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Results for “In vitro maturation”

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PubMed2006

Improvement in bovine embryo production in vitro by treatment with green tea polyphenols during in vitro maturation of oocytes.

The present study examined the effect of green tea polyphenols (GTP) during in vitro maturation (IVM) of bovine oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration and subsequent embryo development. Cumulus-oocyte complexes were aspirated from the ovaries derived from slaughterhouse and cultured in modified synthetic oviduct fluid (m-SOF) supplemented with 0-25 microM GTP for 24h. After IVM, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 15-18 h. Putative embryos were transferred to m-SOF and cultured for 8 days (Experiment 1). In comparison with the absence of GTP, treatment with GTP at a concentration of 15 microM showed a significant increase in the proportion of pronuclear (PN) formation after sperm penetration (65% versus 80%, P<0.05). No significant differences in the rates of sperm penetration and polyspermic fertilization were found among treatments. The cleavage rate at 48 h of in vitro insemination showed no difference in oocytes matured with or without GTP. However, compared to no addition (23.5%), the presence of 15 and 20 microM GTP during IVM significantly (P<0.05) increased the proportion of blastocysts (38.1% and 36.4%) on day 9 of in vitro insemination. A further increase from 20 to 25 microM GTP reduced (P<0.05) the proportion of blastocysts. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P<0.05) level of GSH was found in oocytes matured with 15 microM GTP and compared with 15 microM GTP, GSH was low (P<0.05) at 20 and 25 microM GTP. The results suggest that at certain concentrations of GTP (15 microM) in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in bovine oocytes.

Animals↗

PubMed1981

Thermal stability of reconstituted collagen fibrils. Shrinkage characteristics upon in vitro maturation.

Thermal stability measured as area shrinkage without tension during heating was determined for membranes of collagen fibrils reconstituted from solutions of highly purified rat skin collagens. Shrinkage in per cent of area at 25 degrees C and shrinkage temperature were quantitated in a standardized way and determined as a function of in vitro maturation time for 11 to 104 days after aggregation for the collagen membranes. Similar to reports on intact rat skin, shrinkage temperature remained constant and shrinkage per cent declined with a rate decreasing with time during maturation. Solubility in water at 80 degrees C for 2 hours was 95-96% and remained unchanged for the maturation time (about 2 months) studied. The decreased shrinkage reflecting a lower degree of collapse is ascribed to an increasing thermal stability of the membranes during maturation. Development of heat-stable bonds in the reconstituted collagen fibrils is taken up to be amenable to this increased stability. Similarity in changes of shrinkage characteristics during in vivo and in vitro maturation indicates that maturation changes in reconstituted collagen fibrils reflect those occurring in intact collagen during in vivo aging.

Aging↗

PubMed2005

Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei.

The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (65%) were electrofused, activated and cultured, out of which 4 (10%) developed to 2-cell stage, 3 (7.50%) to 4-cell stage, 2 (5.0%) to early morula stage and 1 (2.50%) to blastocysts stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro matured buffalo oocytes.

Animals↗

PubMed2006

In vitro maturation of human primordial ovarian follicles: clinical significance, progress in mammals, and methods for growth evaluation.

As cancer treatment improves, more young women of reproductive age are surviving, but they suffer from infertility as a consequence of the radiation and chemotherapy. Human ovarian tissue containing immature primordial follicles has been successfully cryopreserved. The ultimate aim of this technique is to induce ovarian function by re-plantation of ovarian tissue or, further into the future, by in vitro maturation (IVM) of the oocytes derived from the cryopreserved-thawed ovarian tissue, followed by routine in vitro fertilization. IVM of primordial follicles from young cancer survivors would avoid the risk of cancer re-transmission by the ovarian grafts. The present review discusses the current achievements in IVM of female germ cells and primordial ovarian follicles and the attempts to improve their development by adding various factors to the culture medium. The established methods for the evaluation of survival and growth in culture are also discussed: follicular counts, immunocytochemical methods, transmission electron microscopy, fluorescent viability markers and endocrine assays. Although the development of IVM systems is still in its infancy, researchers need to pursue their approach step-by-step, especially with regard to factors that might be involved in the activation of the ovarian follicles or female germ cells. The final measure of success will be the ability of the in vitro matured oocytes to fertilize and produce healthy offsprings. The availability of such treatment will probably lead to its demand not only by cancer patients but by other women as well.

Animals↗

PubMed2005

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development.

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.

