of polyspermy in mated animals. Scanning electron microscopy and surgical studies revealed that spermatozoa were arrested and stored in the caudal portion of the isthmus before ovulation, the so-called functional sperm reservoir. There were specific adhesion contacts between the sperm head and endosalpingeal microvilli or cilia. Further experiments indicated that very large numbers of competent spermatozoa could be released from pre-ovulatory binding by microinjections of a solution of progesterone in oil under the serosal layer of the tube: when suitably timed, such treatment led to a high incidence of polyspermic fertilisation. Avid sperm binding in the caudal isthmus before ovulation prevents myosalpingeal activity leading to abnormal fertilisation, as might occur with multiple mating. Temperatures in the reproductive system were assessed and the caudal isthmus was found to be cooler than the ampulla during the pre-ovulatory phase of sperm storage. Finally, the existence of fluid microenvironments within the Fallopian tubes was reported, and the role of suspended cumulus-corona cells in amplifying signals from the zygote examined. An impact of Fallopian tube fluids on embryonic gene expression was also considered--an influence that may be further imposed if such fluids have access to the uterine lumen.

Presence of a 31-kD protein band in human cumulus–corona radiata–conditioned media and pregnancy outcome

Objective: To investigate relationships between cumulus–oocyte complex (COC) morphology, protein patterns of cumulus–corona (CC) cell–conditioned media, and pregnancy outcome in IVF-ET cycles. Design: Retrospective study. Setting: Private university IVF center. Patient(s): One hundred twenty infertile women who underwent IVF-ET procedures. Intervention(s): None. Main Outcome Measure(s): COC grading, analysis of CC cell morphology and conditioned media, and pregnancy rate (PR). Result(s): After IVF and embryo transfer, cultured CC cells were classified as high (HA) or low (LA) on the basis of their adhesive properties. Neither adhesion activity nor fertilization rates and embryo quality were correlated with COC grading. PR in cycles with HA cells was 38%, but 14% of cycles showing LA activity also had positive outcome. To find more meaningful parameters of CC cells useful to predict fertilization and pregnancy, the electrophoretic protein patterns of media conditioned by HA or LA cells were studied. Retrospective analysis showed that all cycles in which replaced embryos were associated with the presence of a 31-kD band in conditioned media failed implantation, whereas 83% of cycles lacking this band resulted in positive, ongoing pregnancy. Conclusion(s): Pregnancy prediction cannot rely simply on CC cell morphological analysis. Screening of conditioned media may provide more reliable parameters.

Effect of timing of oocyte denudation and micro-injection on survival, fertilization and embryo quality after intracytoplasmic sperm injection.

In human in-vitro fertilization (IVF), the oocytes are surrounded by cumulus and corona cells at the time of insemination so that their maturity cannot easily be evaluated. The best IVF results are obtained if the oocytes are inseminated 2-6 h after retrieval. In the intracytoplasmic sperm injection (ICSI) procedure, the oocytes are denuded by enzymatic and mechanical treatment in order to be able to perform the injection. As a consequence, the nuclear maturity of the oocytes can be evaluated and only those that have extruded the first polar body are injected. However, metaphase-II oocytes that have not yet reached cytoplasmic maturity cannot be recognized. The purpose of this study was to investigate the effect of different timing of cumulus-corona cell removal and injection on the outcome of ICSI. For this we allowed the oocytes to complete in-vitro cytoplasmic maturation in two different culture conditions: (i) surrounded by their cumulus and corona cells or (ii) totally denuded. We performed three different studies on sibling oocytes obtained after a standardized buserelin/human menopausal gonadotrophin (HMG) protocol. We investigated the effect of early (1-2 h after retrieval) and late (5-6 h after retrieval) oocyte denudation and injection on the survival and fertilization of the injected oocytes and on embryo cleavage after fertilization. We found no statistically significant differences between early and late injection, indicating that after a standardized buserelin/HMG protocol the metaphase-II oocytes do not need time for further cytoplasmic maturation. Furthermore, a different timing of cumulus-corona cell removal has no effect on the outcome of ICSI, suggesting that the surrounding cells are not necessary for survival, fertilization and cleavage after ICSI.

