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PubMed2004

Nuclear dynamics of parthenogenesis of human oocytes: effect of oocyte aging in vitro.

We investigated in detail the nuclear kinetics of oocyte activation of aged human oocytes following combined activation treatment with calcium ionophore and puromycin. Two types of oocytes were used: (a) 1-day-old oocytes after 20-24 h retrieval, and (b) 2-day-old oocytes after 44-50 h retrieval. A total of 185 unfertilized aged oocytes, 91 1-day-old and 94 2-day-old oocytes, were fixed at 1, 2, 4, 6 and 8 h after activation treatment and then metaphase II (MII), anaphase or telophase II (A/T II) or pronuclear stage were recorded. We demonstrated that a combined calcium ionophore and puromycin treatment induced a high activation rate in both 1-day-old (95.6%) and 2-day-old oocytes (95.2%). Our results also demonstrated that the nuclear progression was faster in 2-day-old oocytes than in 1-day-old oocytes, although nuclear progression in parthenogenetically activated human oocytes requires the longer time periods compared with ICSI fertilization. It is concluded that combined treatment of the calcium ionophore and puromycin allows a high rate of parthenogenetic activation and the nuclear kinetics of parthenogenetically activated human oocytes appears to be more rapid in in vitro aging oocytes.

Calcimycin↗
PubMed2004

missing oocyte encodes a highly conserved nuclear protein required for the maintenance of the meiotic cycle and oocyte identity in Drosophila.

In Drosophila, a single oocyte develops within a 16-cell germline cyst. Although all 16 cells initiate meiosis and undergo premeiotic S phase, only the oocyte retains its meiotic chromosome configuration and remains in the meiotic cycle. The other 15 cells in the cyst enter the endocycle and develop as polyploid nurse cells. A longstanding goal in the field has been to identify factors that are concentrated or activated in the oocyte, that promote meiotic progression and/or the establishment of the oocyte identity. We present the characterization of the missing oocyte gene, an excellent candidate for a gene directly involved in the differentiation of the oocyte nucleus. The missing oocyte gene encodes a highly conserved protein that preferentially accumulates in pro-oocyte nuclei in early prophase of meiosis I. In missing oocyte mutants, the oocyte enters the endocycle and develops as a polyploid nurse cell. Genetic interaction studies indicate that missing oocyte influences meiotic progression prior to pachytene and may interact with pathways that control DNA metabolism. Our data strongly suggest that the product of the missing oocyte gene acts in the oocyte nucleus to facilitate the execution of the unique cell cycle and developmental programs that produce the mature haploid gamete.

Amino Acid Sequence↗
PubMed1996

Influence of oocyte aging on developmental ability of reconstituted embryos produced from oocyte cytoplast and single blastomeres of two-cell stage embryos.

The present study was conducted to investigate the influence of aging of recipient oocyte on the developmental ability of reconstituted mouse embryos produced from the cytoplast of oocytes and single blastomeres of early or late 2-cell stage embryos by electrofusion. Oocytes were obtained at 14 (newly ovulated oocytes), 18, 22 (oocyte that time passed after ovulation; aged oocyte) hr after hCG injection and oocyte cytoplast was produced by manual enucleation using a fine glass needle under the dissecting microscope. The aging of the oocytes significantly influenced on the fusion rate of the reconstituted embryos (14 hr: 42.7-47.2% vs. 22 hr: 75.3-77.6%). Similarly, the cleavage rate of reconstituted embryos increased with aging of the oocytes (14 hr: 50.8-56.3% vs. 22 hr: 82.2-90.7%). The percentage of reconstituted embryos produced from cytoplast of aged oocytes (22 hr post hCG) and single blastomeres of late 2-cell stage embryos developing to the blastocyst (20.8%) was significantly higher than that of reconstituted embryos produced by other combinations (2.0-8.2%: P < 0.01). Although cell cycle stage of donor nuclei influenced to developmental ability of reconstituted embryos, these results are probably related to the aging of the oocytes since aged oocytes can be activated more easily by electrical stimulation than newly ovulated oocytes.

Animals↗
PubMed2000

Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo.

