Presence Of Cumulus Cells Research Articles

Parthenogenetic Activation Induced by Progesterone in Cultured Mouse Oocytes.

Progesterone induced in vitro parthenogenetic activation of ovulated mouse oocytes. Progesterone (100 μM, 31.4 μg/ml) induced a high rate (90%) of activation and progression of the second meiotic division from metaphase to anaphase/telophase and pronuclear formation. Oocytes degenerated at progesterone concentrations of 150 μM and above, whereas 50 μM concentrations or less induced quite little or no activation. Aging of the oocytes prior to treatment and a longer treatment period had significant effects on activation. Oocytes treated with 100 μM progesterone at 16, 18, 20, or 22 h after hCG injection for 6 h showed 50, 85, 89 and 98% activation, respectively, while the effect on activation was less significant when these oocytes were treated for 2 or 4 h. Few oocytes (0-4%) were activated in controls that were incubated in the presence of solvent (0.5% DMSO) only. The presence of cumulus cells did not exert significant effects on the incidence of activation by progesterone. Other steroids tested were cholesteryl sodium sulfate, pregnenolone, 17α-hydroxy-20β-dihydroprogesterone, 17α-hydroxyprogesterone, 20α-hydroxy-progesterone, 20β-hydroxyprogesterone, androstenedione, testosterone, and estradiol-17β. Both 20α-hydroxyprogesterone and 20β-hydroxyprogesterone induced activation, but the remainder induced low or no activation. When culture was continued after progesterone exposure, 60% of oocytes developed to the 2-cell stage, and a small proportion (3%) developed to the blastocyst stage. These results show that when progesterone and its metabolites are present at high concentrations (100 μM) for a long period (6 h), they have the unique property of inducing in vitro parthenogenetic activation in mouse oocytes.

Effects of culture time, ovarian activity, cumulus cells and sera on the nuclear and cytoplasmic maturation of pig oocytes in vitro

Pig cumulus-oocyte complexes (COC) were used in this study to assess the effect of length of culture, ovarian activity, cumulus cells and sera on maturation and fertilization in vitro. In Experiment 1, the incidence of nuclear maturation increased ( P<0.01) from 24 to 48 h of culture and the ability for male pronucleus (MPN) formation peak at 42 h (86.6%). The penetrability of immature COC and those at the metaphase 2 (M2) stage appeared to be not influenced by the stage of meiosis (75.3–89.3%). In Experiment 2, no significant difference in the nuclear maturation (84.8–88.0%) and penetration (82.1–92.0%) rates of COC collected from ovaries with or without corpora lutea (CL) or corpora albicans (CA) was observed, though MPN formation differed ( P<0.05) between O 1 (82.6%) and O 3 (61.5%) oocytes. In Experiment 3, significant differences ( P<0.01) on the nuclear maturation and MPN formation of oocytes cultured with cumulus cells versus those without or lacking cumulus cells was observed. It shows that the presence of cumulus cells is important in supporting maturation in vitro. In Experiment 4, serum supplementation of the medium improved the rate at which COC reached the M2 stage ( P<0.05) and formed MPN except at the 5% level when using fetal bovine serum (FBS) and calf serum (CS). The use of porcine serum (PS) consistently supported the maturation of COC. Serum supplementation is thus necessary for maturation in vitro and the concentration and type of serum to be used is important.

Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics

After labelling DNA with the specific vital fluorophore Hoechst 33342, oocytes, isolated by puncture from antral follicles in adult mice, have two essentially different configurations of their nuclear fluorescence images. These have been called SN (where the nucleolus is surrounded by chromatin) and NSN (where the nucleolus is not surrounded by chromatin). Intermediate configurations are also found, although with a lower frequency. The proportion of each class is on the average equal and depends neither on the presence of cumulus cells nor on the age of the mouse. Electron microscopy confirms several ultrastructural differences between these two nuclear configurations, namely, the structure of the nucleolus, which is vacuolated in NSN-type and compact in SN-type oocytes. Using video-enhanced fluorescence microscopy at low level of excitation light, we could follow directly in vitro the meiotic maturation of both classes, without impairing their viability. We show that in germinal vessicle (GV) state, the chromatin does not change from one configuration into the other and that both classes are able to mature to metaphase II, although the maturation has slightly different characteristics.