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.

Ultrastructural features of bovine cumulus-corona cells surrounding oocytes, zygotes and early embryos.

Integrated transmission and scanning electron microscopic (TEM and SEM) techniques have provided the first detailed description of the ultrastructural features of the bovine cumulus-corona (CC) cells surrounding oocytes at the time of final maturation, zygotes and early cleaving embryos (2/4 to 6/8 blastomeres). TEM revealed the presence of rough endoplasmic reticulum and Golgi complexes in the cytoplasm of CC cells surrounding immature, mature and fertilized eggs, and also revealed an increasing amount of smooth endoplasmic reticulation membranes, lipid droplets and mitochondria with villiform and/or tubular cristae in the cytoplasm of CC cells during maturation and fertilization of the oocyte. In addition, a loss of cell-to-cell junctions between CC cells was evident. TEM also demonstrated that a few residual CC cells were still associated with early embryos and that these cells showed rather degenerative or apoptotic patterns, the latter pattern also observed on cells associated with fertilized eggs. SEM revealed that the complex of CC cells of immature oocytes was compact with narrow intercellular spaces, which progressively enlarged in size around mature oocytes. This phenomenon is mostly due to the production of abundant extracellular matrix. Immature CC cell complexes possessed characteristic long and filiform microvilli whereas the surface of CC cells surrounding mature oocytes showed numerous blebs and occasional large cytoplasmic protrusions as well as microvilli. Zygotes and early embryos were covered with a few polyhedral CC cells possessing scarce and short microvilli and a large amount of pleomorphic blebs. This study demonstrated a precocious luteinization occurring in bovine CC cells at ovulation until zygote segmentation, and this process was associated with a progressive apoptotic mechanism that ended in the complete denudation of the zona pellucida covering the early embryo. The presence of CC cells around the maturing oocyte and fertilized egg could have important functions related to the microenvironmental requirements of ovum maturation as well as facilitating activities related to fertilization.

Follicular cells affect the fertilizability and developmental competency of bovine oocytes in vitro.

The present study examined the time-dependent effects of follicular cells on the fertilizability of oocytes and their subsequent development to blastocysts. The percentages of oocytes reaching the metaphase-II stage of maturation rose from 51.3% after 16 h of culture to 86.2% at 28 h (cumulus-intact oocytes; CIO) and, for the same time points, from 65.4% to 83.3% (corona-enclosed oocytes; CO) and 54.3% to 88.9% (denuded oocytes; DO), respectively. When DO were cultured for more than 24 h before insemination, fertilization rates were significantly lower compared with CIO and CO. The maximum rates of development to blastocysts were observed when the oocytes were cultured for 24 h in the CIO group (22.1%), 20 h in the CO group (19.7%) and 18 h in the DO group (9.2%), respectively. These results suggest that (i) the presence of cumulus cells or corona cells during maturation is not necessary for nuclear maturation of oocytes; (ii) the attachment of corona cells to the oocytes during maturation is important for the further development to the blastocyst stage, and (iii) the presence of attached cumulus and/or corona cells during maturation in vitro extends the maturation period required for further development to the blastocyst stage.

Activation of protein kinase A and protein kinase C mediates the depolarising effect of LH in ovine cumulus-corona cells.

We have studied the changes in membrane potential induced by LH in cumulus and granulosa cells isolated from sheep antral follicles. The investigation was carried out by using a non-invasive technique based on the use of a membrane potential sensitive probe, bis-oxonol. The membrane potential of mural granulosa cells was totally unaffected by LH, while that of cumulus or corona cells showed a marked depolarisation, starting 2-3 min after the addition of the hormone and plateauing after 5-10 min. None of the cells tested reacted to FSH. In the second part of the experiment the role of protein kinase A (PKA) and protein kinase C (PKC) in mediating the effect of LH was studied. The selective activation of PKA or PKC induced in cumulus-corona cells a rapid hyperpolarisation due to increased Cl and K conductance respectively. By contrast, the simultaneous activation of the two kinases induced a rapid membrane depolarisation due to the progressive decrease in K conductance. The activation of each kinase or their combined stimulation did not induce any change in the membrane potential of mural granulosa cells. These data demonstrated that LH has a depolarising effect regionally circumscribed to cumulus-corona cells and that this depolarisation depends on a reduction of K conductance caused by the activation of PKA and PKC.

