Rat Cumulus Research Articles

Reduction of nitric oxide level results in maturation promoting factor destabilization during spontaneous meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured invitro.

Nitric oxides (NO) act as one of the major signal molecules and modulate various cell functions including oocyte meiosis in mammals. The present study was designed to investigate the mechanism of NO action during spontaneous meiotic exit from diplotene arrest (EDA) in rat cumulus oocytes complexes (COCs) cultured invitro. Diplotene-arrested COCs collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h were exposed to various concentrations of NO donor, S-nitroso-N-acetyl penicillamine (SNAP) and inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG) for 3h invitro and downstream factors were analyzed. Our results suggest that SNAP inhibited, while AG induced EDA in a concentration-dependent manner. The iNOS-mediated total NO, cyclic nucleotides and cell division cycle 25B (Cdc25B) levels were reduced significantly. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated cyclin-dependent kinase 1 (Cdk1) level and decreased Thr161 phosphorylated Cdk1 as well as cyclin B1 levels leading to maturation promoting factor (MPF) destabilization. The destabilized MPF finally induced spontaneous EDA. Taken together, these results suggest that reduction of iNOS-mediated NO level destabilizes MPF during spontaneous EDA in rat COCs cultured invitro.

Carbenoxolone reduces cyclic nucleotides level, destabilizes maturation promoting factor and induces meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro

BackgroundDisruption of gap junction and transfer of cyclic nucleotides to the oocyte lead to meiotic exit from diplotene arrest (EDA) in mammals. In the present study, we examined whether a gap junction blocker, carbenoxolone (CBX) could induce EDA by reducing cyclic nucleotides level and destabilizing maturation promoting factor (MPF) in rat oocytes cultured in vitro. MethodsDiplotene-arrested cumulus oocyte complexes (COCs) were collected from ovary of immature female rats after 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48h. These diplotene-arrested COCs were incubated with various concentration of CBX for 3h in vitro. The morphological changes, meiotic status of oocyte, inducible nitric oxide synthase (iNOS), total nitric oxide (NO), adenosine 3′,5′-cyclic monophosphate (cAMP), guanosine 3′,5′-cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), changes in specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels were analyzed. ResultsCBX induced EDA in a concentration-dependent manner. The iNOS expression, total NO and cyclic nucleotides level were significantly decreased. The reduced cyclic nucleotides level resulted in the decrease of Cdc25B expression level. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated Cdk1 level. However, Thr161 phosphorylated Cdk1 as well as cyclin B1 levels were significantly reduced leading to MPF destabilization. The destabilized MPF finally induced EDA in rat COCs cultured in vitro. ConclusionsOur results suggest that CBX blocked gap junctions interrupted the transfer of cyclic nucleotides to the oocyte. Reduction of cyclic nucleotides level destabilized MPF and induced EDA in vitro. Thus, CBX could be used to induce meiotic maturation under in vitro culture conditions during assisted reproductive technology (ART) programs.

A protective role of cumulus cells after short-term exposure of rat cumulus cell-oocyte complexes to lifestyle or environmental contaminants

Ovarian follicular fluid provides a potential reservoir for exogenous compounds that may adversely affect oocyte quality. This study examined the effects of common lifestyle and environmental contaminants, namely bisphenol-A (BPA), caffeine, 3,4-methylenedioxymethamphetamine (MDMA), nicotine and Δ9-tetrahydrocannabinol (THC) on gap junction genes (Gja1, Gja4) and proteins (GJA1), glucose metabolism genes (Gfpt1, Pfkp) and oocyte growth factor genes (Bmp15, Gdf9), as well as gap junction transfer rate, in rat cumulus-oocyte complexes (COCs). In vitro exposure to MDMA and THC accelerated the timing of meiotic resumption and all contaminants altered either gap junction gene expression (BPA, caffeine, MDMA and THC) or transfer rate (BPA and nicotine). In vitro exposure of COCs to MDMA also altered glucose metabolism genes. Overall, oocyte-derived genes were largely unaffected following exposure to any contaminant. In summary, the impact of short-term exposure to lifestyle and environmental contaminants on oocyte function may be diminished due to protective properties of cumulus cells.

The in-vitro effects of cAMP and cGMP modulators on inter-cellular dye transfer and gene expression levels in rat cumulus cell – oocyte complexes

Supplementation of in-vitro maturation medium with reagents that inhibit meiotic resumption whilst supporting normal function of cumulus cell–oocyte complexes (COC) is challenging. This study compared the in-vitro effects of synthetic and physiologically-relevant reagents on meiotic resumption, gap junction activity and gene expression of rat COC. Higher doses of forskolin reduced gap junction activity. Whilst addition of phosphodiesterase inhibitors initially promoted gap junction activity, this decreased with time in-vitro. Moreover despite oocytes remaining in meiotic arrest, there was a concomitant decline in expression of genes critical for oocyte maturation, and evidence of a reduction in overall transcription rate. Similarly, supplementing media with C-type natriuretic peptide and/or oestradiol delayed meiotic resumption and only initially maintained gap junction activity. In contrast, several key genes were stimulated and overall transcription rates remained constant with time in-vitro. In summary, supplementation of media with physiologically-relevant reagents may better enable normal functions of the COC.

