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PubMed2006

Abilities of cumulus and granulosa cells to enhance the developmental competence of bovine oocytes during in vitro maturation period are promoted by midkine; a possible implication of its apoptosis suppressing effects.

We previously reported that when midkine (MK), a heparin-binding growth differentiation factor was used in in vitro maturation (IVM) culture of bovine cumulus-enclosed oocytes (CEOs), their developmental competence to the blastocyst stage after in vitro fertilization (IVF) was enhanced and the effect of MK might be mediated by its action upon mural granulosa cells and cumulus cells that closely surround the oocyte. In the present study, when denuded oocytes (DOs) were matured in IVM medium with or without MK (200 ng/ml) in the presence or absence of isolated cumulus cell masses and subjected to IVF, the enhancing effects of MK on the developmental competence of DOs to the blastocyst stage after IVF were exerted only in the presence of cumulus cells. In addition, we prepared the conditioned media of granulosa cells cultured with or without 200 ng MK/ml (CMMK+ or CMMK- respectively) and examined their effects on the IVM of DOs in terms of their developmental competence to the blastocyst stage after IVF. The supplementation of CMMK+ into IVM medium at 40% (v/v) significantly enhanced the blastocyst development compared with the no additive control and the CMMK- supplemented groups. Furthermore, the effects of MK during IVM of bovine CEOs on the cumulus cell apoptosis were investigated. CEOs were cultured up to 24 h in IVM medium without (control) or with 200 ng MK/ml. The genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect the apoptotic internucleosomal DNA fragmentation. DNA fragmentation was scarcely detected at the start of IVM, whereas it increased time-dependently as the IVM culture progressed. The degree of the fragmentation was significantly lower in the MK-treatment group compared with the control group at 18 and 24 h of IVM. The apoptosis-suppressing effect of MK on cumulus cells was further confirmed in situ by using TUNEL on CEOs. In conclusion, data from the present study further confirmed that MK enhances the developmental competence of bovine oocytes via cumulus and granulosa cells. It was also demonstrated that MK suppresses the apoptosis that occurs in cumulus cells during the period of IVM of bovine CEOs. The putative soluble factor(s) from cumulus cells was suggested from the experiment using CMMK+ . MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.

Animals↗
PubMed2006

Apoptosis of cultured granulosa-lutein cells is reduced by insulin-like growth factor I and may correlate with embryo fragmentation and pregnancy rate.

OBJECTIVE: To correlate apoptosis of cultured human granulosa-lutein cells (GL cells) with the outcome of IVF (embryo fragmentation and pregnancy rate) and to study the effect of insulin and insulin-like growth factor I (IGF-I) on apoptosis. DESIGN: In vitro assays. SETTING: University laboratory and private IVF center. PATIENT(S): Eighty-one women undergoing IVF. INTERVENTION(S): Purified human GL cells from pooled follicles were cultured for 48 hours in serum-free media with or without insulin and IGF-I. Cumulus cells and mural GL cells were evaluated separately. MAIN OUTCOME MEASURE(S): Detection of apoptosis by using caspACE FITC-VAD-FMK, a fluorescent in situ marker for activated caspases; embryo fragmentation; and pregnancy. RESULT(S): Age younger than 38 years and successful pregnancy were associated with less apoptosis (33.0% +/- 17.2% vs. 43.2% +/- 18.0% and 30.2% +/- 14.0% vs. 40.4% +/- 19.5%, respectively). There was a linear correlation between embryo fragmentation and GL cell apoptosis. Insulin-like growth factor I decreased apoptosis in a dose-dependent fashion. A statistically significant effect (17% decrease) was reached at a dose of 10 nM. Insulin (10 nM) caused a small (8%) decrease in apoptosis, but this effect did not reach statistical significance. Cumulus cells consistently had <3% apoptosis. CONCLUSION(S): [1] Apoptosis of cultured GL cells may be associated with IVF outcome and ovarian reserve and [2] IGF-I decreases apoptosis of cultured GL cells.