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles </= 29 mm was significantly higher at A1 than at A2. The number of oocytes at the germinal vesicle stage was significantly higher at A2 (41.5%) than at A1 (17.8%). Meiosis-activating sterols (FF-MAS and T-MAS) were identified in follicular fluid recovered at A2. Follicular concentrations of FF-MAS and T-MAS were unaffected by treatment with crude equine gonadotrophins. The present study demonstrates that follicular aspiration during oestrus allowed a new follicular population to develop and resulted in a higher degree of synchronization of oocyte development with respect to cumulus expansion and nuclear maturation. The availability of a more homogeneous population of oocytes might facilitate a better optimization of in vitro maturation and in vitro fertilization techniques in mares. Administration of crude equine gonadotrophins during early dioestrus did not affect the growth of small follicles, the oocyte yield after aspiration or oocyte maturation in vivo.

Animals↗
PubMed1998

Attitudes of different groups of women in Sweden to oocyte donation and oocyte research.

OBJECTIVES: To evaluate attitudes of Swedish women towards oocyte donation and oocyte research. METHODS: Five different groups of women, with approximately 50 patients in each, were asked anonymously about their attitudes to legislation, tentative roles as donors or recipients, anonymity, suitable donors or recipients, research on fetuses and cadavers as a source of oocytes, age limits and economic aspects. The groups were: 1. Women undergoing IVF treatment (IVF). 2. Infertile women during work-up (INF). 3. Recently delivered women attending a maternity unit (MAT). 4. Women attending a family planning center applying for therapeutic abortion (FPC). 5. Women with Turner's syndrome (TUR). RESULTS: More than 90% of women in all groups investigated advocated amendment of the law in order to permit oocyte donation. The women of infertile groups were more in favor of donating oocytes compared to women of fertile groups (p<0.05). A great majority would prefer anonymity both if they were donors and if they were recipients. If no anonymity was guaranteed, the acceptance of both the donor and recipient groups decreased but more than half of the women in all groups would still donate/accept oocytes. There was a significant difference in attitude towards non-anonymous oocyte donation, with the highest acceptance among Turner patients and the lowest among IVF patients (p<0.01). A majority in all groups were more motivated to donate/accept oocytes from a close relative, with the exception of Turner patients (p<0.01). All groups had a negative attitude to the use of donated fetuses and cadavers as sources of oocytes. IVF patients, close relatives and volunteers were all regarded as suitable donors by a majority of women in all groups. Women of fertile age, with ovarian failure or a genetic disorder, were accepted as recipients by all groups. Postmenopausal women were not accepted as recipients by a great majority in all groups. All groups preferred an age limit for recipients. More than 70% set the limit to the interval 40 to 45 years of age. A majority in all groups believed that donors should be paid to cover medication and loss of income. The recipients were expected, by a majority of women, to pay for some of the costs of the oocyte donation program. CONCLUSIONS: A great majority wanted a change in the Swedish legislation to permit oocyte donation. All groups had a generous attitude to donation of oocytes although anonymity would be preferred. Ovarian dysfunction and genetic disorders among women of fertile age were regarded as major indications for oocyte donation. IVF patients, close relatives and volunteers were all regarded as acceptable donors.

Abortion, Therapeutic↗
PubMed2006

Repeated ultrasound-guided transvaginal oocyte retrieval from cyclic Murrah buffaloes (Bubalus bubalis): oocyte recovery and quality.

The present study was undertaken to explore the potential of the Murrah breed of buffaloes as donors of oocytes and to find out the recovery rate and oocyte quality in cyclic Murrah buffaloes subjected to oocyte recovery once a week. Murrah buffaloes (n = 5) were synchronized for estrus by a single prostaglandin injection schedule. The animals were subjected to transvaginal oocyte retrieval (TVOR) once weekly for 6 weeks, starting from Day 7 of the oestrous cycle (Day 0 = day of oestrus). TVOR was performed using an ultrasound machine with a 5 MHz transvaginal transducer, single lumen 19-gauge, 60 cm long needle and a constant vacuum pressure of 50 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large (> or = 10 mm). The oocytes recovered were classified as grade A, cumulus-oocytes complexes (COCs) with > or = 5 layers of cumulus cells; grade B, those with two to four layers; grade C, partially denuded oocytes; and grade D, completely denuded oocytes. The mean (+/- S.E.M) number of small, medium and large follicles, and the number of total follicles observed per animal per session, which was 2.2 +/- 0.3, 0.6 +/- 0.2, 0.9 +/- 0.1 and 3.7 +/- 0.3, respectively, did not differ between animals or between puncture sessions. Small follicles constituted a major proportion (59%) of the total observed follicles. A mean (+/- S.E.M) number of 3.0 +/- 0.3 follicles were punctured and 2.0 +/- 0.3 oocytes recovered per animal per session, with a recovery rate of 68%. Out of the total 61 oocytes recovered, 36 (59%) were of grades A + B whereas 25 (41%) were of grades C + D. In conclusion, this study describes the potential of cyclic Murrah buffaloes as donors of oocytes collected by repeated TVOR once a week, without any adverse effects on follicular growth and oocyte recovery. It also describes an efficient system for carrying out TVOR in buffaloes.

