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PubMed2000

Cloned mice derived from somatic cell nuclei.

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.

Animals↗
PubMed1990

Effects of culture systems on development of in vitro fertilized bovine ova into blastocysts.

We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P<0.05) increased development into blastocysts (39.0 to 50.7%) when compared with co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were obtained.

Journal Article↗
PubMed1996

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro.

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.

Animals↗
PubMed1991

Localization of luteinizing hormone receptor messenger ribonucleic acid expression in ovarian cell types during follicle development and ovulation.

The action of LH is mediated through specific plasma membrane receptors that are both up- and down-regulated in the ovary during the reproductive cycle. Using immature rats treated with PMSG and hCG as a model system, we have studied the regulation and distribution of LH receptor mRNA in different cell types during follicle development, ovulation, and luteinization by Northern blot and in situ hybridization. In untreated rats, LH receptor mRNA was below the detection level in granulosa cells, cumulus cells, and oocytes, while low levels of LH receptor mRNA were found in the thecal cells. After stimulation with PMSG, expression of LH receptor mRNA was enhanced in the thecal-interstitial cells, while a more dramatic increase in receptor mRNA abundance took place in granulosa cells of large tertiary follicles. In these follicles, the abundance of LH receptor mRNA varied among different subpopulations of granulosa cells, with mural granulosa cells close to the basement membrane exhibiting higher levels than granulosa cells located closer to the antrum, and cumulus cells and the oocyte lacking detectable hybridization signal. The uneven expression of LH receptor mRNA endows different ovarian cells with varying hormonal responsiveness. After an ovulatory dose of hCG, LH receptor mRNA levels were dramatically decreased, particularly in the granulosa cells of preovulatory follicles, to reach the lowest levels just before ovulation. During the transformation of ovulated follicles into corpora lutea, the expression of LH receptor message was again increased. Our results reveal that the previously documented regulation of the LH receptor-binding activity during ovarian development correlates with expression of the LH receptor transcripts, suggesting that the LH receptor gene is regulated in a complex manner during the periovulatory period to achieve cell-specific expression together with gonadotropin induction and suppression of receptor gene activity.

Animals↗
PubMed2002

The synthesis and fate of glycodelin in human ovary during folliculogenesis.

The ontogeny of glycodelin in human ovarian follicles during folliculogenesis was studied. Glycodelin immunoreactivity began to be detected in the granulosa cells and thecal cells of late secondary follicles. Immunoreactivity was also found in both the luteinized granulosa cells and cumulus cells obtained from women undergoing the assisted reproduction treatment. However, only the luteinized granulosa cells, and not the cumulus cells, expressed glycodelin mRNA. Results also showed that the cumulus cells took up radiolabelled glycodelin and partially deglycosylated some of it. Glycodelin (and a partially deglycolsylated form of glycoldelin) appeared to complex with two cytoplasmic or membrane components of the cumulus cells. The data also demonstrated that ZIF-1, a glycoprotein isolated from human follicular fluid, was immunologically similar to glycodelin. In conclusion, we suggest that glycodelin is synthesized in the granulosa cells of ovarian follicles at late secondary follicle stage. It then may be released into the follicular fluid from where it is taken up and partially modified by the cumulus cells.

Binding, Competitive↗
PubMed1991

Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.

Animals↗
PubMed1991

Cytokine involvement in oocytes and early embryos.

