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PubMed1992

Requirement for glucose in ligand-stimulated meiotic maturation of cumulus cell-enclosed mouse oocytes.

In this study, the effect of different energy sources used in Eagle's minimum essential medium on the meiotic maturation of mouse oocytes in culture was examined. The effects of glucose (5.5 mmol 1(-1)), pyruvate (0.23 mmol 1(-1)) and glutamine (2 mmol 1(-1)) in different combinations were tested on the maturation of denuded oocytes in the presence or absence of 300 mumol dibutyryl cAMP 1(-1) during 17-18 h of culture. In the absence of cyclic nucleotide, only oocytes from those groups containing pyruvate resumed maturation at a high frequency (99-100% germinal vesicle breakdown); all other combinations resulted in < or = 54% germinal vesicle breakdown. When dibutyryl cAMP was introduced, all pyruvate-containing groups exhibited maturation frequencies of about 50%, whereas maturation in all other groups was negligible (< or = 10% GVB). Pyruvate was also important for the maintenance of viability in denuded oocytes (> or = 86% viability in pyruvate-containing medium; < or = 35% viability in pyruvate-free groups). When cumulus cell-enclosed oocytes were cultured in medium without inhibitor, all combinations of energy substrates supported high frequencies of maturation (> or = 89% germinal vesicle breakdown) and viability (> or = 91%). The addition of dibutyryl cAMP resulted in inhibition of meiotic maturation (5-33% germinal vesicle breakdown) in all cultures except the pyruvate-alone group (97% germinal vesicle breakdown). Viability in cumulus cell-enclosed oocytes was greatest when two or more energy substrates were present in the medium. Follicle-stimulating hormone (FSH) produced a stimulation of meiotic maturation in all cultures of meiotically arrested cumulus cell-enclosed oocytes, but maximal induction of germinal vesicle breakdown was dependent upon D-glucose. Concanavalin A (ConA)-induced meiotic maturation was also dependent upon D-glucose. Uptake and metabolism of D-glucose by the cumulus cells is important in mediating the stimulatory effects of these ligands on oocyte maturation because (1) both FSH and ConA stimulated uptake of D-glucose and 2-deoxyglucose but not 3-O-methylglucose; (2) phloretin prevented the stimulatory action of FSH and ConA on germinal vesicle breakdown at a concentration that suppressed ligand-induced uptake of D-glucose; (3) 2-deoxyglucose, a hexose that suppresses glycolysis, prevented the induction of meiotic maturation by FSH and ConA and (4) D-mannose, a glycolysable sugar, was as effective as D-glucose in supporting the ligand effects.(ABSTRACT TRUNCATED AT 400 WORDS)

3-O-Methylglucose↗

PubMed1997

Coding sequence, exon-intron structure and chromosomal localization of murine TNF-stimulated gene 6 that is specifically expressed by expanding cumulus cell-oocyte complexes.

Tumor necrosis factor stimulated gene-6 (TSG-6) has been previously shown to be induced in vitro in several cell types by proinflammatory cytokines, and in vivo in pathological conditions such as rheumatoid arthritis. In this study, we report the complete coding sequence for the mouse TSG-6 protein, and the exon intron structure and the chromosomal localization of the gene. We have identified a 1605 nt cDNA sequence from mouse cumulus cell oocyte complexes (COCs) induced to expand in vivo. The sequence contains an open reading frame of 825 nt that codes for the 275 amino acid TSG-6 protein. The gene contains six exons separated by 1.1-5.8 kb introns and has been localized to the murine chromosome 2 by linkage analysis. Comparative reverse transcription-polymerase chain reaction studies have revealed that TSG-6 mRNA is specifically expressed after COC expansion induced in vivo, identifying the first non-pathological process in which TSG-6 may play an important role. Since TSG-6 binds to hyaluronan and interacts with inter-alpha-trypsin inhibitor (IalphaI), molecules that are essential for matrix formation by COCs, this protein may have a structural role in the matrix or may enhance the antiproteolytic effect of IalphaI to protect the matrix from degradation.

Amino Acid Sequence↗

PubMed2001

Expression of endothelial nitric oxide synthase in the porcine oocyte and its possible function.

