• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.3
• /
• pp.419-432
• /
• 1999

Objective: To establish the evaluation system of the quality of oocytes on the basis of the incidence of cumulus cells apoptosis, to investigate the relationships beween the incidence of cumulus cells and the outcomes of IVF-ET. Method: Thirth-four cycles undergoing controlled ovarian hyperstimulation for IVF-ET with tubal infertility (23 cycles) or unexplained infertility (11 cycles) were included in this study. Cumulus cell masses surrounding mature oocyte and co-culture of embryos with autologous cumulus cells during IVF-ET process. The incidence of apoptosis in cumulus cells was assessed by apoptosis detection kit fluorescein. The effect of co-culture using cumulus cells and the incidence of cumulus cells apoptosis. Results: The results were as follows: 1. The incidence of apoptosis in cumulus cells markedly increased in patients aged 40 or over, while the fertilization rate was greatly decreased in those age group. 2. Apoptosis in cumulus cells was found in both the fertilized oocytes and unfertilized oocytes, but the incidence of apoptosis was higher in unfertilized oocytes. 3. There is no clear correlation between apoptosis in cumulus cells and the number of oocytes retrieved. However, the incidence of apoptosis was increased when the number of oocytes retrieved was 5 and fewer in comparison with $6{\sim}10$. 4. Embryo grade was significantly affected by the incidence of apoptosis in cumulus cells. 5. Pregnancy rate of IVF-ET per cycle was 29.4%, and the pregnant group had the higher fertilization rate and a significantly lower incidence of apoptosis in cumulus cells compared with the nonpregnant group. 6. When cumulus cells were used as helper cells in the co-culture of the embryo, in vitro activity of cumulus cells based on morphological change and proliferation did not influence the quality of embryo, but was closely associated with the implantation rate and pregnancy rate, which was enhanced when morphological changes and proliferation of cumulus cells was more active. 7. This difference in the outcome of IVF-ET according to in vitro activity of cumulus cells used for co-cultue was not associated with the incidence of apoptosis in cumulus cells; but rather had likely relations with the different secretion pattern of protein, which may be an embryo trophic factor by cumulus cells. Conclusion: These results suggest that the incidence of apoptosis in cumulus cells can be used in predicting oocyte qualities and the outcomes of IVF-ET. And the effect of co-culture largely depends on the in vitro activity of cumulus cells as well.

• Korean Journal of Animal Reproduction
• /
• v.14 no.2
• /
• pp.93-100
• /
• 1990

These experiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro maturation of bovine follicular oocytes. Antisera to bovine cumulus cell were produced Japanese Ginat rabbit by repeated immunization of intact or solubilized bovine cumulus cell and purified by ammonium sulfate precipitation and Sepharose CL-4B protein-A affinity chromatography. The bovine cumulus cell-specific antibodies were confirmed by indirect ELISA. The results obtained in these experiments were summarized as follows : 1. The titer of the antibodies to cumulus cell determined by indirect ELISA using intact or solubilized bovine cumulus cell coated plates was very high in both intact and solubilized cumulus cells. Namely, the optical density at 1:12,800 dilution of antibodies was still significantly higher than that of non-immunized control serum. 2. When the follicular oocytes were treated with antibody to intact cumulus cells, the maturation rate of cumulus compacted and removed oocytes was ranged 47.6 to 59.1%. These value is significantly lower(p<0.05) than that(78.8%) of follicular oocytes cultured without the antibody. 3. the maturation rate of cumulus compacted and removed oocytes treated with antibody to solubilized cumulus cells was ranged 46.7 to 59.1%, significantly lower(p<0.05) than that(82.1%) of ooyctes cultured in antibody free medium. From above mentioned results, it could be said that cumulus cells promote nuclear maturation of follicular oocytes and that the beneficial effect of cumulus cells to the oocyte maturation is inhibited by the action of antibody to cumulus cells.

• Clinical and Experimental Reproductive Medicine
• /
• v.13 no.2
• /
• pp.201-206
• /
• 1986

These experiments were conducted to know whether RNA syntheis is involved in the cumulus expansion and oocyte maturation of mouse cumulus-oocyte complexes in vitro. Mouse cumulus-oocyte complexes(COC's) were cultured in the presence of cordycepin, an inhibitior of RNA synthesis and its effect on the cumulus expansion and oocyte maturation were examined. The results were as follows. 1. Continuous presence of cordycepin in the medium(200${\mu}g/ml$) inhibited the HCG-induced cumulus cell expansion of mouse complexes. This inhibition was reversible. 2. When the COC'S were preincubated with different concentration of cordycepin plus HCG for 3 hours and then transferred to the plain medium, the HCG induced cumulus expansion was suppressed at $100{\mu}g/ml$ of cordycepin. 3. When the COC'S were cultured with cordycepin after HCG stimulation(3hrs), the cumulus expansion were not suppressed by cordycepin. 4. Oocyte meiotic maturation did not appear to be affected by cordycepin. The data presented here imply that RNA synthesis is involved in the cumulus expansion process and that mouse oocytes are more resistant to RNA synthesis inhibitor than cumulus cells.