OBJECTIVE: The early events of reproduction involve a carefully modulated complex system of oocyte maturation, fertilization, and proliferation. The aim of the study was to measure the presence of cytokines, namely interleukin 1 (IL-1), interleukin 6 (IL-6), colony-stimulating factor 1 (CSF-1), and tumor necrosis factor (TNF) in the conditioned medium (CM) of the oocytes, granulosa cells, cumulus cells, one to eight-cell embryos and sperm. DESIGN: The material was obtained from men and women undergoing in vitro fertilization therapy. MAIN OUTCOME MEASURES: We hypothesized that cytokines might affect embryonic growth and differentiation as they show a pleotropic effect on immune cells. RESULTS: All these cytokines are present in significant quantities in the CM and were shown to be expressed in a sequential manner; thus, some are present in the oocyte and its vestment, the corona-cumulus complex (IL-1, IL-6, and CSF-1), whereas TNF appears only at the stage of six to eight-cell embryos. Inflammatory cytokines could not be detected in sperm samples. CONCLUSIONS: It is possible that these cytokines have a role in the regulation of embryonic development, maternal immunological recognition of pregnancy, and maintenance of proper hormonal environment.

Cytokines↗
PubMed2005

Comparative analysis of development-related gene expression in mouse preimplantation embryos with different developmental potential.

The potential of embryonic and somatic cell nuclear-transferred (NT) mouse oocytes to develop into young is low compared with bovine NT oocytes. To examine the reasons for the low developmental potential of NT mouse oocytes, we analyzed the gene expression patterns of six development-related genes (Oct4, Nanog, Stat3, stella, FGF4, and Sox2) during preimplantation development in manipulated oocytes with different potentials to develop into young using real-time polymerase chain reaction (PCR) methods. The manipulated oocytes were parthenogenetically activated oocytes and embryonic stem cell, cumulus cell, morula blastomere NT oocytes, and in vitro-cultured and in vivo-recovered embryos. The mRNA expression patterns in mouse NT-derived embryos markedly differed from in vivo and in vitro counterparts. Some transcript expression patterns in embryonic stem-cell NT oocytes resembled those of parthenogenetic oocytes. Of the six developmentally important transcripts examined in NT embryos, four had a downregulated expression pattern at the blastocyst stage. Our findings indicate that abnormal expression patterns of development-related genes during preimplantation development correlate with the low potential of NT oocytes to develop into young. Although more detailed information is required, Sox2 mRNA expression pattern in blastocysts seems to closely correlate with the developmental potential of NT embryos.

Animals↗
PubMed1994

Effects of protein source and co-culture on bovine embryo development in synthetic oviductal fluid medium.

Experiment 1 compared the development of 2- to 4-cell bovine embryos cultured in synthetic oviductal fluid with 20% fetal calf serum or 3.2% BSA and in the presence of oviductal cells, cumulus cells, or medium alone. More embryos developed in medium with serum, regardless of culture method (P=0.063). Oviductal cell co-culture resulted in more embryos developing to at least the morula stage (P or=0.400). Addition of serum to oviductal cell co-culture medium increased the number of excellent or good quality embryos (P=0.019). Experiment 2 further compared the development of 2-cell or 3- to 4-cell embryos co-cultured with oviductal cell suspensions in serum-supplemented synthetic oviductal fluid or M-199 medium. More 3- to 4-cell than 2-cell embryos developed to at least the morula stage (P<0.001). More embryos developed to at least the morula stage in synthetic oviductal fluid (P=0.083). Neither initial embryo cell stage nor medium type influenced the percentage of developing embryos that achieved the blastocyst stage or final morphological quality of embryos (P>or=0.535).

Journal Article↗
PubMed2002

Phenotypic effects of somatic cell cloning in the mouse.

