Cumulus Cell Mass Research Articles

Production and transfer of IVF embryos from individual inhibin-immunized cows by ultrasound-guided transvaginal follicular aspiration.

Ultrasound-guided transvaginal follicular aspiration (UTA) was performed on 8 multiparous Japanese Black cows which had not been successful in a conventional superovulation program. These cows were actively immunized with a synthetic peptide replica of porcine inhibition alpha N1-26 (pINH) conjugated with rabbit serum albumin (RSA) using Freund's complete adjuvant. Booster injections of the peptide were given at 6, 10, 14 and 20 wk after the primary injection. Twelve follicular aspirations were performed between 10 and 33 wk after the primary injection. Cumulus-oocyte complexes (COCs) were aspirated from follicles > or = 2 mm in diameter. The recovered COCs were classified into 5 categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers), Grade 4 (denuded) and Grade 5 (expanded and fluffy cumulus cell mass or degenerated ooplasm). The recovered COCs available for in vitro fertilization (IVF; Grades 1, 2, 3 and 4) were matured with granulosa cells (1 x 10(6) cells/ml) for 20 to 23 hr, fertilized with frozen-thawed spermatozoa, and cultured in vitro. At day 7 after insemination, a portion of the embryos were transferred to Holstein heifers. There were great variations among donors in the number of recovered COCs in the numbers of COCs in each grade, and also in the developmental ability of fertilized COCs. IVF procedures were carried out on 685 oocytes and produced 120 embryos (17.5%). The transfer of 36 fresh IVF embryos resulted in 20 pregnancies (55.6%) at 60 days after the transfer. This study has demonstrated first, that the number of COCs collected from individual inhibin-immunized cows by UTA greatly varies among donors. Second, recovered COCs vary greatly in the classification of the morphological categories of surrounding cumulus cell mass among donors and had different developmental abilities when they were cultured after fertilization in vitro. Third, the production of pregnancy in previously nonproductive problem donors is possible using the UTA technique.

Platelet-activating factor acetylhydrolase activity in human follicular fluid.

Platelet-activating factor (PAF) has been implicated in a number of reproductive processes ranging from ovulation to parturition. To examine the role of PAF in the human periovulatory processes, the PAF-acetylhydrolase (PAF-AH) activity was assayed in the follicular fluid (FF) obtained in conjunction with the in vitro fertilization and embryo transfer (IVF-ET) procedure and the activity related to oocyte maturation. The PAF-AH activity was also related to the concentrations of estradiol (E2) and progesterone (P) in FF. PAF-AH activity was significantly lower in the FFs obtained from follicles of more than 20 mm in diameter. The enzyme activity was significantly lower in the FFs of patients with a successful outcome of their pregnancies. E2 concentrations were negatively correlated with PAF-AH activities in the FFs. No correlation was found between the PAF-AH activity and concentration of P in the FF. Significantly more mature oocytes were recovered in the group who subsequently become pregnant compared to the non-pregnant group. It is suggested that PAF may be increased following follicular maturation. The increase in PAF may contribute to oocyte maturation and to the successful outcome of pregnancy following fertilization. An additional function of the increased PAF in FF may also be the stimulation of the contraction of smooth muscle in the ovary, thereby assisting the extrusion of the oocyte cumulus cell mass and signaling the completion of ovulation.

Ooplasmectomy has no influence on FSH-induced expansion of bovine cumulus investment in vitro.

Bovine cumulus-oocyte complexes with or without attached pieces of the mural stratum granulosum were matured with or without FSH in vitro for 24 h. Some complexes were ooplasmectomized (removal of ooplasm) at the onset of culture or after 9 h of culture, and in others the complete oocyte, including the zona pellucida, was removed. At the end of culture, the degree and morphology of corona and cumulus expansion were determined by a subjective scoring system, transmission electron microscopy followed by computerized image analysis, or scanning electron microscopy. In the absence of FSH no expansion was seen. In the presence of FSH, hyaluronidase-sensitive cumulus expansion was observed in intact complexes and complexes ooplasmectomized after 9 h of culture. In complexes ooplasmectomized at the start of culture, hyaluronidase-insensitive expansion of the corona compartment was seen as well. A similar reaction was found in free corona and cumulus cell masses cultured in the presence or absence of oocytes. Mural granulosa in intact and ooplasmectomized complexes expanded only slightly under the influence of FSH and exhibited no hyaluronidase sensitivity. Ooplasmectomy at the start of culture increased the frequency of ruffled cell membranes and decreased the occurrence of intercellular bridges as compared with unmanipulated complexes cultured under corresponding conditions. It is concluded that FSH-induced cumulus expansion and hyaluronidase-sensitive extracellular mucus production of bovine cumulus investment in vitro are not dependent on the oocyte, except for minor changes in the surface morphology of the cumulus cells.