Animals↗

PubMed2006

Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes.

We investigated nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to cytochalasin B (CB) during in vitro maturation (IVM). Nuclear progression was similar in control oocytes and oocytes matured in the presence of 1 microg/ml CB (IVM-CB group) by 37 h IVM; at this time the proportion of oocytes that had reached or passed through the anaphase-I stage did not differ significantly between the IVM-CB and the control groups (61.3 and 69.9% respectively; P < 0.05). After IVM for 37 h, no polar body extrusion was observed in the IVM-CB group. In these oocytes, the two lumps of homologous chromosomes remained in the ooplasm after their segregation and turned into two irregular sets of condensed chromosomes. By 41 h IVM, the double sets of chromosomes had reunited in 89.5% IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached the metaphase-II stage by this time. When IVM-CB oocytes cultured for 46 h were stimulated with an electrical pulse and subsequently cultured for 8 h without CB, 39.0% of them extruded a polar body and 82.9% of them had a female pronucleus. Chromosome analysis revealed that the majority of oocytes that extruded a polar body were diploid in both the control and the IVM-CB groups. However, the incidence of polyploidy in the IVM-CB group was higher than that in the control group (P < 0.05). In vitro development of diploid parthenotes in the control and the IVM-CB groups was similar in terms of blastocyst formation rates (45.8 and 42.8% respectively), number of blastomeres (39.9 and 44.4 respectively), the percentage of dead cells (4.3 and 2.9% respectively), and the frequency of apoptotic cells (7.3 and 6.3% respectively). Tetraploid embryos had a lower blastocyst formation rate (25.5%) and number of cells (26.2); however, the proportion of apoptotic nuclei (7.0%) was similar to that in diploid parthenotes. These results suggest that the proportion of homozygous and heterozygous genes does not affect in vitro embryo development to the blastocyst stage.

Animals↗

PubMed1984

Immunofluorescence distribution of alpha tubulin, beta tubulin and microtubule-associated protein 2 during in vitro maturation of cerebellar granule cell neurones.

In vivo developmental immunocytochemical studies have recently shown that granule cell axons (parallel fibres) undergo progressive modification of alpha tubulin as they form permanent synapses, a change that is not observed in other microtubule-containing structures (e.g. Purkinje cell dendrites). In this study we have used primary cultures of young rat cerebellum enriched in granule cells to follow maturation of their axons under defined in vitro conditions. Our results show that alpha tubulin is not modified in culture as assessed by immunofluorescence using monoclonal antibodies that recognise different forms of alpha tubulin. We have also confirmed in vivo data showing that microtubule-associated protein 2 is absent from granule cell axons at all developmental stages in vitro. The distribution of axonal cytoskeletal proteins is compared and contrasted during in vivo and in vitro maturation, and considered in relation to differences between the two systems.

Animals↗

PubMed1993

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera.

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.

Journal Article↗

PubMed1997

Equine oocyte competence for nuclear and cytoplasmic in vitro maturation: effect of follicle size and hormonal environment.

Equine oocyte competence after in vitro maturation (IVM) was investigated in terms of the diameter of the follicle of origin and the stage of the estrous cycle, with three criteria of maturation: nuclear stage after DNA Hoechst staining, meiotic spindle morphology after tubulin immunocytochemical staining, and cortical granule localization after lectin labeling. Seven successive in vivo ultrasound-guided follicular punctures were performed on 10 cyclic saddle mares, alternatively at the end of the follicular phase (after induction of ovulation with a gonadotropin injection) and in midluteal phase (with or without a gonadotropin injection). Expanded cumulus-oocyte complexes (COCs) were stained at collection, and compact COCs were stained after in vitro culture. They were observed under a confocal microscope. Successive punctures on one mare provided 0.9 preovulatory COCs and 8 immature COCs per 22 days. Among the preovulatory oocytes, 55% had completed nuclear and cytoplasmic maturation, 86% of which displayed a normal meiotic spindle. Of the 262 oocytes cultured in vitro, 37% completed nuclear maturation. The nuclear and cytoplasmic maturation rate significantly increased with follicle diameter. The IVM rate tended to be higher in follicular phase and tended to increase in luteal phase with the gonadotropin injection. The meiotic spindle morphology was not significantly different between the classes of follicular diameters. This study provided the opportunity to increase the number of characterized oocytes collected per cycle and per mare. This is the first report showing the progressive acquisition of meiotic competence in the equine oocyte during antral follicle growth and is the only description of the equine meiotic spindle.