Although a variety of phenotypes and epigenetic alterations have been reported in animals cloned from somatic cells, the exact nature and consequences of cloning remain unclear. We cloned mice using fresh or short-term cultures of donor cells (cumulus cells, immature Sertoli cells, and fetal or adult fibroblast cells) with defined genetic backgrounds, and then compared the phenotypic and epigenetic characteristics of the cloned mice with those of fertilization-derived control mice. Irrespective of the nucleus-donor cell type, about 50% of the reconstructed embryos developed to the morula/blastocyst stage, but about 90% of these clones showed arrested development between days 5 and 8, shortly after implantation. Most of the clones were alive at term, readily recovered respiration, and did not show any malformations or overgrowths. However, their placentas were two- to threefold larger than those of the controls, due to hyperplasia of the basal (or spongiotrophoblast) layer. Although there was significant suppression of a subset of both imprinted and non-imprinted placental genes, fetal gene suppression was minimal. The seven imprinted genes that we examined were all expressed correctly from the parental alleles. These findings were consistent for every cell type from the midgestation through term stages. Therefore, cloning by nuclear transfer does not perturb the parent-specific imprinting memory that is established during gametogenesis, and the phenotypic and epigenetic effects of cloning are restricted to placental development at the midgestation and term stages. Twelve male mice that were born in a normal manner following nuclear transfer with immature Sertoli cells (B6D2F1 genetic background) were subjected to long-term observation. They died earlier than the genotype-matched controls (50% survival point: 550 days vs. 1028 days, respectively), most probably due to severe pneumonia, which indicates that unexpected phenotypes can appear as a result of the long-term effects of somatic cell cloning.

Animals↗
PubMed2001

Refrigeration of donor cells in preparation for bovine somatic nuclear transfer.

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

Animals↗
PubMed2003

Preliminary study on somatic cell nuclear transfer in rabbits in Thailand.

The objective of the study was to develop the somatic nuclear transfer technique by using rabbits as the model. The oocyte recipients aged 16 h post coitus were collected surgically from 20 superovulated rabbit doe with 28 and 40 mg Follicle Stimulating Hormone (FSH) after mating with a vasectomized male. The metaphase II plate and 1st polar body of oocyte was later aspirated by enucleated micropipette under an inverted microscope. A single donor cell; cumulus cell or cultured or frozen fibroblast cell from passage 1 to 9 were transferred to enucleated oocyte and fused with triple DC pulses, 3.2 kv, 20 micros. The fused embryos were cultivated in TCM 199 NaHCO3 + 10 per cent fetal calf serum (FCS) for 4 days. The cleavage rate (2-cell stage) was 37.2 per cent (32/86) from eight experiments, and 18.8 per cent (6/32) developed to the early morula stage. This study also indicated that the enucleation pipette and the somatic cell type influenced the success.

Active Transport, Cell Nucleus↗
PubMed1994

Immunocytochemical localization of aromatase in the ovary of superovulated cattle, pigs and sheep.

The localization of aromatase, an enzyme converting androgens to estrogen, in the ovaries of superovulated cattle, pigs and sheep was studied immunocytochemically in the preovulatory and postovulatory period using anti-human placental aromatase cytochrome P-450 antiserum. Immunostaining for aromatase was detected in the granulosa cells of preovulatory follicles of all species studied. Theca interna cells were stained in preovulatory follicles in the pig but not in cattle and sheep. Interstitial gland cells, cumulus cells and oocytes were unstained in all species. In cattle and pig the corpora lutea were unstained whereas they displayed staining in the sheep. Preantral and small antral follicles were unstained during both the preovulatory and postovulatory period in all species. It is concluded that granulosa cells of preovulatory follicles are the main residence for aromatase activity in superovulated cattle, pig and sheep, whereas the activity of theca interna and corpora lutea is species specific.

Animals↗
PubMed1990

Use of human cumulus granulosa cells for in vitro screening of reproductive toxicants.

