Postnatal development of Leydig cells in the opossum (Monodelphis domestica): an immunocytochemical and endocrinological study

Using a mouse early preantral follicle culture system, mature full grown oocytes, arrested in prophase I of meiosis, were produced after 12 days using a recombinant gonadotrophin-supplemented medium. This culture medium does not mimic the normal extracellular environment of the oocyte and might therefore modify meiotic regulation and more particularly progression to metaphase II (MII). The aim of this study was to optimize the treatment using recombinant stimulatory ligands which were known to induce germinal vesicle breakdown (GVBD) and completion of meiosis I, metaphase II (MII), namely recombinant follicle stimulating hormone (r-FSH), chorionic gonadotrophin (r-HCG) and epidermal growth factor (EGF). Full-grown intrafollicular oocytes could not resume meiosis when the 'ovulatory' stimulus was r-FSH, used at a 100 times higher dose than during culture. r-FSH did not increase progesterone production. When 1.5 IU/ml r-HCG was used as meiotic trigger, germinal vesicle breakdown was obtained in 95% of the oocytes 64% of which extruded a first polar body. r-HCG induced a dramatic increase in progesterone production. When EGF was administered as sole stimulus on day 12 to the attached follicle-enclosed oocytes, only doses > or =5 ng/ml could cause GVBD, although less effectively than r-HCG (45 versus 95%; P < 0.0001). Oocytes undergoing GVBD by the EGF pulse reached metaphase II at a rate of 54% (not significant versus r-HCG). EGF did not stimulate progesterone production. Addition of increasing doses of EGF (0.5; 5; 10; 50 ng/ml) to r-HCG did not increase the GVBD-rate, but EGF doses >5 ng/ml improved MI to MII transition (P=0.027), thereby improving the final yield of MII oocytes by 12.5%. These data show that up to a dose of 50 ng/ml, EGF on its own could only override the somatic inhibitory stimuli in less than half of the cultured follicles. However, in addition to HCG, EGF (25 ng/ml) had a stimulatory effect on completing the first meiotic division. It was concluded that, under the present culture conditions, EGF in combination with HCG provided optimal nuclear maturation.     

Isolation and characterization of maturation inhibiting compound in bovine follicular fluid

The protein fraction which is responsible for the inhibition of maturation of bovine oocytes in vitro was isolated from cow follicular fluid by means of column chromatography on a Sephadex G-200 and a Sepharose 4B, both in 0.1 M ammonium acetate, pH 6.7. The molecular weight of the maturation inhibiting protein fraction is approximately 60 kDa. At a concentration of 2.0 mg/mL in cultivation medium, 100% of the oocytes were arrested at the germinal vesicle stage. At a concentration of 0.25 mg/mL, the protein fraction still had some meiosis inhibiting effects, but 56% of the oocytes were capable of maturing to the metaphase of the second meiosis (MII). Without compact cumulus the inhibiting fraction had no meiosis retarding effect on the oocytes. Cow follicular fluid also exhibited this inhibitory effect on oocyte maturation in vitro. However, the follicular fluid from follicles of 2.5-5.0 mm diameter showed higher meiosis inhibiting effects than the follicular fluid from follicles of 5-10 mm diameter.     

Dynamics of maturation-promoting factor and its constituent proteins during in vitro maturation of bovine oocytes

Maturation-promoting factor (MPF) is known to be a key regulator of both mitotic and meiotic cell cycles. MPF is a complex of a B cyclin and the cyclin-dependent kinase cdkl (p34cdc2). Oocyte maturation and its arrest at metaphase of meiosis II (MII) are regulated by changes in MPF activity. In this study, experiments were conducted to examine the dynamics of MPF activity and its constituent proteins during in vitro maturation of bovine oocytes. Bovine oocytes displayed relatively low levels of MPF (histone H1 kinase) activity at the germinal vesicle stage during the first 8 h of maturation. MPF activity increased gradually thereafter, and its first peak of activity occurred at 12-14 h of maturation (presumptive metaphase I), which was followed by an abrupt reduction in activity at 16-18 h, during presumptive anaphase and telophase. MPF activity then increased, reaching a plateau at 20-24 h of maturation (MII stage). This high level of MPF activity was maintained for several hours but decreased gradually after 30 h of maturation and became barely detectable by 48 h of in vitro maturation (IVM) culture. At each time point, there was a significant variation among individual oocytes in histone H1 kinase activity, which was probably due to asynchronous maturation. Abundance of cdk1 increased gradually during the first 8 h and then remained relatively constant except for an apparent reduction at 18-22 h of IVM. The level of cyclin B2 increased quickly during the initial 2 h of culture, and this high level was maintained until 16 h, after which a significant reduction was observed between 18 and 22 h of IVM. The de novo synthesis of cyclin B2, however, exhibited a biphasic oscillation during maturation, with peaks before the onset of MI and of MII. These results have defined the profiles of MPF activity and its individual components during bovine oocyte maturation in vitro. We conclude that active MPF regulates bovine oocyte maturation and that de novo synthesis of cyclin B2 occurs during the process of maturation.     