Adult↗
PubMed2006

Differential gene expression of bone morphogenetic protein 15 and growth differentiation factor 9 during in vitro maturation of porcine oocytes and early embryos.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) belong to the TGF-beta superfamily and are involved in the regulation of folliculogenesis. Though there are many reports concerning the expression and regulation of GDF9 in the process of oocyte maturation, expression of BMP15 during oocyte maturation is still not clearly understood. It has been reported that BMP15 and GDF9 expression is important in folliculogeneiss and that the regulation of these two proteins is complex and species-specific. In this report, we investigated the expression of BMP15 and GDF9 genes during in vitro maturation (IVM) at 0, 6, 12, 18, 24, 30, 36, 42 and 48 h for porcine oocytes. Porcine GDF9 gene was found to be highly expressed in immature oocytes and declined slowly during the oocyte maturation process. BMP15mRNA and its encoded protein were expressed at low levels in immature oocytes and increased to the highest level at 18 h of IVM, which coincides with the time of cumulus cell expansion. Thus, these two genes were differentially expressed during the oocyte maturation process and BMP15 is specifically expressed during cumulus cell expansion in porcine oocytes.

Animals↗
PubMed1983

Metabolic coupling, cumulus expansion and meiotic resumption in mouse cumuli oophori cultured in vitro in the presence of FSH or dcAMP, or stimulated in vivo by hCG.

The temporal changes of metabolic coupling between the mouse oocyte and the cumulus cells which follow hCG injection in vivo and FSH treatment in vitro were studied by measuring what fraction of [3H]uridine taken up by cumulus cells was transferred to the oocyte. Meiotic resumption and a partial coupling loss (to 35% of the initial value) spontaneously occurred in cumuli cultured in control medium. The addition of 1 microgram FSH/ml in vitro, or the injection of hCG in vivo caused a delay of about 3 h in both phenomena and a near total uncoupling, together with cumulus expansion. FSH caused uncoupling even if cumulus expansion was prevented by the addition of heparin. The presence of 2 mM-dcAMP prevented meiotic resumption in cumulus-enclosed oocytes and maintained a high level of co-operation for at least 6 h. The slow uncoupling observed at later times was due to cumulus expansion, because it was totally prevented by heparin. We suggest that metabolic co-operation with the cumulus oophorus and meiotic resumption are both regulated by FSH through variations of intracellular levels of cAMP.

Animals↗
PubMed2000

Luteal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes.

In this prospective study we investigated whether the maturation and fertilization of immature oocytes can be improved by administration of recombinant follicle stimulating hormone (rFSH) starting in the late luteal phase in two groups of women: group 1 (n = 6) women with regular menstrual cycles; and group 2 (n = 6) women with irregular cycles and polycystic ovaries (PCO) on ultrasound examination. Low-dose (37.5 IU) rFSH was commenced 11 days after LH surge during a spontaneous menstrual cycle and on the ninth day of progesterone administration in an irregular cycle. Recombinant FSH was continued until the leading follicle was approximately 10 mm in diameter. The oocytes were retrieved after withdrawing rFSH for 2-5 days. In total, 136 oocytes were recovered (group 1, 67 oocytes; group 2, 69 oocytes). Nine of the oocytes from PCO women were atretic at retrieval. Oocytes complete with cumulus cells were cultured for 44 h in complex tissue culture medium supplemented with gonadotrophins and fetal calf serum. After maturation, the cumulus cells were removed and metaphase II oocytes were injected with spermatozoa. Respectively, the oocyte maturation and fertilization rates were 64 and 72% in group 1, and 78 and 57% in group 2 (not significant). After fertilization, the zygotes (group 1, n = 22; group 2, n = 11) and cleavage stage embryos (group 1, n = 9; group 2, n = 15) were frozen in propanediol. All women except one (11/12) had approximately five zygotes or cleaved embryos frozen. The viability of in-vitro matured frozen-thawed embryos was generally poorer than that (81%) seen after conventional intracytoplasmic sperm injection, with 61% survival in group 1 and 23% in group 2. Fifteen embryo transfers resulted in one miscarriage at 6 weeks gestation. The late luteal start of low-dose rFSH yielded a good number of immature oocytes in women with both regular and irregular cycles. Two out of three of these oocytes matured and fertilized. However, cryosurvival of the zygotes and cleaved embryos was unsatisfactory and thus cryopreservation of in-vitro matured embryos may not be an optimal procedure.

Adult↗
PubMed1995

Influence of growth factors on the time-dependent meiotic progression of the bovine oocytes during their in vitro maturation.