Platelet activating factor treatment of spermatozoa enhances fertilization rates of rabbit oocytes utilizing subzonal insertion of sperm

We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P<0.05) increased development into blastocysts (39.0 to 50.7%) when compared with co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were obtained.

Localization and synthesis of hyaluronic acid in the cumulus cells and mural granulosa cells of the preovulatory follicle

Mural and cumulus granulosa cells synthesize hyaluronic acid (HA) and expand in vitro in response to follicle-stimulating hormone and a soluble factor(s) produced by fully grown oocytes. In the present study we examined HA synthesis and extracellular matrix organization by the two cell populations in vivo during the preovulatory period. After injection of human chorionic gonadotropin into pregnant mares' serum gonadotropin-primed animals, a progressive increase in HA synthesis was observed by the cumulus cell-oocyte complex (COC), and by the mural granulosa cells adjacent to the antrum (antral granulosa cells). The outermost layers of mural granulosa cells (peripheral granulosa cells) did not synthesize HA. Net HA synthesis was ∼4 pg/cell for COCs isolated after full expansion induced either in vivo or in vitro, whereas the total HA content and cell number in the ovulated COC (∼11 ng HA and ∼3000 cells per COC) were about threefold higher than for COCs expanded in vitro (∼4 ng HA and ∼1000 cells per COC). The increased cell content of ovulated COCs appears to be primarily the result of inclusion of proximal mural granulosa cells which synthesize HA in response to the oocyte factor(s) and become incorporated in the expanded COC extracellular matrix mass. Media conditioned by oocytes enclosed in the cumulus cell mass (intact COCs) contained only 10–20% of the HA-stimulatory activity of media conditioned by an equal number of isolated oocytes when tested on mural granulosa cell cultures. Further, HA-stimulatory activity of media conditioned by isolated oocytes was dramatically reduced (∼70%) by preincubation for 5 hr with cumulus cells compared to preincubation in the absence of cells. The results suggest that differences in HA synthesis between subregions of membrana granulosa depend on a diffusion gradient of the oocyte factor(s).

Bovine cumulus expansion and corona-oocyte disconnection during culture in vitro.

Cumulus-oocyte complexes were aspirated from small antral follicles (3-5 mm in diameter) and divided into 2 groups: complexes in which a dark rim of corona cells were visible around the zona pellucida (group 1); and those in which the corona displayed the same density as the rest of the cumulus cell mass (group 2). Cumulus complexes of both groups were evaluated by acetoorcein staining, scanning electron microscopy (SEM) and 3H-uridine uptake during culture in vitro. Germinal vesicle breakdown was initiated at 7 h of culture in group 1 while more than half of the oocytes in group 2 already displayed germinal vesicle breakdown at the time of aspiration. However, whereas more than 80% of the oocytes in group 1 had reached metaphase II at 24 h of culture, almost half of the oocytes in group 2 were arrested in metaphase I after this time interval. As revealed by SEM the complexes of group 2 showed signs of expansion such as elongation of cumulus cells and presence of extracellular matrix already at the time of aspiration. In group 1 these features were noticed at 7-9 h of culture. A high level of metabolic coupling between corona cells and oocyte was maintained up to 9 h of culture in group 1 followed by a decrease to a constant low level at 13 h. In group 2 a high degree of coupling was maintained up to 7 h followed by a gradual decrease to a constant low level at 13 h. It is concluded that cumulus-oocyte complexes with a dark rim of corona cells as judged by stereomicroscopy mature at a higher rate and maintain unexpanded characteristics and efficient corona-oocyte coupling longer than complexes with even density of the cumulus mass. Consequently, the presence of a dark rim of corona cells may be used as a criterion for selection of oocytes for in vitro embryo production.

