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PubMed1984

Regulation of murine oocyte meiosis: evidence for a gonadotropin-induced, cAMP-dependent reduction in a maturation inhibitor.

We have developed an assay that can detect relative changes in the amount of a non-cAMP inhibitor of maturation present in cumulus cells (Eppig et al., 1983, Dev. Biol., 100:39-49). Using this assay in which accelerated maturation of a group of treated cumulus cell-oocyte complexes relative to untreated complexes indicates a decrease in the amount of inhibitor, results of the experiments described here suggest a possible relationship between elevation of cAMP levels and subsequent decreased amounts of a non-cAMP inhibitor. Mouse oocytes obtained from cumulus cell-oocyte complexes treated with luteinizing hormone (LH) resumed meiosis prior to oocytes obtained from untreated complexes; the degree of acceleration of maturation was dependent on LH concentration. A similar result was obtained with follicle-stimulating hormone (FSH). Correlated with LH- or FSH-acceleration of maturation was an LH- or FSH-induced elevation of cumulus cell cAMP levels. Inhibiting LH-induced elevation of cumulus cell cAMP levels inhibited LH-induced acceleration of maturation. An initial incubation of complexes in medium containing dibutyryl cAMP (dbcAMP) also promoted acceleration of maturation. In contrast, maturation of denuded oocytes was not altered by treatment with either LH, FSH, or dbcAMP. Complexes initially incubated in dbcAMP-containing medium still demonstrated acceleration of maturation after a subsequent 2 h incubation in dbcAMP-free medium. Relative to untreated complexes, none of these treatments disrupted intercellular communication between cumulus cells and the oocyte. Elevating follicle cAMP levels with cholera toxin induced maturation of follicle-enclosed oocytes when cumulus cell-oocyte coupling was still fully maintained. These results are interpreted to indicate that gonadotropin-mediated acceleration of maturation is via a cAMP-dependent reduction in the level of a maturation inhibitor present in granulosa/cumulus cells.

Animals↗
PubMedDate not supplied

Effect of growth factors on proliferation, apoptosis and protein kinase A expression in cultured porcine cumulus oophorus cells.

The proliferation, apoptosis and protein kinase A (PKA) in porcine cumulus oophorus (CO) before and after 40 h of culture together with oocytes in the presence of IGF-I, IGF-II and EGF (all at 10 ng x mL(-1) medium) were compared. Cellular proliferation, apoptosis and PKA contents were evaluated by immunocytochemistry using specific antibodies against PCNA, TUNEL and catalytic (C-alpha) and regulatory (RI) subunits of PKA. The in-vitro culture of oocyte-CO complexes in a basal medium was accompanied by a decrease in the proportion of PCNA-positive CO cells (from 51 to 36%, p < 0.05). The addition of either IGF-I or EGF to the culture medium prevented this process and increased the proliferation rate (64 and 67% respectively, p < 0.001). During culture, the percentage of apoptotic (TUNEL-positive) CO cells increased from 42 to 57% (p < 0.01). The addition of IGF-I or EGF resulted in the inhibition of apoptosis to 36 and 12% respectively (p < 0.001). IGF-II and EGF reduced the amount of PKA catalytic subunits in the CO (percentage of cells with immunoreactive PKA catalytic subunits (28%, p < 0.05 and 27%, p < 0.05 respectively; versus control -41%), whilst the effect of IGF-I on this index was insignificant (31%). The expression of the PKA regulatory subunit was increased by EGF (51% compared with 29% in the control, p < 0.05), but not by IGF-I or IGF-II (30 and 29%). Our observations demonstrate that 40 h of culture of porcine CO resulted in a decrease in the proliferation and development of apoptosis in CO cells. IGF-I or EGF can stimulate proliferation and inhibit apoptosis. The influence of growth factors on the PKA content of the CO suggests that cAMP/PKA may be a mediator of the action of growth factors on these cells. The differential effects of IGFs and EGF on the regulatory subunit of PKA may indicate differences between their mechanisms of action.

Animals↗
PubMed1995

Effect of a serum extender containing growth factors on development of IVM and IVF bovine embryos.