The present study was designed to investigate the localization of endothelial nitric oxide synthase (eNOS) in porcine oocytes and its possible function during in vitro development. RT-PCR and immunoblotting analyses revealed the presence of eNOS in the oocytes prepared from small follicles, with an amplified product of 456 bp and an apparent mol wt of 130 kDa, respectively. The synthesis of oocyte NO was suppressed during a 72-h culture of cumulus-oocyte complexes in the presence of follicle-stimulating hormone (FSH), but not luteinizing hormone (LH). However, the decrease in NO synthesis did not result from the levels of eNOS mRNA and its protein, as revealed by analyses of RT-PCR and Western blot analysis, suggesting that expression of oocyte eNOS is not dependent upon gonadotropin stimulation. In proliferated cumulus cells, LH receptor mRNA expression was detected after a 48-h culture with FSH, as revealed by RT-PCR analysis. mRNA expression was inhibited by an NO-releasing agent (S-nitroso-N-acetyl-DL-penicillamine) after an additional 24-h culture. These results suggest that oocytes may release eNOS-derived NO as a signal for somatic cells to steadily suppress the development of cumulus cells, if not FSH stimulation. Conversely, the synthesis of NO is suppressed during the action of FSH on the cumulus cells with no changes in eNOS expression.

Animals↗

PubMed1999

Cloning the laboratory mouse.

A brief account is given of early attempts to clone mammals (mice) by transferring cells (nuclei) of preimplantation embryos into enucleated oocytes, zygotes or blastomeres of two-cell embryos. This is followed by a brief review of recent successes using adult somatic cells: mammary gland cells for sheep, muscle cells for cattle and cumulus cells for mice. We have developed a technique for cloning the laboratory mouse by transferring cumulus cell nuclei into enucleated oocytes. With this technique, we have produced a population of over 80 cloned animals, and have carried the process over four generations. Development and fertility of these appear normal. However, the yield is very low; only approximately 1% of injected oocytes are carried to term. The challenge is now to understand the reason for this high loss. Is it a problem of technique, genomic reprogramming, somatic mutation, imprinting or incompatible cell cycle phases?

Animals↗

PubMed2001

Protein kinase C, rather than protein kinase A is involved in follicle-stimulating hormone-mediated meiotic resumption of mouse cumulus cell-enclosed oocytes in hypoxanthine-supplemented medium.

It has been reported that protein kinase C (PKC) activation participated in the porcine and bovine oocyte maturation, but not in mouse oocyte maturation in vitro. In the present study, the activators and inhibitors of protein kinase A (PKA) (forskolin, CDPKI and MDL-12230A) or PKC (PMA, staurosporine and sphingosine) were used to investigate the in vitro effect of PKA or PKC on spontaneous murine oocyte maturation, oocyte resumption of meiosis from HX inhibiting medium (medium+HX), and follicle stimulating hormone (FSH)-induced oocyte maturation. The results showed that when cumulus cell enclosed oocytes (CEOs) or denuded oocytes (DOs) were cultured for 24 h in the medium supplemented with forskolin (5 microM), an activator of adenylate cyclase, the spontaneous oocyte maturation were inhibited. A transient exposure (2 h) to forskolin (2-10 microM) in the medium+HX, and then transferred to a new medium+HX for the further culture, stimulated CEO resumption of meiosis. CDPKI (10(-10)-10(-6) M), an inhibitor of PKA, also stimulated oocyte meiotic maturation of CEO in the medium+HX, but not on DO. However, MDL-12230A (10(-12)-10(-9) M), an inhibitor of adenylate cyclase, did not promote oocyte maturation in HX arrested CEO. CDPKI (10(-10)-10(-6) M) or MDL-12230A (10(-12)-10(-9) M) had no effect on FSH-stimulated oocyte meiotic resumption, except at high doses of CDPKI (10(-7)-10(-6) M) or MDL-12230A (10(-9) M) which inhibited the FSH-induced formation of the first polar body (PB1). An activator of PKC, PMA (10(-11)-10(-7) M) dose-dependently inhibited spontaneous oocyte maturation of CEO or DO. Inhibitors of PKC, staurosporine (10(-9)-10(-6) M) or sphingosine (10(-8)-10(-5) M) induced oocytes in CEOs to resume meiosis in the presence of HX in a dose dependent manner, but had no effect on DOs. FSH (50IU/L) stimulated mouse oocytes in CEOs to override the arrest of HX and resume meiosis, while PMA, at the level of 10(-8)-10(-6) M, dramatically inhibited the stimulatory effect of FSH. These results indicate that PKC or PKA may be implicated in the regulation of mouse oocyte maturation. Thus while sustained high level of cAMP or PKA inhibit the resumption of meiosis, a transient rise in cAMP or PKA levels promotes oocyte maturation. The activation of PKC can also block oocyte meiotic resumption. Thus the inactivation of PKC, instead of the transient rise of PKA activity, appears to be involved in the process of FSH-mediated oocyte meiotic maturation.