Animals↗
PubMed1995

Perivitelline space of marsupial oocytes: extracellular matrix of the unfertilized oocyte and formation of a cortical granule envelope following the cortical reaction.

The purpose of this study was to characterize the structure of the vestments surrounding unfertilized and cortical granule-reacted oocytes from a marsupial, the grey short-tailed opossum Monodelphis domestica and to determine if a cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the cortical reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a cortical reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat-tailed dunnart. The cortex of unfertilized oocytes contained cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short-tailed opossum is approximately 1 microns thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes.

Animals↗
PubMed1988

Follicular factors influence oocyte fertilizability by modulating the intercellular cooperation between cumulus cells and oocyte.

In order to investigate whether the follicular tissue influences cumulus-oocyte interaction and, consequently, the fertilizability of the egg, four experiments were carried out. In the first, cumulus-enclosed pig oocytes were cultured for 44 h in control medium (modified TCM-199) or in follicle-conditioned medium, and the intercellular coupling was studied by measuring 3H-uridine uptake. In control medium the intercellular cooperation started to decline immediately, and at 24-32 h the uncoupling was almost complete. By contrast, in follicle, conditioned medium, it remained at high levels until 24-32 h. In the second experiment protein synthesis patterns of oocytes were studied. Oocytes cultured in conditioned medium were characterized by a 45-kD protein band, while those maturing in control medium were identifiable by a marked 56-kD band. In the third experiment mature oocytes were fertilized in vitro. The percentage of penetrated egg was higher in oocytes matured in conditioned medium than in control medium. In addition, only oocytes matured in conditioned medium could consistently decondense spermatozoa and form male pronuclei. Metabolic cooperation, protein synthesis patterns, and fertilizability were also studied in oocytes matured in control medium supplemented with either 17 beta-estradiol or progesterone or testosterone or dihydrotestosterone or androstenedione or ether extract of conditioned medium. Only ether extract and progesterone stimulated cumulus oocyte interaction and sperm decondensation. In the last experiment oocytes denuded at different stage of their maturation in conditioned medium were fertilized in vitro. The longer the eggs were cultured with the cumulus, the higher was their penetrability. Moreover, only oocytes denuded after 40 h of culture could, once fertilized, promote the formation of male pronuclei. These data demonstrate that follicular secretions are fundamental for the maintenance in vitro of a functional intercellular coupling between cumulus and oocyte, which is necessary for the egg to become penetrable by spermatozoa and to acquire the conditions required for the formation of male pronuclei.

Animals↗
PubMed1989

Parthenogenetic activation of unfertilized mouse oocytes by exposure to 1,2-propanediol is influenced by temperature, oocyte age, and cumulus removal.

Cumulus-intact and -denuded unfertilized oocytes from two mouse strains were exposed to 1.5 M ethanol (EtOH) or two cryoprotectant solutions, 1.5 M propanediol (PROH) or 1.5 M dimethylsulfoxide (DMSO), for 4.5 min at 27 degrees C, and the proportion of activating or degenerating oocytes studied. Exposure to DMSO did not significantly increase activation above that of oocytes not exposed to DMSO. Treatment of oocytes in PROH resulted in the activation of up to 87% of viable oocytes. This was significantly higher (P less than .01) than in control oocytes and comparable to the rate of activation after treatment with EtOH (59-96% activation). In solutions at 1 degree C, 47% of control oocytes were activated, which was not significantly different from the rate of activation in EtOH (36%) or PROH (50%) at 1 degree C. Following treatment with PROH, up to 87% of oocytes degenerated within a period of 6 h in vitro. The age of the oocytes (h post hCG) and the time of cumulus removal with the enzyme hyaluronidase, relative to the time of exposure to the chemicals, influenced the level of degeneration in most groups. Significantly fewer oocytes degenerated when cumulus cells were removed before treatment (0-31%) than when the cumulus was left intact throughout the treatment and 6 h culture period (10-87%). Exposure to PROH at 1 degree C reduced oocyte degeneration to 5%. We conclude that PROH causes significantly greater losses of oocytes as a result of parthenogenetic activation and degeneration than of exposure to DMSO.