Heterogeneous distribution of fibronectin, tenascin-C, and laminin immunoreactive material in the cumulus-corona cells surrounding mature human oocytes from IVF-ET protocols--evidence that they are composed of different subpopulations: an immunohistochemical study using scanning confocal laser and fluorescence microscopy.

Monoclonal antibodies and immunofluorescence microscopy, including laser confocal microscopy, were used in this study to point out the production of fibronectin, tenascin-c, and laminin in the cumulus-corona (CC) cells surrounding mature human oocytes from IVF-ET protocols in view of their presumptive importance in the coordination of the processes leading to fertilization and early embryo cleavage, including the final maturation of the ovum, the sperm-egg interaction, and the "complex biochemical dialogue" between the gamete and the oviduct through the tubal luminal environment. One hundred fifty mature oocyte-CC complexes were obtained from IVF-ET protocols and fixed in 4.0% buffered paraformaldehyde. Specimens were incubated with a panel of primary monoclonal antibodies (mabs) recognizing different epitopes of fibronectin, tenascin-c, and laminin and then with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Observations were made by a scanning confocal microscope (Sarastro 2000) and a photomicroscope (Polyvar, Reichert-Jung) equipped with epifluorescence optics. The immunohistochemical data demonstrated that human CC cells are capable of producing fibronectin and tenascin-c but that their production is not homogeneous in the CC population. In fact, fibronectin immunoreactivity was shown mostly by inner CC cells (mainly corona cells), whereas tenascin was produced by some cells scattered in the entire cumulus mass. Moreover, fibronectin and tenascin-c immunoreactive material was observed in the intracytoplasmic areas, at the plasma membrane level as well as in the extracellular matrix. On the contrary, laminin immunofluorescent material was found around plasma membranes of almost all CC cells, but a clear intracytoplasmic reaction was never observed. This leads us to assume that laminin in the extracellular matrix remains entrapped once produced by granulosa follicular cells and that in the postovulatory period no active secretion occurs in CC cells. Even though the functional role of these extracellular matrix proteins remains still unclear, it is reasonable to suggest that they are necessary in various steps of the reproductive process, i.e., from the pick-up of the oocyte, its transport through the oviduct, and fertilization, up until the early cleavage of the embryo. Finally, functional differences between "corona radiata" and "cumulus" cells during the oocyte denudation may be accounted for particular distribution of these adhesive proteins.

Granulosa-Cumulus-Corona Expansion and Aromatase Localization in Preovulatory Follicles in Superovulated Heifers

Granulosa-cumulus and cumulus-corona expansion as well as aromatase localization within ovarian follicles were monitored during the preovulatory period in superovulated cattle that were blood sampled every 2'nd h for LH analyses. Granulosa-cumulus as well as cumulus-corona expansion were studied by means of transmission electron microscopy and computerized image analysis. Localization of aromatase, an enzyme involved in estrogen synthesis, was determined immunocytochemically using anti-human placental aromatase cytochrome P-450 antisera. Nuclear oocyte maturation was determined by aceto:orcein staining. Significant cell dissociation within the granulosa-cumulus stalk occurred before the breakdown of the germinal vesicle, i.e. the oocyte nucleus, during the period up to 5-7 h after the LH peak, i.e. the highest LH concentration during the surge. Significant increase in intercellular spacing between the cumulus-corona cells occurred at 13-15 and 19-21 h after the LH peak. Before the LH peak all layers of granulosa cells were immunocytochemically stained for aromatase. At 5-7 h after the LH peak, however, only the granulosa cell layers located near the basal lamina were stained, and at all later intervals staining was absent. The granulosa cells of primary and secondary follicles, the interstitial gland cells, the theca interna cells and the oocytes in all follicles were immunocytochemically unstained.