We investigated the possibility that human granulosa cells from the cumulus mass obtained during human in vitro fertilization/embryo transfer (IVF/ET) might be useful for screening of potential reproductive toxicants in vitro. The cumulus granulosa cells detached from the zona pellucida after fertilization were allowed to spontaneously adhere to the incubation dish following transfer (removal) of the embryo. These cumulus cells survived in culture for at least four additional days, appeared on simple inspection to be morphologically normal luteinized granulosa cells, and produced large amounts of progesterone (P) over the culture interval. Production gradually declined during culture in the absence of human chorionic gonadotropin (hCG); however, inclusion of hCG (100 ng/mL) in the medium maintained P production at control (day 1) levels. Introduction of estrogenic agents previously shown to suppress P production in porcine or human culture systems using mural granulosa cells showed comparable effects in this human cumulus cell system. 17 beta-estradiol (10(-5) M), clomiphene citrate (10(-5) M), and o,p-DDT (10(-5)) significantly inhibited hCG-supported P production by human cumulus cells in vitro. This system has the advantages that (1) human cumulus granulosa cells are readily available from IVF/ET programs, (2) the techniques for maintaining the cells in culture are extremely simple, (3) a marker of highly differentiated granulosa cell function (P production) can be reliably measured, and (4) the cells respond predictably like other comparable granulosa cell systems. We conclude that human cumulus cells are a readily available and useful resource for in vitro screening of potential female reproductive toxicants.

Chorionic Gonadotropin↗
PubMed2000

Metaphase I arrest and spontaneous parthenogenetic activation of strain LTXBO oocytes: chimeric reaggregated ovaries establish primary lesion in oocytes.

Oocytes of strain LT mice, and related strains such as LTXBO, exhibit a high incidence of arrest in the progression of meiosis at metaphase I (MI) and in spontaneous parthenogenetic activation. Activation of these oocytes within the ovary leads to the formation of ovarian teratomas. In this study, the role of the oocyte's companion granulosa cells, the cumulus cells, was investigated using fully grown oocytes matured in vitro after isolation from LTXBO mice. Results showed that the role of cumulus cells in MI arrest is dichotomous. Cumulus cells temporarily helped to sustain MI arrest, but they also promoted a delayed progression to metaphase II. Cumulus cells also promoted parthenogenetic activation that occurred in association with the delayed progression to metaphase II. Next, the question of whether the lesion(s) promoting MI arrest and spontaneous activation is due to defects in the somatic cells or is intrinsic to the oocyte was addressed using chimeric reaggregated ovaries. An improved method for completely exchanging the germ cell and the somatic cell compartments of ovaries from newborn mice is described. These chimeric reaggregated ovaries, grafted beneath the renal capsule of SCID mice, allowed the complete development of LTXBO oocytes to occur in association with somatic cells from control (B6SJLF(1)) ovaries and development of control oocytes in association with LTXBO somatic cells. Oocyte growth and follicular development appeared generally normal in reaggregated ovaries. High incidences of MI arrest and spontaneous activation of LTXBO oocytes occurred regardless of the genotype of the somatic cells. Moreover, there was a low incidence of MI arrest and spontaneous activation of control oocytes, even though they underwent complete development and maturation associated with LTXBO somatic cells. It is concluded that the phenotypes of MI arrest and parthenogenetic activation in LTXBO oocytes are defects caused by lesions intrinsic to the oocyte. Nevertheless, the oocyte's companion somatic cells play crucial roles in the expression of these lesions.

Animals↗
PubMed2006

Nuclear and cytoskeletal dynamics during oocyte maturation and development of somatic cell cloned pig embryos injected with membrane disintegrated donor cells.

The objectives of this study were to characterize the nuclear and cytoskeletal changes of pig oocytes during in vitro maturation (IVM) and the development of the reconstructed embryos after injection with membrane intact or disintegrated donor cells. Cumulus-oocyte complexes (COCs) were collected from abattoir ovaries by follicle (2-8mm) aspiration. In Experiment 1, COCs were cultured in NCSU-23 medium for 0, 11, 22, 33, and 44 h. Oocytes were fixed at different time points for nuclear and cytoskeletal labeling. Forty-three percent and 75% oocytes progressed to MII stage at 33 and 44 h after IVM culture, respectively. Dynamic shift of spindle and cytoplasmic microtubules was evident. In Experiment 2, matured oocytes were injected with either the whole cumulus cell with or without intact cell membranes after enucleation. The reconstructed oocytes were fixed at 0, 2, or 4 h after cell injection for nuclear and cytoskeletal evaluation. When an intact cumulus cell was injected, the injected cell remained intact within 4h after injection. When a cell with disintegrated membrane was injected, 59-63% (n=146) of the injected cell underwent premature chromosome condensation (PCC). In Experiment 3, the reconstructed pig oocytes received membrane-disintegrated cumulus cells or fetal fibroblasts were cultured in PZM medium. The blastocyst rate of the fibroblast-injected embryos was 10%, which was lower than the non-cloned parthenotes (33%, P<0.05) but higher than the cumulus cell-injected embryos (2.7%). These results suggest that pig oocytes are subjected to nuclear and cytoskeletal reorganization during maturation. Pig oocytes injected with membrane-disintegrated fibroblast cells support better blastocyst development of the cloned embryos.