Cyclin synthesis controls the progression of meiotic maturation in mouse oocytes

To study the mechanisms involved in the progression of meiotic maturation in the mouse, we used oocytes from two strains of mice, CBA/Kw and KE, which differ greatly in the rate at which they undergo meiotic maturation. CBA/Kw oocytes extrude the first polar body about 7 hours after breakdown of the germinal vesicle (GVBD), whilst the oocytes from KE mice take approximately 3-4 hours longer. In both strains, the kinetics of spindle formation are comparable. While the kinetics of MAP kinase activity are very similar in both strains (although slightly faster in CBA/Kw), the rise of cdc2 kinase activity is very rapid in CBA/Kw oocytes and slow and diphasic in KE oocytes. When protein synthesis is inhibited, the activity of the cdc2 kinase starts to rise but arrests shortly after GVBD with a slightly higher level in CBA/Kw oocytes, which may correspond to the presence of a larger pool of cyclin B1 in prophase CBA/Kw oocytes. After GVBD, the rate of cyclin B1 synthesis is higher in CBA/Kw than in KE oocytes, whilst the overall level of protein synthesis and the amount of messenger RNA coding for cyclin B1 are identical in oocytes from both strains. The injection of cyclin B1 messenger RNA in KE oocytes increased the H1 kinase activity and sped up first polar body extrusion. Finally, analysis of the rate of maturation in hybrids obtained after fusion of nuclear and cytoplasmic fragments of oocytes from both strains suggests that both the germinal vesicle and the cytoplasm contain factor(s) influencing the length of the first meiotic M phase. These results demonstrate that the rate of cyclin B1 synthesis controls the length of the first meiotic M phase and that a nuclear factor able to speed up cyclin B synthesis is present in CBA/Kw oocytes.     

Nondisjunction in aging female mice

Oocytes from CBA mice varying in age from 2 to 11 months were cultured to the metaphase II stage of meiosis and the chromosomes analyzed. The oocytes from three maternal age groups were compared with respect to the mean number of oocytes obtained per mouse, the frequency of maturation to metaphase II, and the frequency of numerical chromosomes abnormalities. Both the mean number of oocytes obtained per mouse and the frequency of maturation decreased markedly with maternal age. The frequency of chromosome abnormalities in the oocytes increased with maternal age from the young to the middle-aged mice but dropped off in the oldest maternal age group. No hyperploid (n + 1) oocytes were observed in the young or old group of mice, but 5.2% hyperploidy occurred in the middle-aged group. It is suggested that the lack of hyperploid oocytes in the old CBA females might be due to a threshold effect in which oocytes that are damaged by the number of univalents present at metaphase I become atretic and do not progress to metaphase II. The frequency of diploid (2n) oocytes was 1.7% and was not maternal-age dependent.     