The present study examines the effects of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and the presence of cumulus cells on the meiotic progression of the bovine oocyte maturation. Oocytes were aspirated from 2-8 mm ovarian follicles, and the cumulus-oocyte complexes (COC) obtained (n = 2,729) were divided into COC or denuded oocytes. They were then cultured in four defined treatments comprising TCM-199 and growth-factors: control (no growth factor), IGF-I, EGF, and EGF+IGF-I. Each treatment group of oocytes was cultured during 6, 12, 18, 24, and 30 h; the oocytes were fixed and stained at the end of each 6 hour interval from 0 to 30-h, and were assessed for their nuclear maturation stage. Germinal vesicle (GV) and metaphase II (MII) stages present were used as the final parameters for data analysis. The results indicate that treatments with growth factors enhanced the incidence of GV activation and MII stage in all the culture periods. Maximal stimulation for nuclear maturation occurred with EGF+IGF-I. As regards the denuded oocytes, no positive effects on nuclear maturation rates were observed for any treatment. These results suggest that EGF and IGF-I, singly and combined, stimulate the meiotic progression significantly, and this effect can be induced by a positive signal which is transferred from cumulus cells to oocyte.

Animals↗
PubMed1991

Follicle somatic cells influence pig oocyte penetrability and cortical granule distribution.

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

Animals↗
PubMed2005

Effects of maturation conditions on spindle morphology in porcine MII oocytes.

Incomplete cytoplasmic maturation of in vitro matured (IVM) oocytes has been known to cause microtubule and microfilament alterations, which may result in abnormal pronuclear formation and failed embryonic development. We examined the influences of maturation conditions on meiotic spindle morphology at metaphase of meiosis II (MII) in porcine oocytes. Porcine oocytes were matured under various conditions, i.e., in vitro or in vivo, with different amounts of cumulus cells, with or without hormonal supplements, and with various exposure durations to the hormones, to examine the effects on spindle morphology in MII oocytes by immunofluorescence under confocal laser microscopy. Interpolar spindle length (microm) and spindle area (microm2) were compared among these maturation conditions. The spindle length was significantly shorter in IVM oocytes compared to those matured in vivo. Oocytes collected from cumulus oocyte complexes (COCs), which were poor in cumulus cells, showed smaller spindle areas than those from cumulus-rich COCs. The spindle length and area were both significantly reduced in oocytes grown without hormonal supplements. When oocytes were grown with hormonal supplements for either 6 or 22 hours for the first half of culture, there was no difference in the spindle morphology between these oocytes. These results suggested that maturation conditions significantly influence morphogenesis of MII spindles in porcine oocytes. Oocytes matured in poor conditions were more likely to have a shorter spindle length (long axis) and smaller spindle areas.

Animals↗
PubMed1999

Oocyte regulation of kit ligand expression in mouse ovarian follicles.

Kit ligand (KL), a product of granulosa cells in ovarian follicles, is a putative regulator of oocyte development. However, the factors that regulate KL mRNA levels in granulosa cells remain unclear. This study tested the hypothesis that oocytes regulate granulosa cell steady-state KL mRNA expression levels and that the characteristics of this regulation are dependent on the stage of growth and development of both oocytes and follicles. Levels of mRNA for the KL splice variants (KL-1 and KL-2) were shown to be high in granulosa cells from preantral follicles and then decline after follicular antrum formation. Preovulatory follicular development was associated with a dramatic increase in steady-state levels of KL-1 mRNA in mural granulosa but not cumulus cells. Regulation of these changes was examined in vitro using partly grown oocytes isolated from preantral follicles and fully grown oocytes isolated from preovulatory follicles. FSH increased the steady-state KL mRNA levels in preantral granulosa cells in vitro. Partly grown oocytes either increased or decreased KL mRNA levels in preantral granulosa cells depending on the absence or presence of FSH stimulation, respectively. Fully grown oocytes reduced the KL mRNA level in preantral granulosa cells and increased the ratio of KL-1 to KL-2 mRNA. In mural granulosa cell culture, FSH augmented testosterone-dependent elevation of the steady-state KL mRNA level, but had no effect alone. Fully grown oocytes reduced KL-2 but not KL-1 mRNA levels in mural granulosa cells treated with testosterone plus FSH, whereas fully grown oocytes reduced levels of both KL transcripts in cumulus cell culture. These effects of oocytes on steady-state KL mRNA expression levels in vitro explain the changes in granulosa cell KL mRNA levels observed during follicle development in vivo. The results therefore support the hypothesis that oocytes regulate granulosa cell kit ligand mRNA levels in a way that is characteristic of the stage of growth and development of the oocyte. Moreover, the results suggest that oocytes play a major role in promoting dynamic changes in gene expression by granulosa cells appropriate to the stage of follicular development.

Alternative Splicing↗
PubMed1988

Caffeine promotes in vitro fertilization of mouse ova within 15 minutes.