Animals↗
PubMed2001

Mouse antral oocytes regulate preantral granulosa cell ability to stimulate oocyte growth in vitro.

In this study we evaluated whether mouse oocytes derived from early antral or preovulatory follicles could affect the ability of preantral granulosa cells to sustain oocyte growth in vitro. We found that early antral oocytes with a diameter > or =75 microm did not grow any further during 3 days of culture on preantral granulosa cell monolayers in vitro, while most of the oocytes with a smaller diameter increased significantly in size. Similarly, about 65% of growing oocytes isolated from preantral follicles grew when cultured on preantral granulosa cells. By coculturing with growing oocytes fully grown early antral or preovulatory oocytes, a small proportion (about 10%) of growing oocytes increased in diameter, and changes in granulosa cell morphology were observed. Such effects occurred as a function of the fully grown oocyte number seeded and were not associated with a decrease in coupling index values. By avoiding physical contact between antral oocytes and granulosa cells, the proportion of growing oocytes undergoing a significant increase in diameter was about 36%. These results indicate that fully grown mouse oocytes can control preantral granulosa cell growth-promoting activity through the production of a soluble factor(s) and the maintenance of functional communications with surrounding granulosa cells.

Animals↗
PubMed2002

Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation.

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-alpha-tubulin monoclonal antibodies, polymorphic alpha-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of alpha-tubulin with relatively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic alpha-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic alpha-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic alpha-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic alpha-tubulin isoform. The acidic alpha-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog.

Animals↗
PubMed2006

Effect of oocyte diameter on meiotic competence, embryo development, p34 (cdc2) expression and MPF activity in prepubertal goat oocytes.

The aim of this study was to analyze the relationship between oocyte diameter, meiotic and embryo developmental competence and the expression of the catalytic subunit of MPF, the p34(cdc2), at mRNA, RNA and protein level, as well as its kinase activity, in prepubertal (1-2 months old) goat oocytes. MPF is the main meiotic regulator and a possible regulator of cytoplasmic maturation; therefore, it could be a key factor in understanding the differences between competent and incompetent oocytes. Oocytes were classified according to oocyte diameter in four categories: <110, 110-125, 125-135 and >135 microm and matured, fertilized and cultured in vitro. The p34(cdc2) was analyzed in oocytes at the time of collection (0 h) and after 27 h of IVM (27 h) in each of the oocyte diameter categories. The oocyte diameter was positively related to the percentage of oocytes at MII after IVM (0, 20.7, 58 and 78%, respectively) and the percentage of blastocysts obtained at 8 days postinsemination (0, 0, 1.95 and 12.5%, respectively). The expression of RNA and mRNA p34(cdc2) did not vary between oocyte diameters at 0 and 27h. Protein expression of p34(cdc2) increased in each oocyte category after 27 h of maturation. MPF activity among diameter groups did not vary at 0h but after IVM there was a clear and statistically significant increase of MPF activity in the biggest oocytes.

Animals↗
PubMed2005

Oocyte-specific expression of Gpr3 is required for the maintenance of meiotic arrest in mouse oocytes.

The maintenance of meiotic prophase arrest in mouse oocytes within fully grown follicles, prior to the surge of luteinizing hormone (LH) that triggers meiotic resumption, depends on a high level of cAMP within the oocyte. cAMP is produced within the oocyte, at least in large part, by the G(s)-linked G-protein-coupled receptor, GPR3. Gpr3 is localized in the mouse oocyte but is also present throughout the follicle. To investigate whether Gpr3 in the follicle cells contributes to the maintenance of meiotic arrest, RNA interference (RNAi) was used to reduce the amount of Gpr3 RNA within follicle-enclosed oocytes. Follicle-enclosed oocytes injected with small interfering double-stranded RNA (siRNA) targeting Gpr3, but not control siRNAs, stimulated the resumption of meiosis in the majority of oocytes following a 3-day culture period. Reduction of RNA was specific for Gpr3 because an unrelated gene was not reduced by microinjection of siRNA. Meiotic resumption was stimulated in isolated oocytes injected with the same siRNA and cultured for 1 to 2 days, but at a much lower rate than in follicle-enclosed oocytes that could be cultured for longer. These results demonstrate that GPR3 specifically in the oocyte, rather than in the follicle cells, is responsible for maintenance of meiotic arrest in mouse oocytes. Furthermore, the method developed here for specifically reducing RNA in follicle-enclosed oocytes, which can be cultured for a sufficient time to reduce the level of endogenous protein, should be generally useful for targeting a wide range of other proteins that may be involved in meiotic arrest, the resumption of meiosis, fertilization, or early embryonic development.