Animals↗

PubMed1994

Delivery of a live calf after in vitro maturation and fertilization of oocytes obtained from ovaries removed from a moribund cow.

An aged Holstein cow of high genetic merit and economic value was determined to have a squamous cell carcinoma of the tarsus. Treatment was unsuccessful, as was an attempt at superovulation and embryo collection; thus, the owner elected euthanasia. On day 17 of the estrous cycle, the ovaries were removed via laparotomy and the cow was immediately euthanatized. Oocytes were aspirated from ovarian follicles and subjected to in vitro maturation and fertilization procedures. Seven morulae that developed in culture were transferred nonsurgically to 3 recipient females, resulting in birth of a full-term healthy male calf. In vitro maturation and fertilization procedures can be used to obtain offspring from valuable, terminally ill cows for which conventional embryo collection techniques have proven unsuccessful.

Animals↗

PubMedDate not supplied

Exposure of pig oocytes to PCBs during in vitro maturation: effects on developmental competence, cytoplasmic remodelling and communications with cumulus cells.

Polychlorinated biphenyls (PCBs) are one of the most persistent and widespread groups of endocrine disrupting compounds in the ecosystem. These substances are present in sewage sludge that is spread in increasing amounts on arable land and pasture as fertilizer, and are ingested by farm animals with food and drinking water. This study investigated the effect of different PCB concentrations on pig oocyte in vitro maturation and developmental competence as well as examined the possible mechanisms involved. A concentration ranging from 0 to 1 mg/mL of Aroclor 1254 (A1254), a pool of more than 60 PCB congeners, was added to the maturation medium, as its composition is considered environmentally relevant. A1254 had no effect on maturation of pig oocytes and on the number of oocytes that cleaved following parthenogenetic activation at any of the doses tested. By contrast, a significant decrease in the number of zygotes that developed to blastocyst stage became evident at a concentration of 10 ng/mL. The number of blastocysts obtained decreased significantly, and in a dose response manner with higher concentrations. Exposure to PCBs altered mitochondria relocation during maturation and this was associated with the lack of a cytoplasmic microtubule network. No effect on mitochondria activity was observed. A1254 exposure also perturbed gap-junction mediated communications between oocytes and cumulus cells. In conclusion, PCB exposure of pig oocytes during in vitro maturation significantly decreased oocyte developmental competence, altered both their cytoplasmic remodelling and the communication with the somatic compartment. These data indicated that accumulation of PCBs in the pig organism may have a detrimental effect on the reproductive efficiency in this species.

Animals↗

PubMed1990

Characterization of the in vitro maturation of monocytes and the susceptibility to HIV infection.

The in vitro maturation of peripheral blood monocytes to macrophages can be followed morphologically, and by measurement of cell surface antigens (CD4, HLA-DR, and FcR III) and lysozyme production. We used these markers to correlate monocyte maturation with susceptibility to human immunodeficiency virus (HIV) infection. Maturation of peripheral blood monocytes is associated with a decrease in membrane CD4, while HLA-DR and FcR III expression increase along with lysozyme secretion. Cells at all stages of maturation were susceptible to HIV infection, even mature macrophages without CD4 detectably by immunofluorescent staining. Maximal replication was observed in 7-day-old cells.

Antibodies, Monoclonal↗

PubMed2004

Successful pregnancy and delivery following combined treatment of in vitro maturation (IVM) and testicular sperm extraction (TESE): a case report.

PURPOSE: To report the efficacy of the combined treatment of in vitro maturation (IVM) and testicular sperm extraction (TESE). METHODS: A couple in which the wife had polycystic ovarian syndrome and the husband had severe oligozoospermia. Oocytes were cultured in vitro for maturation followed by oocytes pickup with natural cycle, and TESE was undergone for husband. Matured oocytes were fertilized by intracytoplasmic sperm injection, and two embryos were transferred to wife's uterine. RESULTS: This case was achieved during pregnancy and delivery of a healthy female infant. CONCLUSIONS: The combined treatment of IVM and TESE was effective for this couple's specific infertility factors.

Adult↗

PubMed1998

Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes.

To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium.

Animals↗

PubMed1999

Changes in poly(A) tail length of maternal transcripts during in vitro maturation of bovine oocytes and their relation with developmental competence.