Animals↗

PubMed2006

Denuding and centrifugation of maturing bovine oocytes alters oocyte spindle integrity and the ability of cytoplasm to support parthenogenetic and nuclear transfer embryo development.

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells.

Animals↗

PubMed1993

In vitro development up to hatching of bovine in vitro-matured and fertilized oocytes with or without support from somatic cells.

To verify the importance of somatic cells upon in vitro embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 supplemented with estrous cow serum (10% v/v) and 0.25 mM sodium pyruvate (ECSTCM) under the following treatments: 1) ECSTCM alone; 2) together with bovine oviduct epithelial cells (BOEC); 3) with cumulus cells (CC); 4) in fresh BOEC conditioned ECSTCM; or 5) in frozen-thawed BOEC conditioned ECSTCM. Culturing zygotes encased in cumulus cells significantly reduced the cleavage rate (P<0.05). There was no difference between culture systems in the proportions of embryo development through the 8-cell stage (P=0.42) up to the morula/blastocyst stages (P=0.50) at Day 7 post insemination. However, co-culture with BOEC yielded the highest percentage (21.2% of zygotes; P<0.05) of quality Grade-1 and Grade-2 embryos with the number of blastomeres per embryo (114.4) comparable to that of 7-day-old in vivo-developed embryos of similar grades (102.5), and higher (P<0.05) than those of the other treatments. The ratio of blastocysts to total morulae/blastocysts obtained from frozen-thawed conditioned medium was lower (P<0.05) than that from ECSTCM or after co-culture with BOEC at Day 7 post insemination. On average, 7.5 to 17.5% of the zygotes developed to blastocyst, expanded blastocyst and hatched blastocyst stages by Day 10 post insemination, depending upon the culture system. The difference between treatments, however, was not significant (P=0.68). The results indicate that chronological development up to hatching of bovine IVM-IVF embryos is not favored by somatic cells; however, the presence of viable oviduct epithelial cells in culture significantly improves the quality of 7-day-old embryos.

Journal Article↗

PubMed2000

Quality controls for bovine viral diarrhea virus-free IVF embryos.

Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of in-vivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10(7) cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos > or = 5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos > or = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage to > or = 5 cells (P = 0.026) and development to morula and blastocyst (P = 0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n = 40) and morulae and blastocysts (n = 57) and from all trypsin-treated nonfertile and degenerated ova (n = 80) and morulae and blastocysts (n = 91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.

Animals↗

PubMed2000

FSH suppression of nitric oxide synthesis in porcine oocytes.

The present study was designed to evaluate the regulation of nitric oxide (NO) synthesis in porcine oocytes during follicular development. Cumulus-oocyte complexes were obtained by aspirating the small follicles of immature porcine ovaries and cultured at 39 degrees C for 24-72 h with FSH in a serum-free medium. The oocyte-surrounding cumulus cells markedly proliferated and expressed LH receptor mRNA in response to FSH. The endothelial type of NO synthase (eNOS) (130 kDa) was detected in the oocyte, but not in the proliferated cumulus cells, by immunoblotting. The amount of oocyte eNOS did not significantly alter during culture, but measurement of nitrite and nitrate revealed FSH suppression of NO synthesis by approximately 50%. NO-releasing agents were added to the cultures to examine the effect of NO on the growth of cumulus cells. NO-releasing agents showed inhibitory effects on proliferation of the cumulus cells and expression of LH receptor mRNA. Thus, synthesis of eNOS-derived NO is suppressed in the porcine oocyte during development with no change in the enzyme amount, and it is suggested that it has an inhibitory function in the growth of cumulus cells.

Animals↗

PubMed1997

Expression of tumor necrosis factor-alpha and its receptors type I and type II in human oocytes.