Epididymal sperm were collected from C57Bl6/J X DBA2/J (B6D2) males and allowed to capacitate for 2 hr. When cumulus-free oocytes were exposed to sperm for 15 min in either the presence (6.0 mM) or absence of caffeine, fertilization did not occur. However, when cumulus cells were left intact, 23% of oocytes were fertilized in caffeine-free medium and 62% in caffeine-containing medium. When cumulus-free oocytes were incubated with sperm for 30 min, none was fertilized in the absence of caffeine, but 33% were fertilized when 6.0 mM caffeine was present (P less than .02). These effects of caffeine were on the sperm, as sperm exposed to caffeine and then coincubated with oocytes for 15 min in essentially caffeine-free media fertilized a similar percent of oocytes (93%) as when sperm and oocytes were exposed to caffeine during the fertilization period (86%). When sperm were capacitated in caffeine-containing medium, the percentage of ova fertilized was similar to capacitation without caffeine. We conclude that both cumulus cells and caffeine speed up the fertilization process with mouse gametes and that the effect of caffeine is on the sperm, but not due to more rapid capacitation.

Animals↗
PubMed2003

Role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes.

The role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes was investigated in the present study. To determine the estradiol effect, oocytes were cultured for 42 h in a steroid free medium composed of mTCM-199 supplemented with LH, FSH and 10% charcoal extracted follicular fluid. Estradiol receptor (ER), detected by a binding assay, were present in cumulus cells and oocytes during maturation with higher levels observed at 24 h of culture in the oocytes and at 36 h in the cumulus cells. To block estradiol action an antiestrogen (1-p-dimethylaminoethoxyphenyl-1,2-diphenyl-1-butene (tamoxifen)) was added to the maturation medium at various concentrations. The percentage of treated oocytes that underwent nuclear maturation was similar (P>0.05) to the control group. Cytoplasmic maturation, determined by the ability to form female pronucleus (FPN) and male pronucleus (MPN), was not different (P>0.05) among all groups. The presence of 4-hydroxy-4-androstene-3-17-dione (4-OHA) also did not influence nuclear (P>0.05) or cytoplasmic maturation (P>0.05). The results suggest that estradiol is not involved in maturation of pig oocytes. However, the present experiment used pronuclei formation as the endpoint, no studies were done in regard to estradiol's effects on the embryonic development.

Aromatase Inhibitors↗
PubMed2003

Human oocytes and preimplantation embryos express mRNA for growth hormone receptor.

Human genetic expression of growth hormone receptor (GHR) gene was qualitatively analysed using reverse transcription polymerase chain reaction (RT-PCR) in cumulus cells, immature germinal vesicle (GV) and mature metaphase II (MII) stage oocytes and preimplantation human embryos. The transcripts encoding GHR were detected in cumulus cells and also in naked oocytes, either mature or not. In this case, a nested PCR is needed, as for early embryo preimplantation stages, before genomic activation. The GHR gene is highly expressed from the 4-day morula onwards. This suggests that GHR transcription follows a classical scheme associated with genomic activation. It is probable that, in human, growth hormone plays a role in the final stages of oocyte maturation and early embryogenesis as it does for several other mammalian species.

Blastocyst↗
PubMed2006

Mouse oocyte differentiation during antral follicle development.

During antral follicle development mouse oocytes undergo rearrangement of granulosa cell interactions and the oocytes released from follicles at the beginning or at the end of antral development are either devoid of denuded oocytes (DO) or strictly associated with cumulus-intact (CI) cumulus cells. In this study, these two oocyte classes were analyzed before germinal vesicle (GV) and after in vitro maturation (IVM) to evaluate (a) the ultrastructural aspect of oolemma microvilli by scanning electron microscopy analysis and (b) specific morphological markers of differentiation (chromatin organization, mitochondria, cortical granules, microfilaments, and spindle of metaphase II- MII-). At GV-stage, CI oocytes exhibited remarkable differences (a) in the oolemma microvillar ultrastructure and distribution with respect to DO and (b) in the chromatin organization that was typical of meiotically competent germ cells. By contrast, homogeneous patterns of distribution of mitochondria, cortical granules, and microfilaments characterized both the oocyte classes. At the end of culture, CI oocytes, even when matured without cumulus cells, reached more efficiently the MII stage and acquired an ultrastructural microvillous configuration different from DO. In addition, MII-arrested DO had a higher percentage of meiotic spindles with abnormal morphology in comparison with preovulatory oocytes, while cortical granule and microfilament patterns revealed no appreciable differences between the groups. With regard to mitochondria, a polarized distribution of these organelles was found in 82% of DO and in 97% of CI oocytes. These observations suggested that the achievement of the full antral follicle development is a condition for the acquisition of specific qualitative properties that are essential for the production of fertilizable oocytes, both in in vivo and in vitro models as well.