The present experiments were conducted to determine if supplementation of the culture medium with a serum extender containing growth factors would increase development of bovine embryos into morulae or blastocysts, following in vitro maturation (IVM) and in vitro fertilization (IVF). In Experiment 1, bovine zygotes were cultured in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 2, bovine zygotes were cultured in the presence of cumulus cells in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 3, bovine oocytes were matured in Medium 199 supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 4, oocytes were matured in Medium 199 with 10% fetal bovine serum (FBS) or 5% FBS with serum extender. Following maturation, zygotes were cultured in CR1 medium with 10% FBS or 5 % FBS and serum extender. In all 4 experiments, the embryos were cultured in vitro until Day 7 after IVF, and development to the morula or blastocyst stage was assessed. The findings of the first 2 experiments showed that the serum extender did not directly influence embryo development but did stimulate development when cumulus cells were included in the culture system. The remaining 2 experiments showed that the serum extender did influence development through its interactions with cumulus cells during maturation and/or culture. These findings suggest that although growth factors or other products do not directly stimulate bovine embryo development their effects may be mediated through secondary cell systems.

Journal Article↗
PubMed2005

Atrial natriuretic peptide inhibits the actions of FSH and forskolin in meiotic maturation of pig oocytes via different signalling pathways.

Atrial natriuretic peptide (ANP) as well as its receptors is found in mammalian ovary and follicular cells and its function in oocyte meiotic maturation has also been reported in Xenopus, hamster and rat. But the results are controversial and the physiological mechanism of ANP on oocyte maturation is not clear, especially the relationship between gonadotrophin and ANP as well as the signal transduction, and these need further study. The present study conducted experiments to examine these questions by using drug treatment and Western blot analysis and focused on pig oocyte meiotic maturation and cumulus expansion in vitro. The results revealed that ANP could inhibited FSH-induced pig oocyte maturation and cumulus expansion and prevent the full phosphorylation of mitogen-activated protein kinase in both oocytes and cumulus cells, and that these inhibitory effects could be mimicked by 8-Br-cyclic guanosine 5'-monophosphate (8-Br-cGMP), but blocked by a protein kinase G (PKG) inhibitor KT5823. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, could enhance the inhibitory effect of ANP on oocyte maturation. A specific analogue of ANP, C-ANP-(4-23), which binds to the natriuretic peptide receptor-C (NPRC), had no effect in either FSH-induced or spontaneous oocyte maturation. Treatment with forskolin, a stimulator of adenylate cyclase, had a biphasic effect; 44 h treatment induced cumulus expansion but inhibited oocyte maturation while 2 h treatment induced maturation of cumulus-enclosed oocytes (CEOs). Both ANP and C-ANP-(4-23) could inhibit the effect of forskolin on CEO maturation, and these inhibitory effects of ANP/C-ANP-(4-23) could be blocked by preincubation with pertussis toxin (PT), consistent with mediation by a Gi protein(s) in the cumulus cells. All these results suggest that ANP is a multifunctional regulator of FSH and forskolin on pig CEO maturation by two signalling mechanisms: one is via a cGMP/PKG pathway, the other is via NPRC receptors in cumulus cells and the activation of the PT-sensitive Gi protein(s).

Animals↗
PubMed1998

Ultrastructural features of bovine cumulus-corona cells surrounding oocytes, zygotes and early embryos.

Integrated transmission and scanning electron microscopic (TEM and SEM) techniques have provided the first detailed description of the ultrastructural features of the bovine cumulus-corona (CC) cells surrounding oocytes at the time of final maturation, zygotes and early cleaving embryos (2/4 to 6/8 blastomeres). TEM revealed the presence of rough endoplasmic reticulum and Golgi complexes in the cytoplasm of CC cells surrounding immature, mature and fertilized eggs, and also revealed an increasing amount of smooth endoplasmic reticulation membranes, lipid droplets and mitochondria with villiform and/or tubular cristae in the cytoplasm of CC cells during maturation and fertilization of the oocyte. In addition, a loss of cell-to-cell junctions between CC cells was evident. TEM also demonstrated that a few residual CC cells were still associated with early embryos and that these cells showed rather degenerative or apoptotic patterns, the latter pattern also observed on cells associated with fertilized eggs. SEM revealed that the complex of CC cells of immature oocytes was compact with narrow intercellular spaces, which progressively enlarged in size around mature oocytes. This phenomenon is mostly due to the production of abundant extracellular matrix. Immature CC cell complexes possessed characteristic long and filiform microvilli whereas the surface of CC cells surrounding mature oocytes showed numerous blebs and occasional large cytoplasmic protrusions as well as microvilli. Zygotes and early embryos were covered with a few polyhedral CC cells possessing scarce and short microvilli and a large amount of pleomorphic blebs. This study demonstrated a precocious luteinization occurring in bovine CC cells at ovulation until zygote segmentation, and this process was associated with a progressive apoptotic mechanism that ended in the complete denudation of the zona pellucida covering the early embryo. The presence of CC cells around the maturing oocyte and fertilized egg could have important functions related to the microenvironmental requirements of ovum maturation as well as facilitating activities related to fertilization.