The present study was conducted to examine the expression of tumor necrosis factor-alpha (TNF-alpha) and its receptors (types I and II, designated TNFR-I and TNFR-II, respectively) in human oocytes and cumulus cells at the mRNA and protein levels. mRNA expression was investigated using a reverse transcriptase-polymerase chain reaction (PCR)/Southern hybridization procedure. DNA-free RNA was isolated from the oocytes/cumulus cells, reverse-transcribed, and PCR-amplified using specific oligonucleotide primers based upon genomic/cDNA sequences. The expected bands of 303 bp and 513 bp were observed in oocytes and cumulus cells using primers based on genomic/cDNA sequences of TNF-alpha and TNFR-II, respectively, that hybridized with specific cDNA probes in Southern blot hybridization procedure. The expected band of 368 bp was not observed in oocytes and cumulus cells using primers based on the TNFR-I cDNA sequence. Similar results were observed for expression at the protein level, as seen by the immunoreactivity of the specific antibodies with the paraformaldehyde-fixed oocytes and cumulus cells in the indirect immunofluorescence technique (IFT). These results indicate that human oocytes and cumulus cells express TNF-alpha and its receptor type II (TNFR-II), and not type I (TNFR-I), both at the mRNA and protein levels. These findings provide further evidence and substantiate the proposed physiologic role of TNF-alpha in ovarian function, and may lead to clinical applications in in vitro fertilization programs and in diagnosis and treatment of infertility in women, especially in cases attributed to ovarian dysfunction.

Adult↗

PubMed2005

Factors affecting survival of reconstructed mouse embryos after nuclear transfer.

Reconstruction of mouse embryos was performed by injection of donor genetic material from differentiated cells of various types (cumulus cells, cardiomyocytes, and epithelial cells) into recipient cells (mature oocytes and zygotes). A medium for microsurgery was selected, which enhanced survival of both embryonic and somatic cells during the reconstructive manipulations. Special preparation of somatic cells to transplantation was carried out, which employed factors synchronizing the cells in a certain phase of the cell cycle in order to enhance their capacity to maintain the development of reconstructed embryos. The processes of nucleus reprogramming in specialized cells under the action of cytoplasmic factors of oocytes and zygotes were examined. During in vitro culturing of reconstructed embryos, the most successful development was observed in embryos implanted with donor material from cumulus cells. Mouse embryos reconstructed with a certain genome and subsequent production and use of stem cells are considered as the model system for developing the basic principles of replacement therapy.

Animals↗

PubMed2003

Dual effects of nitric oxide on meiotic maturation of mouse cumulus cell-enclosed oocytes in vitro.

The present experiment used cultured mouse cumulus cell-enclosed oocytes (CEOs) and denuded oocytes (DOs) to study the function of nitric oxide (NO) in mouse oocyte meiotic maturation. Either positive or negative actions of NO on meiotic maturation has been observed when CEOs or DOs were cultured for 24 h in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, or in maturation medium (without HX) supplemented with different doses of sodium nitroprusside (SNP, a NO donor), N-omega-nitro-L-arginine methyl ester (L-NAME) or N(w)-nitro-L-arginine (L-NNA) (two inhibitors of NO synthase, NOS), and L-arginine (the only substrate of NOS). Both NOS inhibitors suppressed the formation of first polar body (PB1) of the oocytes in CEOs in a dose dependent manner, but no effect on germinal vesicle break down (GVBD) was observed. An optimal inhibitory effect was observed with either 10(-3) M L-NAME (P<0.01) or 10(-3) M L-NNA (P<0.01) and the inhibition could be reversed by the addition of SNP (10(-5) M). The above mentioned optimal concentration of L-NAME or L-NNA on CEOs exhibited no effect on oocyte meiotic maturation of DOs. Treatments of low concentrations of SNP (10(-7), 10(-6), 10(-5) M) stimulated significantly the oocyte meiotic maturation of CEOs which were inhibited with HX, but had no effect on DOs in the same culture medium. While, the treatment with high concentrations of SNP (0.1-4 mM) during the CEOs cultured in maturation medium resulted in a lower percentage of oocytes at PB1 stage and a higher percentage of atypical oocytes in a dose dependent manner compared with control. A dose of SNP at 1 mM exhibited significant inhibitory effect on the formation of PB1, but without effect on the number of atypical oocytes compared with control, while, this SNP dosage not only inhibited the oocyte PB1 formation but also increased the percentage of dead oocytes in DOs. Although oocytes of all groups underwent GVBD at the end of the culture in the spontaneous maturation medium, the results of the kinetics showed that the treatment of the optimal concentration of SNP (1 mM) could significantly delay GVBD during the first 5 h culture period. The concomitant addition of L-NAME with SNP did not reverse the inhibitory effect of SNP on CEOs. Similarly, neither pre-incubation nor illumination by ultraviolet ray could balance the inhibitory effect of SNP. Finally, when added alone at a concentration of 4 mM, L-arg caused extensive death of both CEOs and DOs. While, administration of 4 mM L-arg and 1 mM L-NAME to both CEOs and DOs simultaneously resulted in markedly reduced CEOs death percentage as compared with L-arg treatment alone, but not in DOs. These data support the idea that NO could act with a dual action (stimulation or inhibition) in mouse meiotic maturation depending on its concentration.