Animals↗
PubMed2002

Expression of the vascular endothelial growth factor and its receptors and effects of VEGF during in vitro maturation of bovine cumulus-oocyte complexes (COC).

A complete VEGF system consisting of the ligand and two of its receptors has been detected for the first time in the bovine cumulus-oocyte-complex (COC). In the course of a 24 hr in vitro maturation procedure (IVM), expression of the smaller VEGF transcripts and their specific receptors flt and flk changed remarkably in a time-dependent manner as observed by RT-PCR. The transcript concentrations of VEGF declined within 24 hr of culture, whereas both receptor mRNAs were found enriched between 6 and 12 hr of IVM. In the follicular fluid of growing ovarian follicles, immunoreactive VEGF, measured by RIA, increased significantly, reaching highest concentrations immediately before ovulation of the oocyte. The immunohistochemical localization of VEGF in bovine COCs revealed strong signals within the cumulus cell complex clearly extending beyond the oocyte cytoplasm at the beginning of in vitro maturation. After 24 hr, IVM immunoreactive VEGF disappeared remarkably from cumulus cells and the oocyte cytoplasm. An exogenous application of VEGF at the beginning of a 24 hr IVM significantly improved cleavage rates of zygotes and their development into bovine embryos showing obvious synergistic effects in combination with FSH, when compared with untreated control embryos. In addition, the number of blastomeres in deriving blastocysts increased after VEGF supplementation. These results indicate a functional VEGF system controlling important events beside the known angiogenetic effect during in vivo and in vitro maturation of the bovine COC, possibly affecting the early embryonic viability.

Animals↗
PubMed1992

Developmental capacity of mouse oocytes matured in vitro: effects of gonadotrophic stimulation, follicular origin and oocyte size.

Development of mammalian oocytes is usually correlated with ovarian follicular development. This correlation was tested by determining whether gonadotrophic stimulation of follicular development in immature mice resulted in a coordinated increase in the embryonic developmental capacity of the oocytes. Oocyte cumulus cell complexes were isolated at the germinal vesicle stage from small, medium and large antral follicles of 26-day-old mice and matured and fertilized in vitro. The frequency with which embryos from oocytes from small follicles completed the two-cell to blastocyst transition was lower than for embryos from oocytes from large follicles (33% and 79%, respectively). Germinal-vesicle stage oocyte-cumulus cell complexes were isolated from 22-26-day-old mice that were unprimed or primed by injection of equine chorionic gonadotrophin 48 h before isolation. Oocytes were matured in control medium, or in medium containing 1 microgram follicle-stimulating hormone (FSH) ml-1, and then fertilized in vitro. Priming did not increase the number of embryos completing the two-cell stage to blastocyst transition in the 22-day-old group nor did FSH treatment of maturing oocytes when the oocytes were isolated from unprimed 22-day-old mice. In contrast, priming increased the percentage of embryos completing the two-cell stage to blastocyst transition in the 26-day-old group by 20%. FSH treatment of maturing oocytes from the unprimed, 26-day-old group increased the number of embryos completing the transition to the same level as those in the primed 26-day-old group, but FSH did not increase the frequency of transition in the primed 26-day-old group.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed2005

Contribution of high p34cdc2 kinase activity to premature chromosome condensation of injected somatic cell nuclei in rat oocytes.