Animals↗
PubMed2006

The signaling pathways linking to lysophosphatidic acid-promoted meiotic maturation in mice.

The signaling pathways linking to lysophosphatidic acid-promoted meiotic maturation in mice were studied. When mouse oocyte-cumulus cells complexes were cultured with 10(-5) M lysophosphatidic acid (the LPA group), the rate of oocyte nuclear maturation was significantly increased. Additions of pertussis toxin, genistein, U73122, Ro320432, PD98059 or SB203580 significantly suppressed the increase in lysophosphatidic acid-stimulated nuclear maturation rate. These results suggested that Gi/o-coupled lysophosphatidic acid receptors activate phosphatidylinositol-specific phospholipase C, and result in ERK and MAP kinase activation, which is triggered by diacylglycerol-dependent protein kinase C. When intracellular cAMP concentrations of oocytes in the LPA and control groups were measured using the acetylation assay, the intracellular cAMP concentration of an oocyte in the LPA group was significantly lower than the control oocyte (0.117+/-0.04 fmol/oocyte vs. 0.176+/-0.036 fmol/oocyte, p<0.05). In conclusion, our results suggested that lysophosphatidic acid stimulates phospholipase C through a Gi-protein linked receptor on the surface of mouse cumulus cells and stimulates both extracellular signal-regulated kinase and p38 mitogen-activated kinase, resulting in the closure or loose of gap junctions between cumulus cells and the oocyte. The resultant early decrease of oocyte cAMP levels may promote nuclear maturation of mouse oocytes in vitro.

Animals↗
PubMed2006

Cryopreservation of in vitro matured buffalo (Bubalus bubalis) oocytes by minimum volumes vitrification methods.

The aim of this study was to evaluate the efficiency of the solid surface vitrification (SSV) and the cryoloop vitrification (CLV) methods to cryopreserve in vitro matured buffalo oocytes. Another objective of the work was to investigate whether the presence of cumulus cells affects the efficiency of oocyte vitrification in this species. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol, 5% polyvinyl-pyrrolidone and 0.4% trehalose and they were warmed in a 0.3M trehalose solution. In the CLV method, oocytes were vitrified in 16.5% ethylene glycol and 16.5% dimethyl sulfoxide and warmed in decreasing concentrations of sucrose. The oocytes that survived vitrification were fertilized and cultured in vitro up to the blastocyst stage. Although high survival rates were recorded in all groups, when the oocytes were vitrified by the CLV method in the absence of cumulus cells, the survival rate was significantly (P<0.05) lower. However, the CLV gave a significantly higher cleavage rate compared to the SSV with the denuded oocytes (45% versus 26%, respectively; P<0.05), whereas no differences were found between methods with the cumulus-enclosed oocytes (14% versus 15%, respectively). Blastocysts were produced for the first time from in vitro matured oocytes that were vitrified-warmed in buffalo. Nevertheless, vitrification significantly decreased blastocyst yield, regardless of both the method employed and the presence or absence of cumulus cells.

Animals↗
PubMed2006

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: possible role of the hexosamine pathway as a regulator of developmental competence.

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.

Animals↗
PubMed1999

Uptake and metabolism of adenosine mediate a meiosis-arresting action on mouse oocytes.