Animals↗

PubMed2002

Delivery of the oocyte from the follicle to the oviduct: a time of vulnerability.

The multiplicity of genes regulated by the actions of LH in specific granulosa cell and cumulus cell microenvironments of the follicle during ovulation has now revealed how complex and finely tuned the ovulation process is. Although many proteases are expressed in the ovary and are hormonally regulated, the novel proteases ADAMTS-1 and cathepsin L, rather than the MMPs, have gained particular recognition. These may play critical roles in the eventual rupture of the follicle at the ovarian surface. Rupture is only one part of ovulation. Another important aspect is the actual release of the oocyte from the ovulation pore. Several recent studies have shown that the production of the matrix that is evidenced by cumulus expansion is somehow critical for extrusion to occur and for the oocyte to travel into the oviduct. At any point the oocyte may be trapped within a non-ovulating structure. Lastly, if the events of ovulation fail to occur before the events of luteinization are complete, oocytes are destined to degenerate within the non-ovulating structures.

ADAM Proteins↗

PubMed2000

Hyaluronic acid as an anti-angiogenic shield in the preovulatory rat follicle.

Angiogenesis in the preovulatory follicle is confined to the theca cell layers, and penetration of capillaries through the basement membrane into the granulosa cell layers does not occur until after ovulation. However, elevated expression of the angiogenic growth factor (VEGF) has been reported in the cumulus cells surrounding the oocyte, which are expelled from the follicle during ovulation. This spatial and temporal discrepancy between VEGF expression and angiogenesis was studied here in the rat ovarian follicle, and we showed that cumulus cells secrete to the follicular fluid, in addition to VEGF, material with antiangiogenic activity that blocks endothelial cell proliferation, migration, and capillary formation in vitro. Hyaluronic acid produced by the cumulus cells can account for this antiangiogenic activity. Degradation of hyaluronic acid by hyaluronidase restored proliferation and migration of endothelial cells directed toward the cumulus. Inhibition of hyaluronic acid synthesis with 6-diazo-5-oxo-1-norleucine restored endothelial proliferation and migration in vitro, and it also resulted in early penetration of capillaries across the follicular basement membrane in vivo. These results support the role of hyaluronic acid produced by the cumulus cells as a high-molecular-weight, antiangiogenic shield that prevents premature vascularization of the preovulatory follicle by blocking endothelial cell migration and proliferation.

Animals↗

PubMed1998

Energy substrates and amino acids provided during in vitro maturation of bovine oocytes alter acquisition of developmental competence.

Energy substrates and amino acids were evaluated for supporting acquisition of developmental competence by bovine cumulus-oocyte complexes during in vitro maturation. The basic culture medium (Basic Medium-3) used for in vitro maturation of oocytes was modified to produce six media containing glucose or glutamine with lactate or pyruvate, or glucose + glutamine, or glucose + 11 amino acids; a seventh (control) medium was TCM199. All media contained polyvinyl alcohol, gonadotropins, epidermal growth factor and oestradiol. Following maturation, oocytes were incubated in medium TALP for fertilisation, then cumulus cells were removed and presumptive embryos cultured for 48 h in a chemically defined medium (HECM-6) followed by 120 h in medium TCM199 + bovine calf serum. Six substrate treatments yielded similar first cleavage responses (66-78%) at 72 h post-insemination; however, blastocyst development at 192 h varied significantly. Oocytes matured in medium with glucose + 11 amino acids gave the best blastocyst development: 21% of inseminated oocytes or 25% of 2-cell embryos. Cumulus expansion in HECM-6 required glucose with either glutamine, 11 amino acids or lactate, or glutamine + lactate. We conclude that (1) the type of energy substrate or nutrient supplied during in vitro maturation of oocytes profoundly affects subsequent developmental competence; (2) oocyte maturation in simple medium containing glucose with lactate or 11 amino acids or glutamine, or lactate + glutamine, can support development equally as well as the complex medium, TCM199; and (3) media supporting at least moderate cumulus expansion during oocyte maturation also support subsequent blastocyst development.