Molecules of mRNA are stored in the oocyte cytoplasm in order to be used during the initial phases of embryonic development. The storage takes place during oocyte growth and the extent of poly(A) tail at the 3' end of the transcripts has emerged as an important regulatory element for determining their stability. The objective of the present study was to analyse changes in polyadenylation levels of mRNA transcripts, stored in bovine oocytes, during in vitro maturation and their possible relation with developmental competence. Oocyte developmental competence was predicted on the basis of the morphological appearance of their originating ovary as previously established (Gandolfi et al. 1997a. Theriogenology 48:1153-1160) and were divided into groups H (high competence) and L (low competence). The length of the poly(A) tail of the following genes, beta-actin (beta-Act), connexin 43, glucose transporter type 1, heat shock protein 70, oct-4, plakophilin, pyruvate dehydrogenase phosphatase (PDP), and RNA poly(A) polymerase, was determined at the germinal vesicle (GV) and metaphase II (MII) stage. The results indicated that the poly(A) tail of all genes except for beta-Act and PDP, is shorter after in vitro maturation (IVM) in both groups. Moreover, group L oocytes showed a shorter poly(A) tail than group H oocytes in all genes except for beta-Act and PDP, both at GV and MII stage. We conclude that most of the examined transcripts follow the default deadenylation pattern described during oocyte maturation in other species and that a shorter poly(A) tail is correlated with low developmental competence.

Animals↗

PubMed1992

Development of bovine in vitro-matured follicular oocytes activated with ethanol.

Bovine follicular oocytes cultured in vitro for 30 hours were activated by ethanol and then the developmental ability of the oocytes was assessed. When the activated oocytes were treated with cytochalasin B at concentrations of 0, 1.25, 2.5, 5 and 7.5 mug/ml, the percentages of oocytes having two pronuclei were 8, 18, 54, 66 and 60%, respectively. The percentage of the activated oocytes with two pronuclei also increased with increased exposure time to cytochalasin B. When the activated oocytes were treated with cytochalasin B (5mug/ml) for 10 hours, 20% of them cleaved after 36 hours and 5% of them developed to the eight-cell stage after 60 hours of culture. These results indicated that bovine in vitro matured oocytes can be activated by ethanol and develop to the eight-cell stage following treatment with cytochalasin B.

Journal Article↗

PubMed2001

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

Animals↗

PubMed2005

Spindle positions and their distributions in in vivo and in vitro matured mouse oocytes.

BACKGROUND: This study was carried out to compare spindle locations and their developmental competencies both in vivo and in vitro in matured mouse oocytes. Spindle locations were identified using a polscope. Since meiotic spindles in living oocytes are highly birefringent, their structures can be viewed non-invasively by using a polscope. METHODS: In vivo matured metaphase II oocytes were collected from the oviducts of mice. Immature oocytes were collected from mouse ovaries, and then cultured in YS medium until the first polar body (PB) extrusion. In vitro and in vivo matured oocytes were classified into four categories according to their spindle positions relative to the first PB (0 degrees , 0-90 degrees , 90-180 degrees and without a spindle image), and rates of fertilization and blastocyst formation were assessed. In vivo matured oocytes with a 0 degrees spindle disposition relative to PB were cultured in vitro for 24 h, and then their spindle positions were re-assessed. RESULTS: Most in vivo matured oocytes (89.1%) had a 0 degrees spindle position. Only 6 and 3% of oocytes had spindle positions of 0-90 degrees and 90-180 degrees , respectively. No spindle image was observed in 2%. However, most in vitro matured oocytes (83.1%) had a 0-90 degrees spindle position and, in contrast, only 6.5% of these oocytes had a 0 degrees spindle position. The rate of fertilization and blastocyst rate were significantly higher for in vivo matured oocytes than in vitro matured oocytes (87.1 versus 64.9% and 76.1% versus 66.0%, respectively, P<0.05 for each). We also observed that 71.7% of the in vivo matured oocytes with the 0 degrees spindle position showed a spindle position change to 0-90 degrees after 24 h of culture. These oocytes had a poor fertilization rate (43%) and a zero blastocyst rate. CONCLUSION: In vitro matured mouse oocytes showed quite different spindle positions compared with in vivo matured oocytes. Moreover, in vivo matured oocytes cultured for 24 h had a spindle position distribution that was similar to that of in vitro matured oocytes. The different spindle positions observed in in vivo and in vitro matured oocytes may reflect differences in their cytoplasmic maturation processes. These findings have implications regarding the lower developmental competency of in vitro matured oocytes.

Animals↗