Fertilization and in vitro development of cryopreserved human prophase I oocytes

OBJECTIVE: To determine the potential for in vitro maturation, fertilization, and cleavage after cryopreservation of immature, prophase I human oocytes. DESIGN: Immature oocytes obtained in excess of the number required by the patient were randomized and cryopreserved at the prophase I stage or cultured as control. After thawing and maturation in vitro, test and control oocytes were inseminated with husband's sperm and evaluated for fertilization and cleavage in vitro. SETTING: In vitro fertilization program. PATIENTS: Consenting patients undergoing controlled ovarian hyperstimulation for the purposes of IVF. MAIN OUTCOME MEASURES: Rates of maturation to metaphase II, fertilization, and cleavage were compared between control and cryopreserved oocytes. RESULTS: Upon thaw, 58.5% (72/123) of prophase I oocytes were viable. Control oocytes demonstrated a 74.8% (98/131) maturation rate to metaphase II, a 56.5% (52/92) fertilization rate, and an 11.5% (6/52) blastocyst rate. Cryopreserved oocytes showed a 83.3% (60/72) rate of maturation, a 57.7% (30/52) fertilization rate, and a 3.3% (1/30) blastocyst rate. No significant differences were noted between any of these parameters. CONCLUSIONS: These results demonstrate that prophase I oocytes from stimulated IVF cycles are able to survive cryopreservation and resume meiosis to achieve full nuclear maturation post-thaw. In addition, cryopreserved oocytes retain the same capacity for fertilization and development as control oocytes.     

Analysis of human T-cell antigen receptor variable beta gene usage following vaccination with recombinant HBsAg

Exposure of mammalian oocytes to the protein phosphatase (PP)-1 (PP1) and PP2A inhibitor okadaic acid (OA) stimulates oocyte meiosis. However, treated oocytes do not develop beyond metaphase I (MI), and they display morphological aberrations. Experiments were conducted to define inhibitor treatment conditions for macaque oocytes that would result in germinal vesicle breakdown (GVB) stimulation and completion of meiosis without significant cytoplasmic abnormalities. As described above for OA, continual exposure of macaque oocytes to 50 nM calyculin-a (CL-A) significantly enhanced GVB at 24 h compared to that in controls, and the majority of the treated oocytes displayed cytoplasmic abnormalities. However, transient exposure (10 min) of rhesus macaque oocytes to either 50 nM CL-A or 1.0 microM OA enhanced GVB rates compared to that in controls and did not increase the incidence of cytoplasmic abnormalities. Meiotic maturation from germinal vesicle-intact oocytes to MII was enhanced following transient treatment with CL-A or OA compared to that in controls; however, development from MI to MII occurred at a similar frequency. In vitro-matured oocytes transiently exposed to OA and CL-A were capable of fertilization. In addition, ovarian immunohistochemical analysis revealed that both PP1 and PP2A were present in macaque oocytes. PP1 was localized throughout the cytoplasm with a predominance in the nucleus, whereas PP2A was evenly distributed throughout the cytoplasm with a reduction in the nuclear area. These results taken together-differential developmental responses to inhibitor treatment and intracellular enzyme localizations-may be indicative of multiple regulatory roles of PP1 and/or PP2A during meiosis.     

Oocyte competence for in vitro maturation is associated with histone H1 kinase activity and is influenced by estrous cycle stage in the mare

The in vitro maturation rate of equine oocytes remains low, regardless of culture conditions. Our objective was to determine the reasons for failure of equine oocytes to resume meiosis during in vitro maturation and to ascertain the influence of the estrous cycle stage on meiotic competence. In 10 cyclic mares, 7 ultrasound-guided follicular punctures were performed alternately during the follicular phase (group DF; n = 3 punctures), at the end of the follicular phase (group EF; n = 2), and during the luteal phase (group DL; n = 2). We evaluated the competence of the oocytes for in vitro maturation and measured their maturation-promoting factor activity by histone H1 kinase assay. Puncturing once at the end of the follicular phase and once during the luteal phase, or three times during the follicular phase, yielded about 11 cumulus-oocyte complexes per 22 days. The maturation rate was different between the groups, 51% in group EF, 34% in group DL (p < 0.05), and 15% in group DF (p < 0.01), and it increased with an increase in follicular diameter (p < 0.05). After in vitro culture, the H1 kinase activity was lower in oocytes that remained in germinal vesicle or dense chromatin stages than in oocytes that reached metaphase I or metaphase II (p < 0.05). The H1 kinase activity was not different between oocytes in germinal vesicle stage after in vitro maturation and immature oocytes that were not cultured in vitro, and was higher in preovulatory oocytes that reached metaphase II in vivo than in the oocytes that reached metaphase II after in vitro maturation (p < 0.001). This is the first report on kinase activity in the equine oocyte.     