Amino Acids↗

PubMed1995

Beneficial effect of oocyte activation prior to and during nuclear transfer in cattle using in vitro matured oocytes 24 h of age.

This research was designed to study in vitro development of bovine nuclear transferred embryos using enucleated young in vitro matured oocytes 24 h of age as recipient cytoplasts activated prior to nuclear transfer. The oocytes were enucleated and then activated with electric pulse at 24 h of age followed by incubation with cycloheximide for 6-7 h before nuclear transfer and membrane fusion with a blastomere of the 16-32 cell stage. This new protocol effectively improved cleavage (68 vs 17%, p < 0.05) and morula/blastocyst development of reconstructed embryos (29 vs 6%, p < 0.05), compared to similar nuclear transfer procedure without prior activation of recipient oocytes. Corresponding rates of cleavage and morula/blastocyst development for oocytes activated similarly without nuclear transfer were 49 and 19%. Factors affecting nuclear transfer were also compared. Two electric pulses for fusion increased rates of fusion (76 vs 60%, p < 0.05) and subsequent development of cloned embryos (32 vs 11%, p < 0.05). Cytochalasin B treatment following nuclear transfer manipulation seemed not to be beneficial in improving development of cloned embryos (p > 0.05). Both co-culture systems with buffalo rat liver (BRL) cells and cumulus cells promoted development of cloned embryos compared to the controls (28, 21 vs 0%, p < 0.05). The BRL cell system seemed to be better for manipulated embryos by reducing embryolysis.

Animals↗

PubMed1995

Differential distribution of gangliosides in adult rat ovary during the oestrous cycle.

Gangliosides are ubiquitous membrane components in mammalian cells and are suggested to play important roles in various cell functions, such as cell-cell recognition, differentiation and transmembrane signalling. Rat ovary contained GM3, GD3 and GD1a as major gangliosides, and GM1 as a minor one. In order to study their distribution in the rat ovary and its possible changes during the oestrous cycle, frozen sections were stained with specific monoclonal antibodies against 11 ganglio-series gangliosides including those mentioned above. GM3, GM1 and GD1a were expressed in a spatio-temporally different manner during the oestrous cycle, but GD3 and other gangliosides were not immunohistochemically detected. In primary and secondary follicles, GM3, GM1 and GD1a were expressed in theca cells, but not in granulosa cells. The oocyte in primary, but not secondary, follicles was positive to the anti-GD1a antibody. In Graafian follicles, GM1 and GD1a were similarly expressed as in secondary follicles, however, the expression of GM3 spread gradually from theca cells to granulosa cells. In early Graafian follicles, only GM3 was expressed to a detectable extent from the outer part of the granulosa layer to the inner part. Shortly before ovulation, all granulosa cells and cumulus cells became positive to anti-GM3 antibody. After ovulation, differential distribution of GM3, GM1 and GD1a was also observed in luteal cells. GD1a was localized in thread-like structures, while GM3 was distributed throughout the cytoplasm, but not in the nucleus. GM1 was localized only in the plasma membrane and/or its close vicinity. Other ganglio-series gangliosides, including GD3, were not detected to an appreciable extent in the ovaries by immunohistochemistry.

Animals↗

PubMed1992

Reliability of detection by polymerase chain reaction of the sickle cell-containing region of the beta-globin gene in single human blastomeres.

Human preimplantation embryos at various stages of development have been analysed using the polymerase chain reaction to amplify a 680 base pair fragment of the beta-globin gene. Successful amplification was achieved more frequently with DNA from intact embryos containing between one and 11 cells, single cumulus cells, oocytes which had failed to fertilize and polar bodies than from single blastomeres disaggregated from intact embryos and treated in an identical manner. The distribution of nuclei demonstrated using the nuclear chromophore diamino-phenyl-indole showed considerable inter-blastomere variation; however, no clear correlation between staining pattern and successful amplification was observed. The reason for the unreliable amplification of DNA from single blastomeres is unclear but this finding has important implications for preimplantation diagnosis of genetic disease.

Blastomeres↗