Animals↗
PubMed2003

Disrupted function of tumor necrosis factor-alpha-stimulated gene 6 blocks cumulus cell-oocyte complex expansion.

During ovulation, the oocyte and surrounding somatic cumulus cells contained within a specialized, mucoid matrix are released from the ovary. One matrix component, TNF-alpha-stimulated gene 6 (TSG-6), is a hyaluronan binding protein induced in cumulus cells of preovulatory follicles by the LH surge and is decreased in cumulus cells of COX-2 and prostaglandin E2 (PGE2) receptor subtype EP2 null mice that exhibit impaired ovulation and cumulus expansion. To determine if TSG-6 was hormonally induced in cumulus cells in vitro and was functional during the formation of the expanded matrix, we established a cumulus cell-oocyte complex (COC) culture system. This system was used to analyze the effects of FSH, PGE2, EP2 receptor, and selected protein kinase inhibitors on TSG-6 production as well as specific antibodies to the TSG-6 link module on TSG-6 function. We document that TSG-6 message and protein are induced by cAMP/protein kinase A/MAPK signaling pathways and that blocking these cascades prevents expansion and the production of TSG-6. FSH but not PGE2 rescued expansion and production of TSG-6 in the EP2 null COCs, indicating that generation of a cAMP signal is essential. Furthermore, disruption of the functional interactions between TSG-6, inter-alpha trypsin inhibitor, and hyaluronan with specific antibodies severely altered matrix formation and cumulus expansion, as recorded by time-lapse imaging. Collectively, these results indicate that TSG-6 mRNA is induced in cumulus cells in culture by cAMP and that the secreted TSG-6 protein is a key structural component of the mouse COC matrix.

Animals↗
PubMed2005

Proviral DNA of caprine arthritis encephalitis virus (CAEV) is detected in cumulus oophorus cells but not in oocytes from naturally infected goats.

The aim of this study was to determine whether oocytes taken from ovarian follicles in 123 naturally infected goats were carrying the proviral CAEV genome. Examination of DNA isolated from 190 batches of oocytes with intact cumulus cells and 190 batches of oocytes whose cumulus cells had been removed, taken from follicles of the same ovaries, demonstrated that 42/190 batches of oocytes with intact cumulus cells had the proviral CAEV genome, whereas none of the 190 batches of oocytes without cumulus cells were positive for the provirus. To confirm that the proviral genome was present in the cumulus cells and not in the oocyte cells, 586 oocytes from 56 different ovaries, were separated from their cumulus cells. The DNA was then extracted from each fraction and examined. The purity of the oocyte fraction was verified by searching for granulosa cell-specific mRNA, using RT-PCR; this was negative in all the batches of oocytes in which the cumulus cells had been removed. PCR analysis demonstrated that none of the oocytes without cumulus cells were positive, whereas 22/56 of the batches with cumulus cells were found to be positive. This study clearly demonstrates that despite being surrounded by infected cumulus cells, the oocytes are not infected, and that the enzymatic and mechanical technique for removing the cells surrounding the oocyte, as used in this study, is effective, thus enabling CAEV-free oocytes to be obtained from infected goats.

Animals↗