This study was carried out to evaluate the possible role of adenosine uptake and metabolism in mediating the inhibitory actions of this nucleoside on spontaneous mouse oocyte maturation. Uridine blocked 3H-adenosine uptake by oocyte-cumulus cell complexes (OCCs) and cumulus cell-enclosed oocytes (CEOs) by 82-85%, whereas uptake by denuded oocytes (DOs) was suppressed by 97%. Uridine had no effect on germinal vesicle breakdown (GVB) in CEOs when meiotic arrest was maintained with hypoxanthine or hypoxanthine plus adenosine but reversed the combined inhibitory action of these purines in DOs. Five of six adenosine analogs that bind to purinoceptors demonstrated meiosis-arresting activity but not in relation to their relative affinities for inhibitory or stimulatory adenosine receptors and only at high concentrations. Moreover, in DOs, uridine reversed the inhibitory effect of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, two receptor agonists that are poor substrates for adenosine-metabolizing enzymes. Results of experiments with adenosine kinase inhibitors showed that methylmercaptopurine riboside (MMPR) and tubercidin, but not 5'-amino-5'-deoxyadenosine, reversed meiotic arrest maintained by hypoxanthine +/- adenosine, but this required an additional inhibitory action on de novo purine synthesis. Inhibition of de novo purine synthesis alone was not sufficient because azaserine failed to reverse meiotic arrest. MMPR was a very potent meiosis-inducing agent, completely reversing meiotic arrest in CEOs and DOs in the presence of a variety of meiotic inhibitors. The adenosine deaminase inhibitor deoxycoformycin had opposite effects on oocyte maturation depending on the presence or absence of adenosine: the inhibitory action of hypoxanthine alone was bolstered, but the meiosis-arresting action of adenosine was reversed. These data therefore indicate that at low adenosine concentrations phosphorylation predominates, but at higher adenosine concentrations deaminated products contribute to the meiotic inhibition. This idea was borne out by the ability of inosine to mimic the synergistic interaction of adenosine with hypoxanthine. The action of adenosine is not due to deamination to inosine and conversion to nucleotides through the hypoxanthine salvage pathway because adenosine-mediated inhibition was not compromised in oocytes from mutant mice unable to salvage hypoxanthine.

Adenosine↗
PubMed2003

In vitro fertilization and polyspermy in the pig: factors affecting fertilization rates and cytoskeletal reorganization of the oocyte.

Polyspermy is a common phenomenon in the pig. Extensive information has become available from in vitro studies on not only the quality of oocytes but also the quality of spermatozoa. However, little information is available on the relative penetration rates of fresh and frozen spermatozoa from the same ejaculate from boars of different breeds. The present results, based on a total of 15 boars of three different breeds, revealed that the inter-breed variation in fertilization and polyspermic rates is larger than intra-breed variation. It was also shown that the incidence of polyspermy as well as penetration rate was greatly decreased by freezing and thawing, even if a higher number of sperm was coincubated with cumulus-free oocytes for a longer period compared to fresh sperm of the same ejaculate. This study focuses on the cytoskeletal organization of the oocyte with respect to the status of cumulus investment, and monospermic and polyspermic fertilization. The status of cumulus cells correlated with the density of transzonal cumulus-cell processes and with the maturation rate of oocytes and, to some degrees, the incidence of polyspermy. Polyspermic zygotes formed multiple microtubule domains in association with individual male pronuclei (PN), but in a high degree of polyspermy (more than trispermy), the pronuclear apposition did not proceed. The effect of multiple PN of paternal and maternal origin on the cytoskeletal reorganization is also discussed.

Animals↗
PubMed2003

Immunohistochemical localization and expression of the hyaluronan receptor CD44 in the epithelium of the pig oviduct during oestrus.