Animals↗
PubMed1986

Cryopreservation of hamster oocytes: effects of vitrification or freezing on human sperm penetration of zona-free hamster oocytes.

Three experiments were conducted for evaluation of the efficacy of conventional freezing or vitrification of hamster oocytes for use in a human sperm penetration assay (hSPA). In experiment 1, oocytes were cryopreserved and evaluated for survival on the basis of morphologic criteria. Survival of vitrified oocytes and that of frozen oocytes were not different, whereas all cryopreserved groups had lower survival than noncryopreserved controls. In experiment 2, oocytes were conventionally frozen or vitrified and evaluated in an hSPA. Vitrified oocytes had a lower frequency of sperm penetration than frozen oocytes, and all cryopreserved groups had lower penetration rates than untreated controls. In experiment 3, oocytes were exposed to the cryoprotectant used to vitrify (VS1) or freeze (DMSO) but not cooled prior to evaluation in an hSPA. Exposure to DMSO but not VS1 reduced hSPA values. It is concluded from these experiments that while all cryopreserved oocytes do not survive, at current stages of development conventionally frozen oocytes perform better than vitrified oocytes in the hSPA and losses associated with conventional freezing procedures may be related to cryoprotectant exposure, whereas vitrification losses are more probably due to events associated with rapid cooling and/or warming of the oocytes.

Animals↗
PubMed2001

Effects of maturational stage, cumulus cells and coincubation of mature and immature cumulus-oocyte complexes on in vitro penetrability of porcine oocytes.

The in vitro penetrability of porcine oocytes is conditioned by several factors, some of which remain unclear. Knowledge of the different effects of the cellular components involved in penetrability would no doubt serve to simplify laboratory IVF methods. This study was designed to evaluate the effects of the following factors on penetrability: oocyte maturational stage, the presence of isolated or oocyte-attached cumulus cells, and coincubation of in vitro-matured and immature oocytes. Immature oocytes and oocytes matured in Waymouth medium were obtained from non atretic follicles and fertilized in TCM 199 medium. Sperm-rich fractions were collected by the gloved hand method and semen was used for IVF at a final concentration of 1 x 10(6) cells/mL in all experiments. Under the same conditions of IVF, the penetrability of the immature cumulus-oocyte complexes (COCs) was significantly lower than that of mature COCs, in terms of penetration rate and mean number of sperm per penetrated oocyte. This difference was abolished when the oocytes were denuded, leading to similar penetration rates. Coincubation of mature and immature COCs reduced the penetrability of immature COCs compared with that observed when these were incubated in isolation. However, neither the addition of isolated cumulus cells from decumulated mature oocytes nor the addition of denuded mature oocytes to immature COCs modified the penetration rate. These findings suggest that the presence of surrounding cumulus cells is mainly responsible for the differences observed in penetrability, regardless of the maturational stage of the oocyte. Moreover, when mature and immature COCs are coincubated, penetrability of immature COCs is diminished by the effects of the mature COC and not by the independent actions of the cellular components.

Animals↗
PubMed1999

Reconstruction of mouse oocytes by germinal vesicle transfer: maturity of host oocyte cytoplasm determines meiosis.