Development of bovine embryos in chemically defined medium at different levels of polyvinyl alcohol

This work was undertaken in order to study the developmental competence of nuclear transfer feline embryos with regard to the recipient-cytoplast’s age and type of somatic cells used as donor nuclei.Oocytes were recovered by mincing the ovaries in HEPES-buffered TCM-199. Selected cumulus–oocyte complexes (COCs) with compact cumulus cell mass and a dark, homogenous ooplasm were cultured for maturation in the modified medium TC-199 for 24, 35, and 43 h, and after enucleation were used as a source of recipient cytoplasts for exogenous somatic nuclei.Two experiments were carried out. In Experiment 1, the source of recipient cytoplasts was oocytes matured in vitro for 24 h (Group 1), 35 h (Group 2), and 43 h (Group 3), while the source of donor nuclei was cycling fetal fibroblasts. Somatic cell–cytoplast complexes (SC–CCs) were fused electrically by double DC pulses of 2.0 kV/cm for 15 μs. The reconstructed embryos were cultured in B2 medium for 72 h after NT, then co-cultured with BRL cells in the same medium supplemented with 10% FBS at 38.5 °C under 5% CO2 in air. In Groups 1, 2, and 3, the fusion rates were 71.4 (25/35), 74.6 (47/63), and 57.5% (46/80), respectively. The cleavage rates in Groups 1, 2, and 3 were 80.0 (20/25), 55.3 (26/47), and 60.8% (28/46), respectively. The development to morula and blastocyst stages was higher in Groups 1 and 2 compared to Group 3 (morula stage 14/25 (56.0%), 16/47 (34.0%), and 13/46 (28.2%); blastocyst stage 2/20 (8.0%), 4/47, (8.5%), and 0/46, respectively). In Experiment 2, the oocytes matured for 24–35 h were used as a source of recipient cytoplasts and cycling fetal fibroblasts and cumulus cells derived from mature COCs were used as a source of donor nuclei. The fusion rates were 115/193 (59.6%) versus 65/143 (45.4%) for fetal fibroblasts and cumulus cells, respectively. The cleavage rate was 72/115 (62.6%) versus 48/65 (73.8%), and the development to blastocyst stage 6/115 (5.2%) versus 5/65 (7.7%), for fetal fibroblast and cumulus cells, respectively. In conclusion, a prolonged maturation period of cat oocytes decreases developmental competence of reconstructed embryos, especially the ability to reach the blastocyst stage. The in vitro development of reconstructed embryos with either nuclei of fetal fibroblasts or cumulus cells was at approximately the same level.

Effects of culture systems on development of in vitro fertilized bovine ova into blastocysts

We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P<0.05) increased development into blastocysts (39.0 to 50.7%) when compared with co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were obtained.

Effect of cumulus cell mass and follicle quality on in-vitro maturation of cynomolgus monkey oocytes.

Cynomolgus monkey oocytes were recovered from healthy or atretic follicles greater than 1000 micron in diameter at day 8 of gonadotrophin stimulated cycles and cultured in vitro, cumulus enclosed or cumulus free, for 2 days. Germinal vesicle breakdown (GVBD) occurred in 26.7% of healthy cumulus enclosed oocytes (n = 60) compared to 52.6% of atretic cumulus enclosed oocytes (n = 38). The mechanical removal of the cumulus cell mass increased the GVBD rate of the healthy oocytes (56.5%, n = 23) and accelerated the passage of the atretic oocytes (n = 23) from metaphase I (4.3%) to metaphase II (47.8%). In the healthy primate follicle, the dictyate stage could be maintained by an inhibitory substance secreted by the cumulus; follicular atresia could either decrease the synthesis of this substance and/or induce the metabolism of a stimulatory substance.