Effect of follicle cells on in vitro fertilization of pig follicular oocytes

We investigated the effect of cumulus and granulosa cells (follicle cells) on in vitro fertilization of pig follicular oocytes matured in vitro. Oocytes surrounded by cumulus and connected with a piece of parietal granulosa cells (complexes) were matured in vitro for 46hours and were then divided into 4 groups: Group I oocytes were surrounded by expanded cumulus and granulosa cells; Group II oocytes were surrounded by expanded cumulus cells; Group III were denuded oocytes; and Group IV were denuded oocytes with cumulus cells from other complexes. After incubation for 4 hours and 40 minutes with frozen, thawed and preincubated pig epididymal spermatozoa, the oocytes were cultured for 5 hours and 20 minutes. When oocytes were inseminated in the presence of cumulus cells, the penetration rates were higher (92.5% for Group II and 89.5% for Group IV) than when cumulus cells were not used for insemination (Group III, 66.8%) or when oocytes with follicle cells were inseminated (Group I, 72.3%). Denudation of follicle cells before insemination (Group III) decreased the percentage of male pronuclear formation (50.8%) compared with that of oocytes surrounded by follicle cells (66.7% for Group II and 80.2% for Group I). These results support the ability of a moderate number of follicle cells to facilitate sperm penetration of pig follicular oocytes and male pronuclear formation.

Follicle somatic cells influence pig oocyte penetrability and cortical granule distribution

The influence of somatic cells on oocyte penetrability was studied during in vitro maturation. Four experiments were carried out. In the first, pig oocytes were fertilized in vitro immediately after collection (immature oocytes) or after being cultured for 44 hr with cumulus cells connected to the whole wall of the extroverted follicle (follicle oocytes) or without cumulus cells (denuded oocytes) (Mattioli et al.: Gamete Res 20:177-183, 1988a). In follicle and immature oocytes, 12 hr after insemination, the sperm were equally distributed between zona and ooplasm; in denuded oocytes, the majority (90.5%) of the sperm were located in the zona. In the second experiment, zonae pellucidae of follicle and denuded oocytes, obtained after 44 hr of culture, were incubated with a sperm suspension for 2 hr at 39 degrees C to evaluate the zona binding. The different number of sperm found on the zona pellucida of follicle or denuded oocytes (49.3 +/- 2.97 vs. 37.8 +/- 1.77) did not achieve statistical significance (P greater than 0.05). In the third experiment, follicle and denuded oocytes were denuded of their zonae after maturation and then fertilized in vitro. The number of sperm recorded in the ooplasm of zona-free follicle oocytes was significantly higher than that recorded in zona-free denuded oocytes (9.2 +/- 0.93 vs 1.19 +/- 0.28) (P less than 0.01). In the last experiment the influence of somatic cells on the distribution of cortical granules was evaluated. Pig oocytes were denuded at different stages of culture and, after completion of maturation, processed for electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of cumulus cells and serum on the in vitro maturation, fertilization, and subsequent development of rat oocytes.

Immature oocytes were collected from immature female rats (60-65 g) 40 h after injection with 6 IU pregnant mare's serum gonadotropin (PMSG). Oocytes were matured cumulus-intact (CI) or cumulus-free (CF) in medium supplemented with 0.5% bovine serum albumin (BSA) or 5-20% serum for periods of up to 24 h. After assessment for nuclear maturation, the oocytes were exposed to epididymal sperm for fertilization in vitro. In vitro-matured and ovulated oocytes undergoing fertilization were transferred to unilaterally pregnant recipients for embryonic and fetal development. The presence of cumulus cells and serum shortened (by 2 h) the time required for polar body emission by in vitro-matured oocytes and also helped to increase significantly the penetrability of the oocytes by spermatozoa. A high proportion (45.6%) of fertilized oocytes showed evidence of abnormal fertilization following maturation in the absence of cumulus cells. Oocytes matured CI before fertilization were able to develop to viable fetuses (57.8%) in proportions similar to ovulated oocytes (55.0%) after in vitro fertilization. These findings indicate an essential role for cumulus cells in promoting normal cytoplasmic maturation of oocytes necessary for pronuclear formation and subsequent developmental capability.

Effects of timing of ovum recovery, cumulus cells, sperm preincubation time, and pH on in vitro fertilization in C57BL/6 mice.

The effects of the time of ova recovery following hCG injection, the presence of cumulus cells, duration of sperm preincubation time, and pH on in vitro fertilization in C57BL/6 mice were investigated. Significantly more ova were recovered at 14 h than at 12 h post-hCG injection. Although the number of ova recovered at 16 h was similar to that at 14 h, the percentage of ova showing degeneration increased. The presence or absence of cumulus cells had no effect on ovum fertilization rates, although sperm incubated with cumulus-intact ova underwent the acrosome reaction sooner than those incubated with ova lacking cumulus cells. Sperm motility was sustained slightly longer in the presence of cumulus-free ova than in the presence of cumulus-intact ova. The average percent fertilization of eggs combined with sperm preincubated 1 h was higher than that of sperm preincubated 0 and 0.5 h. Longer preincubation times resulted in a linear decrease in the percent motility and an increase in the percent acrosome reactions. A plot of the number of sperm attaching to the egg vs. coincubation time produced a bell-shaped curve in each case. The greatest number of sperm attaching to the egg occurred between 45 min and 1 h. When the medium was at pH 7.4, fertilization rates were higher than at pH 7.0, 7.2, or 7.6, as were the percent sperm motility and the number of sperm attached to ova. A pH of 7.6 induced 60% of the sperm to undergo the acrosome reaction immediately, and within 1 h all motility was lost.