Amino acids in maturation medium and presence of cumulus cells at fertilization promote male pronuclear formation in porcine oocytes matured and penetrated in vitro

The present study was conducted to examine the ability of porcine oocytes to achieve male pronuclear (MPN) formation when they are matured and penetrated in vitro under various culture conditions. When cumulus-enclosed oocytes were cultured for 24-48 h in modified Whitten's medium (pH 7.4) supplemented with 10% porcine follicular fluid, 10 IU eCG/ml, and 10 IU hCG/ml (designated mWM-FG), nuclear maturation of oocytes reaching metaphase II was completed by 36 h after the start of culture. However, there were no differences in the proportions (94-95%) of oocytes penetrated in vitro by cryopreserved ejaculated spermatozoa or in the rates (35-45%) of MPN formation between oocytes cultured for 36 and 48 h. When cumulus-enclosed oocytes were cultured for 36 h in mWM-FG supplemented with 2% (v:v) minimal essential medium (MEM) essential amino acids (EAA) with the addition of 0.1 mM glutamine and/or 1% (v:v) MEM nonessential amino acids (NEAA) and inseminated in vitro, 93-97% of oocytes were penetrated regardless of the presence of amino acids during maturation, but the rates of MPN formation were higher in the presence (79-84%) than in the absence (51%) of any amino acids. The addition of EAA+NEAA and/or 0.57 mM cysteine to mWM-FG also did not affect sperm penetration in vitro, while it promoted MPN formation (76-83%) in penetrated oocytes as compared with those matured in the absence of amino acids and cysteine (53%). When oocytes were freed from cumulus cells after culture in mWM-FG, sperm penetration rates were not different between cumulus-enclosed (100%) and cumulus-free (92%) oocytes, but the rate of MPN formation was higher in cumulus-enclosed (53%) than in cumulus-free (28%) oocytes. When EAA+NEAA+cysteine was added to mWM-FG, MPN formation was not improved in cumulus-free oocytes but was much improved (78%) in cumulus-enclosed oocytes. These results indicate that MPN formation in porcine oocytes is promoted by the addition of amino acids and/or cysteine in simple maturation medium and by the presence of cumulus cells at fertilization in vitro.     

A review of major nursing vocabularies and the extent to which they have the characteristics required for implementation in computer-based systems

Fertilised mouse eggs develop the oolemma block to sperm penetration within 1 h. This block makes zona-free eggs at the pronuclear stage (zygotes) fully resistant to sperm penetration. In this study we investigated whether this block can spread--as a result of cell fusion--to the oolemma of eggs that are competent to be penetrated by spermatozoa. Preovulatory (GV) oocytes, ovulated oocytes in metaphase II (MII) and 1-cell parthenotes were fused with zygotes and the hybrid cells inseminated at various intervals after fusion. Sperm penetration was assessed on the basis of the presence of Giemsa-positive sperm heads in the air-dried preparations. The oolemma block to sperm penetration develops in all types of hybrids although at different speeds: it develops fast (2-3 h) in oolemma derived from MII oocytes and artificially activated eggs, and slowly in oolemma derived from GV oocytes. In the GV oocyte-zygote hybrids the time of formation of the block varied: while 50% of cells lost the ability to fuse with sperm by 2 h after fusion, in the remaining cells the block must have developed some time between 5 and 18 h after fusion. How these sperm-induced modifications of the oolemma of fertilised egg spread in the hybrid cell and render the 'virgin' part of oolemma resistant to sperm penetration remains unknown.     

Transcription factor E2F and cyclin E-Cdk2 complex cooperate to induce chromosomal DNA replication in Xenopus oocytes