Oocyte and embryo quality as well as pregnancy rate in intracytoplasmic sperm injection are not affected by high follicular phase serum progesterone.

The effect of elevated serum progesterone concentrations (> 1 ng/l) on or before the day of human chorionic gonadotrophin (HCG) injection on the outcome of women receiving gonadotrophin-releasing hormone analogue (GnRHa)/human menopausal gonadotrophin (HMG) for ovarian stimulation prior to intracytoplasmic sperm injection (ICSI) was evaluated. A total of 1275 ICSI cycles were analysed retrospectively. In 53 cycles (4.5%), serum progesterone concentrations were > 1 ng/ml. Patients in the high progesterone group had significantly higher oestradiol and luteinizing hormone concentrations on the day of HCG injection. The characteristics of the cumulus-corona cell complexes and the nuclear maturity of the oocytes were similar in the groups of patients with high and low serum progesterone levels. Fertilization and cleavage rates as well as embryo quality were not different in the two groups. No difference in implantation or clinical pregnancy rates was observed between the high progesterone and low progesterone groups. Moreover, the cumulative exposure to progesterone during the follicular phase, as expressed by the area under the curve (AUC), and the duration of exposure to high serum progesterone levels (> 1 ng/ml) were not significantly different between pregnant and non-pregnant women in the high progesterone group. We conclude that in ICSI cycles pretreated with GnRHa, increased serum progesterone concentrations on or before the day of HCG injection do not affect ICSI outcome.

A study of the cumulus-corona cell complex in in-vitro fertilization and embryo transfer; a prognostic indicator of the failure of implantation.

In-vitro morphological changes and proliferation of cells of the cumulus oophorus, in particular cells of the corona radiata, were seen to differ between individuals and between oocytes of the same cohort in an unselected series of 159 IVF cycles. Cell proliferation did not relate to indications for treatment, type and length of ovarian stimulation, age of patient, ovarian response and quality of oocyte, fertilization rate or embryo morphology. A direct relationship, however, was demonstrated between the proliferative capacity of these cells and the incidence of clinical pregnancy. No pregnancies resulted when there was an absence of morphological change or proliferation of these cells. In cycles where the proliferative capacity of these cells varied between oocytes of a single cohort the pregnancy rate was 40% per cycle, and in cycles where cells associated with all oocytes underwent extensive proliferation the pregnancy rate was 49% (P < 0.001). Thus, regardless of the apparent quality of gametes or embryo, no pregnancies arose if the cumulus-corona complex was incapable of replication. We have, therefore, identified two populations of oocytes based on the proliferative capacity of cumulus-corona cells in vitro, one being associated with a failure of implantation.

Co-Expression of Cytokeratins and Vimentin in Sheep Cumulus-Oocyte Complexes. Alteration of Intermediate Filament Distribution by Acrylamide: (granulosa cells/vimentin/cytokeratin/acrylamide/maturation).

The association between germ cells and somatic granulosa cells persists throughout the growth of the oocyte by means of foot processes of the cumulus corona cells that cross the zona pellucida. During meiotic maturation important nuclear and cytoplasmic events occur in cumulus-oocyte complex suggesting implication of cytoskeletal elements. Immunoblotting analysis of cytoskeletal proteins of the cumulus cells revealed the presence of vimentin polypeptide and of at least two cytokeratin polypeptides. Using immunofluorescence techniques on cryostat sections through frozen tissue, we provided evidence for the presence of cytokeratins of the simple epithelial type in addition to vimentin in sheep cumulus cells. These two types of intermediate filaments were localized throughout the cytoplasm and especially in the foot processes which cross the zona pellucida. The contact area between the two cell types was also labelled with the antibodies. Acrylamide treatment of cumulus-oocyte complexes involved a drastic disorganization of the intermediate filament network and triggered the isolation of the oocyte from its cumulus cells. This isolation resulted in resumption of meiosis. From these results it appears that intermediate filaments could participate in the process of gap junction loss and indirectly in the control of meiosis resumption.