We evaluated the maturational competence of mouse oocytes reconstructed by the transfer and electrofusion of germinal vesicles (GV) into anuclear cytoplasts of GV stage oocytes (both auto- and hetero-transfers), metaphase II stage oocytes or zygotes. Following in-vitro culture, the maturation rates of the reconstructed oocytes to metaphase II did not significantly differ between auto- and hetero-transfers (40/70 versus 95/144 respectively); these rates also did not differ from those of control oocytes (57/97) which were matured in vitro without micromanipulation and electrofusion. In contrast, when a GV was transferred into an enucleated metaphase II oocyte or a zygote, only a few reconstructed oocytes underwent germinal vesicle breakdown (5/30 and 2/21 respectively); moreover, none reached metaphase II stage. Cytogenetic and immunofluorescence analyses were conducted on hetero-GV oocytes that extruded a first polar body. Each oocyte showed two groups of chromosomes, one in the cytoplast and one in the polar body, as well as a bipolar spindle with twenty univalent chromosomes. Our findings suggest that oocytes reconstructed by GV transfer into a cytoplast of the same developmental stage mature normally in vitro through metaphase II. Such oocytes may be a useful research model to elucidate the cytoplasmic and nuclear mechanisms regulating meiosis and the relationships between meiotic errors and age-related changes in the oocyte.

1-Methyl-3-isobutylxanthine↗
PubMed2005

Treatment option for sperm- or oocyte-related fertilization failure: assisted oocyte activation following diagnostic heterologous ICSI.

BACKGROUND: Failed fertilization occurs in 2-3% of ICSI cycles and is mainly due to lack of oocyte activation. Heterologous ICSI of patient's sperm in mouse oocytes allows discrimination between sperm- and oocyte-related aetiologies of activation failure. Assisted oocyte activation (AOA) by Ca-ionophore treatment can initiate fertilization in subsequent therapeutic ICSI. We report on diagnosis and clinical treatment in 17 patients with previously failed fertilization. METHODS: Sperm from patients were injected into mature mouse oocytes. Activation capacity was assessed by 2-cell formation (mouse oocyte activation test, MOAT). When no activation occurred, it was assumed that the spermatozoon was deficient; otherwise an oocyte-related factor was suspected. In a subsequent ICSI cycle, AOA was done by ICSI with CaCl2 followed by a Ca2+ ionophore exposure. Fertilization was checked 16-20 h later. Embryo transfer was on day 2 or 3. RESULTS: MOAT showed sperm-related activation deficiency in six globozoospermic patients and two patients with extreme oligoasthenoteratozoospermia. One patient with small sperm acrosomes had a normal activation percentage. In eight other patients, the MOAT revealed a relatively normal activation capacity of the sperm, indicating an oocyte-related defect. After AOA, fertilization rates were 77 and 71% in the sperm- and oocyte-related groups respectively. Five pregnancies were achieved in the globozoospermia group and three in cases of oocyte-related activation failure. CONCLUSIONS: Assisted oocyte activation enables normal fertilization and pregnancy in sperm- and oocyte-related fertilization failure.

Acrosome↗
PubMed1991

Assisted fertilization of mouse oocytes and preliminary results for human oocytes using zona drilling.

The zona-drilling procedure was investigated in mouse oocytes prior to a study on human oocytes. The procedure involved the injection of 5-nl volumes of acidic Hepes-buffered medium at pH 2.5 using a microinjection instrument. Zona-drilled mouse oocytes had significantly higher rates of fertilization (60/99; 61%) than zona-intact oocytes (6/103; 6%) at an insemination concentration of 1 x 10(4) sperm/ml (P less than 0.001). The procedure did not induce parthenogenetic activation of oocytes and more than 97% of zygotes developed to the blastocyst stage. A similar rate of live progeny was observed when zona-drilled (38.0%) and control embryos (38.5%) were transferred to pseudopregnant recipients. Chromosome analyses were performed on zona-intact, zona-free, and zona-drilled oocytes inseminated with varying concentrations of sperm and analysed at the first cleavage division. Zona-free oocytes had high rates of polyploidy (greater than or equal to 40%) with varying insemination numbers but the zona-drilled oocytes did not reveal significant increases in the rate of polyploidy or aneuploidy when compared to controls. In the human studies, zona-drilled oocytes achieved higher rates of fertilization than zona-intact oocytes, with sperm numbers as low as 1 x 10(4)/ml (6/8; 75%). Polyspermic fertilization was observed in 1/2 and 2/6 of fertilized oocytes inseminated with 1 x 10(5) and 1 x 10(4) sperm/ml, respectively. With the low sperm concentration 2/4 of those which were normally fertilized developed to healthy blastocysts. These studies suggest that the zona-drilling technique as described can be performed without apparent harm to oocytes and generate normal embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