Although no chromosomal DNA replication actually occurs during Xenopus oocyte maturation, the capability develops during the late meiosis I (MI) phase in response to progesterone. This ability, however, is suppressed by Mos proteins and maturation/mitosis promoting factor during the second meiosis phase (meiosis II; MII) until fertilization. Inhibition of RNA synthesis by actinomycin D during early MI prevented induction of the replication ability, but did not interfere with initiation of the meiotic cell cycle progression characterized by oscillation of the maturation/mitosis promoting factor activity and germinal vesicle breakdown. Microinjection of recombinant proteins such as dominant-negative E2F or universal Cdk inhibitors, p21 and p27, but not wild type human E2F-1 or Cdk4-specific inhibitor, p19, into maturing oocytes during MI abolished induction of the DNA replication ability. Co-injection of human E2F-1 and cyclin E proteins into immature oocytes allowed them to initiate DNA replication even in the absence of progesterone treatment. Injection of cyclin E alone, which was sufficient to activate endogenous Cdk2 kinase, failed to induce DNA replication. Moreover, the activation of Cdk2 was not affected under the conditions where DNA replication was blocked by actinomycin D. Thus, like somatic cells, both activities of E2F and cyclin E-Cdk2 complex are required for induction of the DNA replication ability in maturing Xenopus oocytes, and enhancement of both activities enables oocytes to override DNA-replication inhibitory mechanisms that specifically lie in maturing oocytes.     

Effects of ovarian source, patient age, and menstrual cycle phase on in vitro maturation of immature human oocytes

OBJECTIVE: To determine the efficiency of in vitro maturation, expressed by nuclear maturation, of oocytes aspirated during gynecologic surgeries or collected from excised ovaries. To assess the effect of patient age and cycle phase at collection on the oocyte's ability to mature in vitro. To examine the time course of oocyte maturation in vitro. DESIGN: Nuclear maturation based on patient criteria compared. SETTING: University-based IVF program and research center. PATIENT(S): Consented patients undergoing gynecologic surgeries or patients undergoing oophorectomy. INTERVENTION(S): Oocytes were maintained in culture for 48 hours and evaluated for maturation. MAIN OUTCOME MEASURE(S): Nuclear maturation evaluated as germinal vesicle breakdown (GVBD) or progression to the metaphase II (MII) stage. RESULT(S): A significantly higher percentage of oocytes collected during the follicular phase of the menstrual cycle underwent GVBD than did oocytes collected during the luteal phase (60% versus 48%, respectively). The percentage of oocytes reaching the MII stage, from these two groups, was not different. No statistically significant differences in maturation were observed in oocytes from different ovarian sources or from patients >40 or <40 years of age. CONCLUSION(S): These data suggest that oocytes collected during the follicular phase are more likely to undergo GVBD than oocytes collected during the luteal phase. In this study, ovarian source, age, or cycle phase did not influence the final meiotic maturation of oocytes to metaphase II.     

Inhibition of phosphoinositide metabolism or chelation of intracellular calcium blocks FSH-induced but not spontaneous meiotic resumption in mouse oocytes

Mammalian oocytes are arrested at the diplotene phase of the first meiotic division until ovulation. In the mouse, germinal vesicle breakdown (GVBD) and progression to metaphase II is thought to be triggered by a positive signal originating in the follicular cells following stimulation by the luteinizing hormone (LH) surge. Isolated, fully grown oocytes can also undergo spontaneous reinitiation of meiosis in vitro in the absence of gonadotrophin stimulation. To investigate the mechanism of meiotic resumption, inhibitors of phosphoinositide metabolism and an intracellular calcium chelator were used during maturation in vitro under different conditions. In a series of experiments, isolated cumulus cell-oocyte complexes (COCs) maintained in meiotic arrest by hypoxanthine were induced to resume meiosis by treatment with follicle-stimulating hormone (FSH). Under these conditions, both LiCl and neomycin, which inhibit phosphoinositide hydrolysis, produced a dose-dependent inhibitory effect on meiotic resumption. Similar results were obtained when FSH-induced meiotic resumption was observed in the presence of the acetoxymethyl ester form of 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), an intracellular calcium chelator. In hypoxanthine-arrested oocytes, GVBD induced by epidermal growth factor (EGF), which mimics FSH action in in vitro maturation, was also repressed by LiCl and neomycin. Conversely, meiotic resumption triggered by a pulse of 8-bromo-cyclic adenosine monophosphate (8-Br cAMP) was not affected by these two inhibitors. In experiments in which oocytes were cultured under conditions which permit spontaneous meiotic maturation, resumption of meiosis was not affected by either inhibition of phosphoinositide hydrolysis or chelation of intracellular calcium. Therefore, it appears that meiotic resumption induced by hormone stimulation requires activation of the phosphoinositide pathway and mobilization of intracellular calcium. In contrast, spontaneous maturation probably occurs through a different mechanism because it is not affected by inhibition of this signaling pathway.     