Hyaluronan is related to essential reproductive processes in pigs. Hyaluronan produced by cumulus cells builds, via specific cell surface receptors, an extracellular matrix responsible for cumulus cell cloud expansion during final oocyte maturation, a preparatory event for ovulation and fertilization. In addition, hyaluronan that has been localized in the pig oviduct both in the intraluminal fluid and on the surface of the lining epithelium of the preovulatory sperm reservoir, has proven beneficial during in vitro fertilization and embryo culture, thus indicating that it has a role in vivo. This study monitored the immunolocalization, protein determination and gene expression of the major cell surface hyaluronan receptor CD44 in the epithelial lining of the pig oviduct during selected stages of standing oestrus, in relation to spontaneous ovulation. The CD44 immunostaining in the lining epithelium was localized to the surface membrane and the supranuclear domain of mainly the secretory cells, particularly in the sperm reservoir of both treatment (inseminated) and control (non-inseminated) specimens. Up to four hyaluronan-binding protein (HABP) bands (60, 90, 100 and 200 kDa) were detected in the tubal epithelium, and the 200 kDa band was determined as CD44 by immunoblotting. The expression of CD44 mRNA was higher before than after ovulation (P < 0.05), most conspicuously in the uterotubal junction (UTJ). In addition, CD44 expression in the preovulatory UTJ and the ampullary-isthmic junction (AIJ) of control animals was higher than in those that were inseminated (P < 0.05 and P < 0.01 for UTJ and AIJ, respectively). The results demonstrate for the first time that the specific hyaluronan receptor CD44 is expressed by the oviduct epithelial cells during spontaneous oestrus, and is particularly abundant in the sperm reservoir before ovulation. Presence of spermatozoa in this segment seemed to downregulate the receptor. The variation in the expression of CD44 in relation to spontaneous ovulation and the presence of spermatozoa indicate that the hyaluronan CD44-signalling pathway may play a role in oviduct function during sperm storage and fertilization in pigs.

Animals↗
PubMed2000

Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro.

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P<0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P<0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P<0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P<0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P>0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.

Animals↗
PubMed1982

In the absence of protein, estradiol suppresses meiosis of porcine oocytes in vitro.

The effect of estradiol on the spontaneous maturation of porcine oocytes was investigated. Cumulus-enclosed (intact) and cumulus-free (denuded) oocytes were cultured in the presence of estradiol-17 beta (0 to 10 microgram/ml) in a chemically defined bicarbonate-buffered medium that contained either dextran or BSA, or in a complex Hepes-buffered medium that was supplemented with serum. After 24 hr, chromatin spreads were prepared and meiotic maturation was scored. The biochemical integrities of the cumulus cells were assessed by determination of the estradiol and progesterone content of spent media after culture of intact oocytes in the presence of 0.5 X 10(-6) M testosterone and 10 microgram/ml follicle-stimulating hormone. Estradiol did not significantly affect the onset of maturation of either intact or denuded oocytes that were cultured in medium containing either BSA or serum. In serum-supplemented medium, however, the progression of maturation beyond metaphase I was significantly affected by the steroid in a dose-dependent manner. The steroid significantly inhibited the release from meiotic arrest of both types of oocyte cultured in medium supplemented with dextran. Supplementation of all media with testosterone and FSH significantly stimulated the synthesis of estradiol by the cumulus cells, compared with that of control groups. The synthesis of progesterone, however, was significantly stimulated by testosterone and FSH only in the BSA and serum-supplemented media. It is concluded that exogenous estradiol has the capacity to arrest meiosis in vitro but that this capacity can only be expressed if no exogenous protein(s) is present. In the absence of exogenous protein, progesterone synthesis by the adherent cumulus cells is minimal.

Animals↗
PubMed2002

Transgenic strategy for demonstrating nuclear reprogramming in the mouse.

Totipotency can be restored to the nuclei of somatic cells by reprogramming the nucleus via the technique of nuclear transfer. As genes expressed in somatic cells differ from those expressed in early embryos, a change in gene expression must accompany nuclear reprogramming. In this study, nuclear reprogramming of somatic cell nuclei following nuclear transfer (NT) was demonstrated by the reactivation of developmentally regulated lacZ reporter genes. NT embryos were generated by direct injection of adult cumulus cell nuclei into mature mouse oocytes from which maternal chromosomes were subsequently removed. Cumulus cells were collected from transgenic mice which show developmentally regulated lacZ reporter gene expression as a result of integration and functional coupling of reporter gene expression with the endogenous Oct4 or COB54 genes. As both genes are transcriptionally silent in somatic cells but are expressed during early embryonic development, reactivation of reporter gene expression in cumulus cell-derived NT embryos was assessed as a means of monitoring nuclear reprogramming. The pattern of X-gal staining observed in individual NT embryos derived from both transgenic lines revealed that coordination of reactivation appeared to be specific for each gene, and that the timing of expression was consistent with that seen in control non-manipulated transgenic embryos. However, the staining in some NT embryos appeared to be diminished or absent. This reduction in staining may indicate a failure to reprogram gene expression in these embryos. Similar transgenic strategies employing lacZ vital stains, or alter-native reporters such as GFP, may facilitate real-time monitoring of reprogramming and a potential selection strategy to increase cloning efficiency through the identification and selection of reprogrammed, preimplantation stage NT embryos prior to embryo transfer.