Daily differential expression of melatonin-related genes and clock genes in rat cumulus-oocyte complex: changes after pinealectomy.

This study investigated the maturational stage (immature and mature ovaries) differences of mRNA expression of melatonin-forming enzymes (Aanat and Asmt), melatonin membrane receptors (Mt1 and Mt2) and putative nuclear (Rorα) receptors, and clock genes (Clock, Bmal1, Per1, Per2, Cry1, Cry2) in cumulus-oocyte complexes (COC) from weaning Wistar rats. We also examined the effects of pinealectomy and of melatonin pharmacological replacement on the daily expression of these genes in COC. qRT-PCR analysis revealed that in oocytes, the mRNA expression of Asmt, Mt2, Clock, Bmal1, Per2, and Cry1 were higher (P<0.05) in immature ovaries than in the mature ones. In cumulus cells, the same pattern of mRNA expression for Asmt, Aanat, Rorα, Clock, Per1, Cry1, and Cry2 genes was observed. In oocytes, pinealectomy altered the daily mRNA expression profiles of Asmt, Mt1, Mt2, Clock, Per1, Cry1, and Cry2 genes. In cumulus cells, removal of the pineal altered the mRNA expression profiles of Mt1, Mt2, Rorα, Aanat, Asmt, Clock, Bmal1, Per2, Cry1, and Cry2 genes. Melatonin treatment partially or completely re-established the daily mRNA expression profiles of most genes studied. The mRNA expression of melatonin-related genes and clock genes in rat COC varies with the maturational stage of the meiotic cellular cycle in addition to the hour of the day. This suggests that melatonin might act differentially in accordance with the maturational stage of cumulus/oocyte complex. In addition, it seems that circulating pineal melatonin is very important in the design of the daily profile of mRNA expression of COC clock genes and genes related to melatonin synthesis and action.

196 EXPRESSION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR IN OOCYTES AND CUMULUS CELLS FROM MOUSE, PIG, AND COW

Tissue-type plasminogen activator (tPA) is one of the components of the plasminogen-plasmin (PLG-PLA) system, better known as fibrinolytic system for its role in the blood clot lysis. It has been demonstrated recently that the activation of plasminogen into the protease plasmin during the sperm-oocyte interaction in the pig and cow decreases the percentages of penetration and increases monospermy (Mondéjar et al. 2012). However, in the mouse species, it was showed that PLG-PLA system enhances fertilization (Huarte et al. 1993). Expression of tPA has been described in rat oocytes (Bicsak et al. 1989) and cumulus cells (Ny et al. 1987; O’Connell et al. 1987), but no clear evidence about its expression in mouse, pig, and cow oocytes or cumulus cells is available. We hypothesised that differences in the effect of PLG-PLA system on fertilization results between the species mentioned above could be related to differences in tPA expression. The aim of this study was the detection of mRNA encoding tPA in oocytes and cumulus cells in mouse, pig, and cow by molecular analysis. Total RNA was obtained from oocytes and cumulus cells and cDNA was synthesised with an oligo-dT as primer. These cDNAs were used as template in RT-PCR amplifications using specific primers designed based on the GenBank sequence for Mus musculus, Sus scrofa, and Bos taurus tPA (NM_ 008872, NM_214054, NM_174146, respectively). The results of this study showed a different expression in the 3 studied species. In mouse, amplicon encoding tPA was detected in oocytes and cumulus cells. In cow and pig, tPA transcripts were obtained only in cumulus cells. The relation between the differences in the tPA expression pattern and the role of PLG-PLA system on fertilization remains to be investigated. This study was supported by MICINN (AGL2009-12512-C02-01-02).

Factors Influencing Chromosome Condensation and Development of Cloned Rat Embryos

Factors influencing premature chromosome condensation (PCC) in transferred rat nuclei have been examined. Chromosome condensation of rat cumulus cell nuclei did not occur when the cell nuclei were injected into enucleated rat oocytes. By contrast, chromosome condensation did occur after transfer to enucleated mouse oocytes or intact rat oocytes. In the first serial NT experiment, rat somatic cell nuclei were injected into enucleated mouse oocytes, and the reconstructed oocytes were activated by strontium chloride. From these reconstructed embryos, karyoplasts containing pronucleus-like vesicles were transferred into pronuclear zygote-derived cytoplasts by a DC pulse. Transfer of a total of 340 serial NT zygotes into recipient females, including 206 two-cell embryos, resulted in only seven implantation sites. In the second serial NT experiment, rat somatic cell nuclei were injected into intact rat oocytes; the recipient metaphase-plate was then aspirated under UV light from the NT oocytes in which PCC of injected nuclei was observed. After activation of the NT oocytes, karyoplasts were introduced into zygote-derived cytoplasts. Transfer of a total of 115 serial NT zygotes, including 37 two-cell embryos, resulted in four implantation sites but no live offspring. These results establish a mean of inducing chromosome condensation in rat oocytes and demonstrate that reconstructed rat zygotes can be prepared by serial NT procedures. Developmental competence of these embryos remains to be clarified.