• Korean Journal of Veterinary Service
• /
• v.26 no.4
• /
• pp.329-337
• /
• 2003

The functional role of the cumulus cells on sperm penetration and polyspermy during in vitro fertilization was examined. The penetration rate was significantly higher(p<0.01) in oocytes with(61%) than without(25%) cumulus cells. No significant differences, however, was observed in polyspermy. When the hyaluronidase was supplemented to the fertilization medium with different concentrations, penetration rates in oocytes with cumulus cells were higher than oocytes without cumulus cells at 0(61 vs 34% ; p<0.05), 0.01(56 vs 35% ; p<0.05), 0.1(66 vs 30% ; p<0.05) and 1.0 mg/$m\ell$(39 vs 27%). On the other hand, the polyspermy rates were lower oocytes without than with cumulus cells, and had a tendency to decrease with high concentrations of hyaluronidase. In another experiment, the penetration and polyspermy rates had a tendency to increase as time of sperm-oocytes culture was prolonged. At 16 and 20hrs after insemination, the penetration rates were significantly higher(p<0.05) in oocytes with(48 and 62% for 16 and 20hrs) than without(25 and 31% for 16 and 20hrs) cumulus cells in medium with hyaluronidase. However, the polyspermy rates were significantly(p<0.05) lower in oocytes without(3 and 16%) than with(37 and 48%) cumulus cells at 16 and 20hrs after insemination. In cumulus-free oocytes inseminated in medium with or without hyaluronidase at different concentrations of cumulus cells, the penetration rates were significantly(p<0.05) higher in medium with than without hyaluronidase at different concentrations of cumulus cells. The proportions of polyspermy were lower in medium without than with hyaluronidase at 0 (10 vs 0%), 10$^2$(25 vs 0%), 10$^4$(24 vs 14%) and 10$\^$6/(29 vs 10% ; p<0.05) cumulus cells/ml. These results suggest the advantage of culture in medium with cumulus cells and denuded oocytes to inhibit polyspermy with no decrease in the penetration rates during the fertilization in vitro in the porcine.

• The Korean Journal of Zoology
• /
• v.30 no.2
• /
• pp.117-139
• /
• 1987

The present experiments were carried out to investigate the mode of cAMP regulation of cumulus expansion in pig. Intracellular level of cAMP in the cumulus cells was modulated by culturing porcine cumulus oocyte complexes (COC's) with forskolin, an adenylate cyclase stimulator and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. The role of calcium in the hormone induced cumulus expansion process was also studied. Forskolin in the medium stimulated cumulus expansion from the concentration of 0.01 $\mu$M and induced full expansion at l-10 $\mu$M In contrast, IBMX in the medium (20-180 $\mu$M) failed to induce the expansion. Verapamil, a calcium ion transport blocker, suppressed follicle stimulating hormone(FSH)-induced cumulus expansion in a dose dependent fashion (0.002-0. 2 mM) when the COC's were exposed to the drugs during culture period (32 hr). But verapamil did not interfere with the triggering action of FSH during early four hours of culture period. The data presented here showed that adenylate cyclase in the porcine cumulus cells may play a key role in the regulation of the intracellular cAMP level and calcium ion may be involved in the later period of cumulus expansion process.

• Clinical and Experimental Reproductive Medicine
• /
• v.17 no.2
• /
• pp.185-196
• /
• 1990

These experiments were performed to know ultrastructural changes of the cumulus expansion in virot. SEM:In expanded oocyte-cumulus complex, the cell surface are characterized by the presence of many evaginations:they are relatively short and round shape. The mucous extracellular material were deposited between cumulus cells. TEM:In compact cumulus cells, golgi apparatus and rough endoplasmic reticulum developed. In expanded cumulus cells, rough endoplasmic reticulum decreased and the smooth endoplasmic reticulum increased. Also, there were numbers of mitochondria. Extracellular mucous material which is presumed to be hyaluronic acid appears when cumulus cell were expanded. In expanded cumulus cell, numbers of smooth endoplasmic reticulum help cumulus cell to develop in steroidogenic cell.

• The Korean Journal of Zoology
• /
• v.31 no.2
• /
• pp.81-86
• /
• 1988

The relationship between cumulus cell expansion, cocyte maturation and metabolic cooperativitiy was investigated by using mouse and pig cocyte-cumulus complexes in vitro. Cocyte germinal vesicle breakdown (GVBD) and cumulus expansion were manipulated with hormones or reagents which increase intracellular cAMP leveL Metabolic cooperativity between oocyte and cumulus cells was assessed by determination of the fraction of radiolabelled uridine marker that was transferred from the cumulus mass to the oocyte. Uptake of uddine marker by mouse and pig cumulus mass was increased by about fourfold of basal level with the stimulation of hormones (human choriononic gonadotrophin, HCG; follicle stimulating hormone, FSH) or cyclic AMP sttmulators (3-isobutyl-1-methylxanthine, IBMX; forskolin) during culture. However, the fraction of uridine that was transferred from the cumulus mass to the cocyte (transfer ratio) was gradually decreased during culture, irrespective with the presence of hormones or stimulators. The decrease of the transfer ratio was not correlated with the state of occyte whether they have GV or not, or with the degree of cumulus expansion. In mouse complexes, HCG induced more significant reducton of transfer ratio than other treatments. These results do not support the idea that modulations of metabolic cooperativity between cumulus cells and oocytes are important for the regulation of meiotic resumption in mammals.