Methods for collecting follicular oocytes from mares

A series of experiments was conducted to develop a procedure for consistent, repeatable collection of oocytes from the preovulatory follicle of the mare. In one experiment, in situ follicular aspiration with a needle and syringe was performed on 19 mares. From 37 aspirations, four oocytes were recovered (10% recovery rate). In a second experiment, ovaries were visualized via standing flank laparotomy during which two different aspiration techniques were used. Use of a needle and syringe as in the first experiment resulted in successful oocyte recovery in one of seven (14%) attempts. Aspiration via a continuous irrigation vacuum system (CIV), developed for use during laparotomy, resulted in collection of oocytes from six of 10 (60%) attempts. In the third experiment, oocytes were recovered from seven of 18 (38%) attempts at in situ follicular aspiration using a double-lumen needle attached to the CIV. In each experiment, some mares were subjected to stimulation of follicular maturation by exogenous hormones. Oocyte recovery was significantly increased in treated mares as compared with nontreated mares. Results indicate that collection of equine follicular oocytes by in situ aspiration is possible with moderate success. Oocytes apparently are not physically damaged by the procedure, as most retained either the corona radiata or the entire cumulus cell mass.

Cleavage stage of human embryos two days after fertilization in vitro and their developmental ability after transfer into the uterus

In 126 selected patients treated with clomiphene and human menopausal gonadotrophin, 154 embryos were transferred into the uterus 41-50 h after in-vitro insemination. Fifty per cent of embryos implanted. The rate of cleavage was inversely related to the success of embryo transfer; implantation failures occurred for 69 and 46% of embryos with more than or less than four cells, respectively (p less than 0.05). Cleavage rate was not related to the viscosity of the cumulus cell mass, serum supplementation of culture medium, nor to the age or genetic factors of the woman. There was a trend towards smaller follicles, and a significant (p less than 0.05) tendency for patients developing numerous follicles to yield faster-cleaving embryos. These relationships were not found in 50 other embryos obtained from patients treated with pure FSH.

Factors affecting human in vitro fertilization: a multifactorial study

The influence of four factors on the cleavage rate of 705 mature oocytes submitted for in vitro fertilization (IVF) was assessed with the use of a multifactorial method, i.e., logistic regression. The studied factors were (1) the regimen for cycle stimulation, (2) cumulus cell mass appearance, (3) semen quality, and (4) the time of incubation of oocytes before insemination. The logistic function permitted testing the respective influence of each factor on the cleavage rate, with the level of the other factors taken into account. The most important factor was the stimulation treatment, the association of human menopausal gonadotropin with clomiphene citrate (CC) giving better results than CC alone. Time of incubation was demonstrated to have no influence on the cleavage rate, whereas semen quality had an influence. A problem was raised by the existence of an interaction between the stimulation treatment used and the appearance of the cumulus cell mass. It can be concluded that adequate cycle treatment and eventually couple selection with respect to the semen quality of the male partner are to be considered in view of obtaining better results in IVF attempts.

Ultrastructure of the cumulus cell mass surrounding a human egg in the pronuclear stage.

The cumulus cell mass enclosing a penetrated human egg was studied. The egg, recovered from the Fallopian tube approximately 80 h after luteinizing hormone peak and 35 h after insemination, was surrounded by a large, expanded and dissociated cumulus. Dispersions of the outermost cumulus cell layers occurred during processing, the innermost cell layers remained attached enclosing the egg. The photomicrographs showed that the follicular cells were embedded in an intercellular matrix and contact via gap-junction-like structures between neighboring cells existed. Cumulus cell processes traversing the zona pellucida were not found. Two types of follicular cells coexisted within the cumulus, light and dark cells. These cellular types, were different in morphology and size. Light cells displayed cytoplasmic organelles normally associated with protein synthesis and steroidogenesis. Dark cells with long cytoplasmic processes were involved in sperm phagocytosis. It is suggested from the characteristics of the cytoplasmic organelles that dark cells seem to be modified light follicular cells.

Modulation of cell-to-cell communication in the cumulus-oocyte complex and the regulation of oocyte maturation by LH

Prior to ovulation, cell-to-cell communication between the oocyte and the cells of the cumulus oophorus is terminated ( N. B. Gilula, M. L. Epstein, and W. H. Beers, 1978, J. Cell Biol. 78, 58–75; R. M. Moor, M. W. Smith, and R. M. C. Dawson, 1980, Exp. Cell Res. 126, 15–29). In this paper we report that LH, Bt 2-cAMP, and the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, are all capable of interrupting communication in vitro in rat follicle-enclosed cumulus-oocyte complexes. Moreover, the breakdown of communication appears to be closely correlated with the ability of hyaluronidase to disperse the cumulus cell mass. This observation allows simple screening of the effects of various agents on cumulus-oocyte communication. The in vitro system employed in this study has also been used to investigate the relationship between communication and oocyte maturation. The findings presented indicate that the interruption of communication between the cumulus and the oocyte leads to relief of meiotic arrest, and are consistent with the possibility that cAMP, transmitted from the cumulus to the oocyte, may be the inhibitor of oocyte maturation in vivo.