Development of normal mice from metaphase I oocytes fertilized with primary spermatocytes

Primary spermatocytes are the male germ cells before meiosis I. To examine whether these 4n diploid cells are genetically competent to fertilize oocytes and support full embryo development, we introduced the nuclei of pachytene/diplotene spermatocytes into oocytes that were arrested in prophase I (germinal vesicle stage), metaphase I, or metaphase II (Met II). Both the paternal and maternal chromosomes then were allowed to undergo meiosis synchronously until Met II. In the first and second groups, the paternal and maternal chromosomes had intermingled to form a large Met II plate, which was then transferred into a fresh enucleated Met II oocyte. In the third group, the paternal Met II chromosomes were obtained by transferring spermatocyte nuclei into Met II oocytes twice. After activation of the Met II oocytes that were produced, those microfertilized at metaphase I showed the best developmental ability in vitro, and three of these embryos developed into full-term offspring after embryo transfer. Two pups (one male and one female) were proven to be fertile. This finding provides direct evidence that the nuclei of male germ cells acquire the ability to fertilize oocytes before the first meiotic division.     

Chromatin decondensation, pronucleus formation, metaphase entry and chromosome complements of human spermatozoa after intracytoplasmic sperm injection into hamster oocytes

Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO2) and (ii) omission of Ca2+ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set 2, n=278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca2+-free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca2+ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.     

Timed analysis of the nuclear maturation of oocytes in early preantral mouse follicle culture supplemented with recombinant gonadotropin

OBJECTIVE: To analyze the effects of combined FSH and variable doses of LH on the nuclear maturity and capacity to resume meiosis in oocytes from preantral follicles from prepubertal mice. DESIGN: Prospective, randomized, and controlled in vitro laboratory experiment. SETTING: Academic research environment. INTERVENTION(S): Meiosis was studied after somatic cell removal or after stimulation with hCG plus epidermal growth factor in three culture conditions: maturation medium with FSH alone and with two different doses of LH. MAIN OUTCOME MEASURE(S): The nuclear maturation of the oocytes and the E2, progesterone, and alpha-specific inhibin content of the conditioned medium. RESULT(S): Somatic cell removal and hormonal stimulation were equally effective in inducing germinal vesicle breakdown, but the hormonal stimulus was essential for the completion of meiosis, which was maximal (70%) on day 13 of culture. Continuous addition of LH to FSH during the oocytes' growth made them more prone to spontaneous resumption of meiosis I but resulted in a higher proportion of oocytes reaching the completion of meiosis. Estradiol and progesterone measurements demonstrated that the presence of LH influences luteinization. CONCLUSION(S): In contrast to oocytes grown in vivo, cumulus cell removal by itself is an insufficient stimulus for oocytes cultured in vitro to complete meiosis. Timed stimulation with hCG and epidermal growth factor increases nuclear maturation rates. A maximum number of metaphase II oocytes are obtained after a 13-day in vitro growth period when LH is added to the maturation medium.     

Perivitelline space granularity: a sign of human menopausal gonadotrophin overdose in intracytoplasmic sperm injection

The significance of the presence of coarse dark granules in the perivitelline space of oocytes has not been studied before. The study included 2288 intact oocytes [2063 in metaphase II (MII), 136 in metaphase I (MI), and 89 in germinal vesicle (GV)] retrieved in 206 intracytoplasmic sperm injection cycles stimulated by a long agonist protocol. The incidence of granules varied with oocyte maturity. It was detected in 34.3% and 4% of the MII and MI oocytes respectively, while none of the GV oocytes contained granules. The woman's age, hormonal values (oestradiol and progesterone), human chorionic gonadotrophin/oocyte retrieval interval, number of oocytes retrieved, and oocyte retrieval/injection interval were not related to the percentage of granular oocytes. Moreover, there was no correlation between the percentage of granular oocytes and the fertilization and cleavage rates, pregnancy outcome, as well as the implantation rate. Patients were divided into three groups according to the total human menopausal gonadotrophin (HMG) dose they received. There was a statistically significant difference between the three groups in the percentage of granular oocytes [17.4 +/- 5.2% versus 26.7 +/- 3.2% versus 45.4 +/- 4.2% in the low-dose (< 30 ampoules), intermediate dose (31-45 ampoules), and high-dose (> 45 ampoules) groups respectively]. We conclude that granularity in the perivitelline space is probably a physiological phenomenon related to the maturational events in oocytes and enhanced by exposure to high dosages of HMG.     