Factors affecting premature chromosome condensation of cumulus cell nuclei injected into rat oocytes.

To date, production of cloned rats by somatic cell nuclear transfer (NT) has not yet been successful. Inducing premature chromosome condensation (PCC) of injected cell nuclei in recipient cytoplasm is considered essential for successful mouse cloning by the Honolulu method. In the present study, some factors affecting PCC of rat cumulus cell nuclei injected into rat oocytes were examined. Wistar female rats (young: 4 to 5-week-old, mature: > or =10-week-old) were superovulated by injections of eCG and hCG, and oocytes recovered 14 or 17 h after hCG injection were received with cumulus cell nuclei using piezo-driven micromanipulator. When the oocytes were recovered 14 h post-hCG injection from young rats and the nuclear injection into oocytes was completed within 45 min, PCC was observed in 44-49% of NT oocytes. In the case of oocytes from mature rats, PCC occurred in 11-19% of the NT oocytes. Oocytes recovered 17 h post-hCG injection did not support PCC of the injected nuclei (0-7%) regardless of the donor age. Treatment of oocytes with a neutral cysteine protease inhibitor, N-acetylleucylleucylnorleucinal, slightly increased the incidence of PCC (48 vs 37%). Comparison of rat strains for oocyte donors indicated that proportions of NT oocytes undergoing PCC in Wistar and LEW oocytes (41-46%) were higher than those in Donryu and F344 oocytes (17-25%). Thus, ability of rat oocytes to promote PCC of the injected nuclei is dependent on the characteristics of oocytes, such as age or strain of donor rats, and timing of oocyte recovery.

Preimplantation and postimplantation development of rat embryos cloned with cumulus cells and fibroblasts.

In this report we demonstrate the successful in vitro culture of fertilised embryos from 1-cell to blastocyst stage, albeit in a strain-dependent fashion. We report procedures for the enucleation of rat oocytes; nuclear transfer by injection of nuclei (NT) from adult rat cumulus cells, rat primary embryonic fibroblasts and genetically modified rat fibroblasts; and activation resulting in advanced preimplantation development. Blastocyst stage rat embryos were obtained after in vitro culture of nuclear transfer zygotes at similar frequencies with each of these nuclear donor cell types. Transfer of NT embryos to surrogate mothers leads to implantation of 24% of the zygotes. These results suggest that the nuclei of cultured rat cells, even following genetic modification, can be reprogrammed to support early embryonic development, which is a prerequisite to cloning the rat.

Identification of extracellular proteins in the rat cumulus oophorus

We have examined the proteins associated with the mucous matrix of the rat cumulus oophorus and compared them to the composition of rat serum, follicular fluid, ampullary fluid, and oocyte-cumulus cell extract. The cumulus matrix was dispersed using Streptomyces hyaluronidase, and the proteins were analyzed by high-resolution two-dimensional polyacrylamide gel electrophoresis and compared with proteins of the serum, proestrous follicular fluid, and postovulatory ampullary fluid and extracts of oocytes and cumulus cells. In addition to albumin and transferrin, which were common to all the fluids analyzed, the cumulus material contained many proteins in common with the follicular fluid and the ampullary fluid. However, the protein extract of the cumulus matrix also contained four major proteins not present in the other fluids analyzed. Two of these proteins were acidic and heterogenous in charge and size (MW approximately 81,000 and 100,000). The other two proteins were more basic and occurred at MW approximately 90,000 and 150,000. Our results show that the extracellular matrix of the cumulus contains proteins that are not present in the fluids that surround the oocyte.

Gonadotrophins stimulate lactate production by rat cumulus and granulosa cells.

Oocyte-cumulus complexes and mural granulosa cells, respectively, isolated from pre-ovulatory rat follicles and cultured in lactate-free medium showed a continuous accumulation of lactate during a 1 to 7 h incubation. Lactate production was stimulated by gonadotrophins, both when administered in vivo and in vitro, with the exception that LH given in vivo did not affect granulosa cell lactate production. Since the oocyte is known to have specific demands on energy substrate, it is suggested that the lactate produced is important for the growing and maturing oocyte.