Intrafollicular cumulus cell transformations associated with oocyte maturation following gonadotrophic hormone stimulation of adult mice.

AbstractEffects of gonadotrophic hormones on the intrafollicular morphological transformation of cumulus cells were examined in adult mice. Formation of transformed cumulus masses was examined during both the pre‐ and post‐ovulatory course of gonadotrophic (PMS or PMS + HCG) induction of follicular synchronization and ovulation. Within nine hours of PMS injection and prior to ovulation, transformed cumulus cell masses were present within ovaries. Injection of HCG within 48 hours of the PMS injection stimulated the formation of additional and increased numbers of pre‐ovulatory transformed cumulus cell masses. Ovulation occurred within 24 hours of both the PMS injection and the HCG injection when given to animals previously stimulated with PMS. Oocytes isolated from transformed cumulus cell masses following PMS or HCG injection had invariably undergone meiotic maturation. Transformed cumulus masses were also recovered from ovaries following induced ovulation; however, their numbers were decreased. Follicles stimulated by PMS remained sensitive to HCG (underwent ovulation) for approximately four days following a single injection of PMS. Results indicate that identifiable morphological and physiological changes in cumulus cells precede ovulation and are strongly correlated with the induction of the meiotic maturation process in the enclosed oocytes. The presence of mature oocytes in transformed cumulus masses following ovulation furthermore indicates that asynchrony of the oocyte maturation and ovulatory events are induced by the gonadotrophic hormones. Intrafollicular cumulus cell transformations are discussed in relation to dissociation of the somatic and germ cells, the mechanisms of meiotic induction and the process of atresia. Results suggest that the simple morphological changes described provide a quantitative and selective indicator of gonadotrophic action within the ovary.

SPERM PENETRATION OF LIVE AND DEAD OOCYTES WITHIN OVIDUCTS OF GILTS

Sperm penetration of live and dead (rapidly frozen to −196 °C) porcine follicular oocytes was studied by transferring combinations of fluorescein isothiocyanate-stained (S) and nonstained (NS) live and dead eggs into the oviducts of tubally-inseminated gilts. All oocytes to be transferred were recovered from preovulatory follicles of gonadotropin-treated gilts via ovariectomy or follicular aspiration in situ. Only oocytes with a full complement of expanded cumulus were used. They were rendered nonviable by plunging semen straws containing S or NS oocytes into liquid nitrogen and storing them for at least 7 days before rapid thawing. The freeze–thaw cycle, without cryopreservative, resulted in numerous ruptures of the vitelline membrane and presumably the death of all oocytes examined. However, the zona pellucida and cumulus cell mass remained intact. Neither freezing nor staining affected ova recovery post-transfer or sperm penetration. Upon recovery, sperm slits and similar numbers of accessory sperm were observed in the zonae of both frozen–thawed and freshly transferred oocytes. Polyspermy was not observed.

RNA and Protein Synthesis in Control and Follicularly-Aged Rat Oocytes

SummaryAutoradiographic analysis revealed that follicularly-aged oocytes show a significant reduction in RNA synthesis, but the capacity to synthesize protein was unaltered. Abnormally low amounts of RNA were synthesized by 54% of the oocytes obtained from delayed-ovulatory rats and 7% of the control oocytes. The reduced RNA synthesis characteristic of the aged oocyte could be associated with metabolic changes which are responsible for the developmental anomalies.Evaluation of RNA and protein synthesis in the cumulus cell mass, which surrounds the tubal oocyte, demonstrated that neither the percentage of cells labeled, nor the amount of RNA or protein synthesized by the cumulus cells, is altered during the follicular aging process. However, the cumulus cells which enclose the oocyte are more loosely associated with the aged oocyte.