Oocyte maturation and intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI), treatment of severe male infertility allows an accurate evaluation of oocyte maturity at recovery after corona-cell removal. In cycles comprising a GnRH analog desensitization and a stimulation by hMG or FSH, 12% of oocytes aspirated from follicles (> 14 mm), 34 hours post-hCG are still immature, in prophase or metaphase 1. They are able to achieve meiosis in vitro in 66% of the cases and will be fertilized (2 PN) by ICSI in 51% of the cases as the in vivo mature oocytes of the same cohort. Nevertheless, the quality of cytoplasmic maturation and consequently of embryonic viability remains to be assessed as there still are few pregnancies arising from in vitro matured oocytes. ICSI also represents the only way to obtain normal fertilization in some exceptional but observed anomalies of oocyte maturation, particularly when there is a lack of zona reaction leading to repetitive polyspermy in conventional IVF.     

p53 tumor-suppressor gene: clues to molecular carcinogenesis

Pig oocytes were examined to test their ability to undergo cortical granule exocytosis upon penetration by spermatozoa during meiotic maturation. Immature or maturing oocytes (cultured in vitro for 0 h, 26 h and 46 h) were inseminated with ejaculated boar spermatozoa in vitro. Before and after insemination, oocytes were stained with peanut agglutinin labelled with fluorescein isothiocyanate and the cortical granule distributions were examined under the fluorescent microscope and the laser confocal microscope. Before insemination, all the oocytes at the germinal vesicle stage showed a uniform distribution of cortical granules throughout the cortical cytoplasm. The granules migrated centrifugally during maturation and were distributed just beneath the oolemma in the oocytes after germinal vesicle breakdown, forming a monolayer in metaphase I or metaphase II. Cortical granules were still present in all penetrated oocytes at the germinal vesicle stage 18 h after insemination; in contrast, 26% and 84% of the oocytes inseminated at the stages of germinal vesicle breakdown or at metaphase I and II, respectively, completely released their cortical granules. Nuclear activation rates of penetrated oocytes were 0%, 38% and 96% in oocytes cultured for 0 h, 26 h and 46 h, respectively. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 88% (oocytes cultured for 46 h) released cortical granules completely. Complete cortical granule exocytosis was not observed in nuclear-inactivated oocytes. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 80% (oocytes cultured for 46 h) of monospermic oocytes and 67% (oocytes cultured for 26 h) and 91% (oocytes cultured for 46 h) of polyspermic oocytes released cortical granules, and no statistical difference was observed between oocytes cultured for 26 h or 46 h, or between monospermic and polyspermic oocytes. The proportion of oocytes with cortical granule exocytosis increased as insemination time increased and was greatest 18 h after insemination in oocytes cultured for 26 h and 46 h; no obvious changes were observed when the insemination time was prolonged to 24 h. These results indicate that pig oocytes develop the ability to release cortical granules after penetration by spermatozoa following germinal vesicle breakdown, and that this ability is not fully developed until metaphase II. Cortical granule exocytosis is accompanied by nuclear activation, suggesting that both nuclear and cytoplasmic maturation are responsible for the cortical reaction. Polyspermy may be a result of a complete failure of cortical granule exocytosis in immature oocytes and delayed CG exocytosis in matured oocytes.     

Chemical modification and chemical cross-linking for protein/enzyme stabilization

The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 +/- 12% of the oocytes were at the germinal vesicle stage compared to 97 +/- 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 +/- 18% were at MII stage, compared with 93 +/- 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 +/- 8% vs 89 +/- 7%; P < 0.01), oocyte activation higher (11 +/- 5% vs 2 +/- 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 +/- 7% vs 4 +/- 4%; P > 0.05), compared with the control group. Cleavage was lower (61 +/- 13% vs 81 +/- 6%; P < 0.001), as well as day 8 blastocyst formation (17 +/- 7% vs 36 +/- 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development.