Effect of luteinizing hormone and follicle-stimulating hormone on progesterone synthesis by cultured rat cumulus cells.

This study examined the responsiveness of the rat cumulus oophorus to gonadotropin in terms of progesterone (P) secretion. Immature female rats pretreated with a single injection of PMS gonadotropin were used. Oocyte-cumulus complexes were isolated aseptically from large preovulatory follicles or from the oviduct shortly after ovulation and cultured in modified Eagle's medium. Ten cumuli from each rat were cultured for 24 or 48 h, with change of medium after 12 and 24 h. P accumulation in the culture medium was determined by RIA, and cell morphology was examined. Cumuli isolated from rats killed before the endogenous LH/FSH surge and cultured in hormone-free medium secreted low amounts of P (<10 pg/cumulus/ day) throughout the culture period. By contrast, cumuli isolated 3–5 h after the surge secreted high amounts of P (300–400 pg/day). This increment in P secretion was abolished by pentobarbital treatment of the rats during the critical period. Postovulatory cumuli isolated from the oviduct 5–9 h after o...

Progesterone and prostaglandin secretion by ovulated rat cumulus cell-oocyte complexes.

Hormone production (progesterone and prostaglandin) by oocyte-cumulus cell complexes was studied in the postovulatory period. Cumulus-oocyte complexes were collected from oviducts of prepuberal rats after the synchronization of follicle development and ovulation with PMS gonadotropin and hCG injections. Cumulus masses from individual animals were cultured in roller vessels containing Eagles' Minimal Essential Media with or without added heat-inactivated rat sera (5% CO2-air; 37 C). Culture media were collected after 4.5 h of incubation and analyzed by RIA for prostaglandin E2 (PGE), PGF, and progesterone. Extensive progesterone secretion occurred during the culture period and was markedly stimulated by the addition of rat serum; however, cumulus masses did secrete some progesterone in the absence of rat serum and without exogenous gonadotropins. The addition of aminoglutethimide, a steroidogenesis inhibitor, suppressed progesterone secretion but did not alter the secretion of PGF or PGE. Both PGF and PGE were detected in the culture medium, and in all cases, PGE was the predominant PG detected. Indomethacin, an inhibitor of PG synthesis, suppressed PGE and PGF secretion but had no effect on progesterone synthesis. Small amounts of PGs were also detected in nonincubated cumulus-oocyte masses. Hormone secretion in culture was measured after cumulus mass dissociation by enzymatic digestion. Somatic cumulus cells, rather than germ cells, were the primary cellular source of progesterone and PGs. The results demonstrate that cumulus-oocyte complexes can synthesize and secrete steroid and PG hormones subsequent to ovulation, and thus, these masses should be considered functional endocrine tissues. The physiological significance of the endocrine activities of the cumulus-oocyte complex are discussed in terms of oviductal and gametic processes. (Endocrinology 108: 457, 1981)

Cyclic AMP, prostaglandin E2 and steroids: possible mediators in the rat cumulus oophorus mucification.

The possible role of cyclic AMP (cAMP), prostaglandin E2, progesterone and estradiol-17β in in vitro induction of the rat cumulus oophorus mucification was studied with the scanning electron microscope. Follicular cumulus-oocyte complexes were isolated at early proestrus and cultured for 24 h. Cyclic AMP levels in the incubated complexes were elevated by addition of cAMP derivatives (dibutyryl cAMP or 8 bromo cAMP), the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX), or the adenylate cyclase stimulator, cholera enterotoxin. Under all these culture conditions the cumulus cells were stimulated to secrete a hyaluronidase-sensitive mucoid material which coated the cumulus-oocyte complexes. These complexes were similar in appearance to those incubated in the presence of gonadotropins. In the absence of the above agents extracellular material was not observed. In vitro cumulus mucification was not induced by prostaglandin E2; indomethacin, an inhibitor of prostaglandin synthetase, failed to block mucification in complexes that have been incubated in the presence of LH. Cumulus complexes were not stimulated to mucify in vitro by progesterone or estradiol-17β. Therefore, the response of the cumulus cells to the gonadotropic stimulus appears to be mediated by cAMP. Prostaglandin E2 and steroids seem not to be involved in this process.

Progesterone secretion by the post‐ovulatory rat cumulus oophorus

AbstractThis study was designed to determine whether cumulus oophorus cells secrete progesterone. Immature PMS‐primed, hCG‐treated rats were used. Their cumuli were isolated from pre‐ovulatory (no hCG) and peri‐ovulatory (after hCG) follicles, and from post‐ovulatory oviducal ampullae. Treatment with hCG increased progesterone secretion by almost two‐and‐one‐half‐fold. It was speculated that the cumulus oophorus secretion of progesterone modifies the accumulation of fluid in the ampulla, thus providing the medium in which fertilization takes place.