• Korean Journal of Animal Reproduction
• /
• v.14 no.2
• /
• pp.101-106
• /
• 1990

These mxperiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro fertilization and following development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6mm of diameter. Bovine oocytes were matured in vitro for 24~26hrs in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$ and subsequently cultured in medium containing cumulus cell antibody for 1 hour. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hrs in BO solution 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then cultured for 7 days. The results obtained in these experiments were summarized as follows : 1. When the follicular oocytes matured in vitro were treated with antibody to intact cumulus cells, the fertilization rate of cumulus intact and removed oocytes was ranged to 45.0 to 53.7%. These value is slightly lower than that(64.3%) of follicular oocytes not treated with the antibody, and increased frequency of both male and female pronuclear formation was found in cumulus intact oocytes cultred in medium without the antibody(p<0.05). 2. The fertilization rate of cumulus intact and removed oocytes treated with antibody to solubilized cumulus cells was ranged 45.0 to 52.5%, significantly lowre than that(62.8%) of oocytes cultured in antibody free medium, and increased frequency of ova with male and female pronuclei was found when cumulus cells were present(p<0.05). 3. The rates of cumulus cell intact and removed oocytes developed to 8-, 16-cell and morula or blastocyst after treatment of intact and solubilized cumulus cell antibody were ranged 7.1 to 14.5, 2.9 to 5.9 and 1.5 to 2.9%, respectively, slightly lower than 18.6, 10.0 and 8.6% of cumulus intact oocytes cultured in medium without the antibody. The results of this stduy indicate that cumulus cells promote not only normal fertilization with proper pronuclear formation, but embryo development and that the beneficial effect of cumulus cell to the pronuclear formation and embryo development is blocked by the action of antibody to cumulus cell.

• Reproductive and Developmental Biology
• /
• v.34 no.3
• /
• pp.169-173
• /
• 2010

Correlations between cumulus cells and germinal vesicle (GV) chromatin configuration were examined in porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from 2~6 mm follicles and divided into three categories according to cumulus cell morphology. "A" group was compacted COCs with more than three cumulus cell layers. "B" group was COCs with less cumulus cell layers than "A" group. "C" group was COCs with one or less layer of cumulus cells. Cumulus cells were removed 0.1% hyaluronidase, and denuded oocytes were stained with Hoechst 33342. GV chromatin configuration was classified into GV-Con and GV-Dis. GV-Con meant that a nucleus was surrounded by condensed chromatin in a ring. GV-Dis meant that filamentous chromatin clumps were distributed in nucleus. The proportion (80.2%) of GV-Con in "A" group was significantly higher than "B" (62.0%) or "C" (44.9%). The proportion (55.1%) of GV-Dis in "C" group was significantly higher than "A" (19.8%) or "B" (38.0%). The meiotic competence of COCs was examined after 44 h culture. The proportion (90.0%) of oocytes reaching to metaphase II (M-II) in "A" group was significantly higher than "B" (76.5%) or "C" (45.5%). In conclusion, oocytes with good quality cumulus cell layers are synchronized early GV stage, and early GV stage is important for meiotic competence in pigs.

• Clinical and Experimental Reproductive Medicine
• /
• v.48 no.4
• /
• pp.362-367
• /
• 2021

Objective: The impact of early mechanical removal of cumulus cells on fertilization and embryonic development is not yet precisely known. This study aimed to investigate the effects of early and late cumulus cell removal on fertilization, polyspermy, embryonic development potential, blastocyst development, and clinical outcomes. Methods: A prospective study was conducted of patients who underwent in vitro fertilization between September 2019 and October 2020. Sibling oocytes were randomly allocated after insemination to early cumulus cell removal at 6 hours (group I) and late cumulus cell removal at 16-18 hours (group II). If total fertilization failure (TFF) was determined to have occurred at early cumulus cell removal, rescue intracytoplasmic sperm injection (ICSI) was performed. Fertilization, embryonic development, and pregnancy outcomes were compared. Results: A total of 912 oocytes were assigned to group I (458 oocytes) and group II (454 oocytes). Fertilization, polyspermy, embryo quality, and pregnancy outcomes were not significantly different between both groups. Rescue ICSI enabled fertilization of 79.2% of the TFF oocytes. Conclusion: Early cumulus cell removal at 6 hours had no significant difference in fertilization, polyspermy, embryo development, or obstetric and perinatal outcomes compared to late removal. Early cumulus cell removal combined with early rescue ICSI may have the potential to help couples with TFF.