The present study was undertaken to clarify the relationship between the p34cdc2 kinase activity of in vitro-aged or enucleated rat oocytes and the premature chromosome condensation (PCC) of microinjected cumulus cell nuclei. Wistar rat oocytes were placed in vitro up to 120 min after the animal was killed. The p34cdc2 kinase activity of the oocytes decreased in a time-dependent manner. The incidence of PCC was higher when nuclear injection into intact oocytes was completed in 15-45 min rather than 46-120 min. When rat oocytes were enucleated for subsequent nuclear injection, the p34cdc2 kinase activity transiently increased soon after enucleation but drastically decreased after 30 min. Removal of the cytoplasm instead of the meta-phase-plate did not affect the p34cdc2 kinase activity even after 60 min. PCC occurred in intact and cytoplasm-removed oocytes but not in enucleated oocytes. In contrast, oocytes from BDF1 mice exhibited a p34cdc2 kinase level twice that of rat oocytes and supported PCC despite enucleation. The p34cdc2 kinase level of intact rat oocytes was reduced to the equivalent level of aged (120 min) or enucleated (+60 min) oocytes by a 45 min treatment with roscovitine, an inhibitor of p34cdc2 kinase. None of the roscovitine-treated oocytes supported PCC while half of the control oocytes did. When rat oocytes were treated with MG132, a proteasome inhibitor, delayed inactivation of the p34cdc2 kinase was observed in the MG132-treated oocytes. A significantly higher proportion of the MG132-treated oocytes supported PCC when compared with the control oocytes. Moreover, a higher proportion of MG132-treated and enucleated oocytes carried two pseudo-pronuclei after cumulus cell injection and developed to the two-cell stage when compared with the enucleated oocytes at the telophase-II stage. These results suggest that the decreased level of p34cdc2 kinase activity in aged or enucleated rat oocytes is responsible for their inability to support PCC of microinjected donor cell nuclei and that inhibition of p34cdc2 kinase inactivation by chemicals such as MG132 is in part effective for rat oocytes to promote PCC and further development.

Animals↗
PubMed1993

Cellular localization of luteinizing hormone receptor immunoreactivity in the ovaries of immature, gonadotropin-primed and normal cycling rats.

In this study we used two monoclonal antibodies against purified LH receptor (LHR) to localize and quantify LHR in ovarian compartments during follicular development, using gonads from immature, gonadotropin-primed, and normal cycling rats. In early preantral follicles, LHR immunoreactivity (LHRI) was identified in vascular endothelium and subsequently appeared in vascular pericytes. In healthy small antral (200-550-microns) follicles, LHRI continued to be present in thecal pericytes, but not in cells of the theca interna. However, in small antral follicles undergoing atresia, a dramatic decrease in thecal vessel LHRI with a concomitant increase in LHRI in hypertrophied theca was observed. In healthy antral follicles, LHRI of thecal cells was not observed until the cells reached medium (550-microns) size. High LHRI was occasionally observed in macrophage-like cells adjacent to the oocyte of large preantral follicles and among granulosa layers of medium-sized antral follicles. In the membrana granulosa, LHRI first appeared in cumulus cells of medium-sized antral follicles and subsequently spread to the entire granulosa cell population of large (750 microns) antral follicles. Treatment of immature rats with eCG markedly enhanced LHRI in theca and granulosa cells of all antral follicles, while eCG/hCG-treated (pseudopregnant) rats showed lack of LHRI in follicles and interstitial glands, but not in corpora lutea (CL). Within degenerating CL in the cycling ovary, compared to fresh and mature CL, a significant decrease occurred in intracellular LHRI. Our observations indicate that 1) vascular pericytes may play a role in follicular development; 2) LHR expression in granulosa may require an interaction of macrophages, oocytes, and cumulus cells; and 3) thecal hypertrophy accompanied by enhanced LHR expression occurs on follicles undergoing atresia.

Aging↗
PubMed2006

Donor centrosome regulation of initial spindle formation in mouse somatic cell nuclear transfer: roles of gamma-tubulin and nuclear mitotic apparatus protein 1.

During the process of spindle-chromosome complex depletion in the oocyte, it is unclear whether both gamma-tubulin and nuclear mitotic apparatus protein 1 (NUMA1), which are required for mitotic organization and spindle assembly, are removed. The role of the donor cell centrosome and donor nuclear NUMA1 in the initial spindle morphogenesis and chromosome remodeling also remains unclear. In the present study, we show that in the mouse, the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only approximately 10% of NUMA1 is removed during spindle-chromosome complex depletion in the recipient oocyte. This process does not impede initial spindle morphogenesis and is regulated by the centrosome of the donor cumulus cell. Retaining the donor cell centrosome establishes a monopolar spindle, whereas prior removal of the centrosome by a narrow-bore micropipette leads to bipolar spindle formation. Our data show that the centrosome of the donor cell regulates initial spindle morphogenesis and that the donor cumulus cell NUMA1 compensates for the deficiency in recipient NUMA1 during the formation of metaphase-like structures after nuclear transfer. Full-term offspring of cloned mice were obtained after injection of donor cells only with a pipette having an inner diameter of 7-8 microm, which retained the donor cell centrosome. In contrast, removing the donor cell centrosome with a small pipette impaired preimplantation development and prevented full-term development. In conclusion, the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell in the mouse.

Animals↗