The ultrastructure of human cumulus-corona cells at the time of fertilization and early embryogenesis. A scanning and transmission electron microscopic study in an in vitro fertilization program.

We observed the ultrastructure of the cumulus-corona cells (CC cells) surrounding: 1) human preovulatory oocytes unfertilized after in vitro insemination and 2) in vitro-fertilized polypronuclear ova (PO) at the pronuclear stage (3 pronuclei) and during early cleavage, at the 3-8 cell stage (cleaving PO). All the samples were obtained from women who underwent pharmacological hormonal stimulation during in vitro-fertilization procedures. Both cell groups were composed of irregularly rounded CC cells, showing an oval nucleus with one or more reticular nucleoli. Spermatozoa in close contact with CC cells were also seen. Linear and annular gap junctions between neighbouring cells were present, particularly in Group 1. Lipid droplets were present in both groups, appearing slightly more numerous and electron-dense in Group 2. In Group 1, mitochondria were numerous, polymorphic, and provided with cristae varying from lamellar to tubular. In Group 2, mitochondria also showed polymorphism, with bacilliform organelles with tubular cristae being predominant. In both groups cisternae and associated vacuoles and vesicles belonging to the Golgi complex were scattered in the cytoplasm of CC cells. Similarly, tubular and vesicular profiles of smooth endoplasmic reticulum were abundant and uniformly distributed throughout the cytoplasm of CC cells of both groups. In contrast, the abundant rough endoplasmic reticulum in Group 1 was formed by parallel stacks of flattened cisternae, whereas it was less plentiful and not arranged in stacks in Group 2. The CC-cell surface appeared covered by numerous membrane expansions in both groups. The expansions in Group 1 were mainly composed of blebs of various sizes and a few short microvilli, whereas in Group 2 numerous microvilli covered the cell surface. These observations demonstrate that a gradual establishment and maintainance of a steroidosynthetic capability (luteinization) takes place in CC cells, particularly during and shortly after fertilization, as occurs contemporaneously in the granulosa cells of the postovulatory follicle. Our results may be considered as ultrastructural confirmation of the capability of the CC cells to produce small amounts of steroids (estrogens and mainly progesterone). These hormones, alone or together with other substances (proteins, nutrients, growth factors?), might--around the fertilization time--act positively upon the early embryo itself, as well as on the microenvironment in which the embryo develops, both in vivo and in vitro conditions.

Asynchrony between human cumulus-corona cell complex and oocyte maturation after human menopausal gonadotropin treatment for in vitro fertilization

One hundred seventeen oocyte-cumulus-corona cell complexes (CCCs) were obtained from 15 women undergoing in vitro fertilization after human menopausal gonadotropin/human chorionic gonadotropin follicular stimulation. In each woman, five oocyte-CCCs were left intact, and the remaining one to five oocytes were freed of their CCCs by hyaluronidase (300 IU/ml) dispersal treatment. Dispersal time for individual CCCs correlated well with their degree of mucification and was significantly shorter in 20 mature CCCs as compared with 24 intermediate CCCs (1.20 +/- 0.05 versus 2.35 +/- 0.13 minutes, respectively; P less than 0.001). Oocyte maturation at collection and after 8 hours of in vitro incubation was not related to the CCC type present at harvest. Maturation of oocytes progressed regardless of CCC type; so that after 8 hours of preincubation, 73% of the oocytes attained a polar body while 20% were still at the germinal vesicle breakdown stage and 7% at the germinal vesicle stage. Overall, the 44 denuded oocytes and 72 intact oocyte-CCCs were comparable in rate of fertilization (64% versus 68%) and subsequent cleavage (75% versus 82% of all fertilized oocytes). It is concluded that in human menopausal gonadotropin-stimulated cycles, asynchrony between individual CCC mucification and oocyte maturation may occur, and that the absence of CCC does not seem to affect in vitro fertilization and cleavage rates.