The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal

The aim of this project was to compare the developmental capacities of mouse oocytes matured in vivo and in vitro. The frequencies of fertilization, preimplantation development, and birth of live offspring after transfer of morulae to uteri of pseudopregnant foster mothers were compared after germinal vesicle stage oocytes underwent spontaneous maturation in vitro, and after gonadotropin-induced maturation in vivo and ovulation. Both groups of matured ova were fertilized in vitro, and preimplantation development was carried out in vitro. Equivalent developmental capacities were observed for all comparisons between the two groups of oocytes. The acquisition of normal developmental capacity depended on the presence of serum in the oocyte maturation medium. The expansion (mucification) of the cumulus oophorus was not required for fertilization or normal development. The frequency of fertilization was lower in oocytes that matured while denuded of cumulus cells. However, when fertilization did occur in these oocytes, a normal percentage developed to live offspring. It is concluded that a normal developmental program occurs during spontaneous maturation of mouse oocytes, and that the presence of cumulus cells during spontaneous maturation may affect the oocyte's fertilizability rather than its subsequent developmental capacity.

The effects of amino acids, cumulus cells, and bovine serum albumin on in vitro fertilization and first cleavage of hamster eggs.

Three factors were studied for their effects on the first cleavage division of in vitro fertilized hamster eggs. Eggs from superovulated females were inseminated with precapacitated epididymal hamster sperm in medium containing either fatty acid-free (FAF) or fraction V (V) bovine serum albumin (BSA), in the presence or absence of cumulus cells. After incubation for 3 to 4 h with sperm, eggs were transferred to culture medium containing FAF- or V-BSA, with or without amino acids (glutamine, isoleucine, methionine, and phenylalanine), and the percentages of eggs cleaving to two cells were recorded after a further 20 h of incubation. Fatty acid-free BSA was found to support a significantly higher percentage of eggs cleaving than V-BSA and was used in all further experiments. The presence of cumulus cells during fertilization was found to have a beneficial effect on cleavage when no amino acids were added to the culture medium, but when amino acids were included, eggs fertilized with or without cumulus present cleaved equally well. These results represent another step toward defining the optimal environmental conditions for obtaining the first cleavage division of hamster eggs in vitro.

Cell interactions and actin synthesis in mammalian oocytes.

In this study, we have examined the profiles of proteins synthesized by mammalian oocytes in the presence and absence of cumulus cells. The results show that the patterns of protein synthesis are broadly similar in cumulus-enclosed and denuded oocytes, but that as a consequence of removing the cumulus cells, the presence of a 45,000-dalton band is greatly reduced. This band, identified by comigration studies on two-dimensional polyacrylamide gels as actin, is strongly synthesized in both isolated cumulus cells and in oocytes labeled in the presence of cumulus cells, but is only weakly synthesized by oocytes incubated in the absence of cumulus cells. We suggest that the presence of newly synthesized actin in the oocyte is dependent upon cellular cooperation between the cumulus cells and oocytes.

Glucose Requirement for Mouse Sperm Capacitation in vitro

The requirements for glucose lactate and pyruvate which are present in Whittens medium for fertilization of mouse ova in vitro by epididymal sperm were examined to determine to what extent fertilization occurred when complete medium was used when all energy sources were omitted and when the only energy source was glucose (5.55 mM) or fructose (5.5 mM). The possibility that pyruvate was also requir ed for sperm capacitation was determined by examining the effect of preincubating the sperm in media with and without pyruvate upon their subsequent ability to fertilized ova. A possible effect of sodium lactate on sperm capacitation was examined by removing ova at various times after mixing with sperm to observe any differences in the proportion of ova fertilized in Whittens medium with or without sodium lactate and with pyruvate and glucose. Capacitation of sperm during a 3-hour preincubation period occurred equally well in complete medium and in medium from which lactate and pyruvate ahd been omitted. Glucose is thus the major source of energy for capacitation of epidiymal mouse sperm but the presence of cumulus cells or pyruvate is required to maintain ova viability during the incubation with sperm. The substitution of fructose for glucose in Whittens medium resulted in very low fertilization rates similar to those found when glucose was omitted from the medium. Measured rates of carbon dioxide production in this situation indicates the possibility that the failure of fructose to support fertilization probably involves deficiencies in its transport or intracellular metabolism to provide adequate energy for sperm motility and capacitation.