Animals↗
PubMed2006

Membrane integrity and development of immature murine cumulus-oocyte complexes following slow cooling to -60 degrees C: the effect of immediate rewarming, plunging into LN2 and two-controlled-rate-stage cooling.

Cryopreservation of murine germinal vesicle (GV) stage cumulus-oocyte complexes (COCs) has been shown to result in poor development and cumulus cell damage. In an attempt to determine the stage of the cryopreservation protocol at which damage occurs, three cooling profiles were compared: slow-cooling (0.3 degrees C/min) to -60 degrees C (protocol A); slow-cooling to -60 degrees C and plunging to -196 degrees C (protocol B); or slow-cooling to -60 degrees C followed by further cooling at 10 degrees C/min to -150 degrees C, then plunging to -196 degrees C (protocol C). GV-stage COCs were collected from hormone-primed mice by repeated puncturing of ovarian follicles. COCs were exposed to 1.5 M Me(2)SO prior to cooling to -60 or -196 degrees C. Membrane integrity was assessed immediately after thawing using carboxy fluorescein and propidium iodide. A greater proportion of cumulus cells were damaged following protocol B than protocol A. Damage was less extensive following protocol C than following protocol B. For assessment of development, COCs were matured and fertilised in vitro. Morphological normality was significantly reduced following cooling to -60 or -196 degrees C compared with non-cryopreserved controls. Fertilisation of oocytes assessed as normal post-treatment was not significantly different between any of the groups. Development to blastocyst was least from oocytes exposed to protocol B, being significantly worse than for oocytes exposed to protocol A, but not significantly different to protocol C. A protocol comprising two stages of controlled-rate cooling decreased damage to the membranes of cumulus cells but did not significantly improve embryo development.

Animals↗
PubMed2001

Impaired maturation, fertilization, and embryonic development of porcine oocytes following exposure to an environmentally relevant organochlorine mixture.

The reproductive health risks related to exposure to persistent organic pollutants in the environment remain controversial. This debate is partly because most studies have investigated only one or two chemicals at a time, whereas populations are exposed to a large spectrum of persistent chemicals in their environment. Using the pig as a toxicological model, we hypothesized that exposing immature cumulus-oocyte complexes to an organochlorine mixture during in vitro maturation (IVM) would adversely affect oocyte maturation, fertilization, and subsequent embryo development. This organochlorine mixture mimics that which contaminates the Arctic marine food chain. Cumulus-oocyte complexes were cultured in IVM medium containing increasing concentrations of the organochlorine mixture, similar to that found in women of highly exposed populations. Organochlorines reduced the quality of cumulus expansion and the viability of cumulus cells in a dose-response manner. The proportion of apoptotic cumulus cells also increased due to organochlorine exposure. Half of the oocytes were fixed after insemination, and the remainders were cultured for 8 days. Concentrations of organochlorines did not affect the rates of oocyte degeneration, sperm penetration, and development to morula. However, incidence of incompletely matured oocytes increased and polyspermy rate decreased, both in a dose-response manner with increasing organochlorine concentrations. Blastocyst formation and number of cells per blastocyst declined with organochlorine concentration. Exposing porcine cumulus-oocyte complexes to an environmentally pertinent organochlorine mixture during IVM disturbs oocyte development, supporting recent concerns that such pollutants harm reproductive health in humans and other mammalian species.

Animals↗
PubMed1989

Enhanced viability after in vitro fertilization of bovine oocytes matured in vitro with high concentrations of luteinizing hormone.

The purpose of this study was to better understand requirements for oocyte maturation to yield viable embryos after bovine in vitro fertilization (IVF). High proportions (95% to 100%) of cumulus-surrounded oocytes matured in vitro in all treatments, but subsequent development was enhanced after maturation with high concentrations of purified bovine luteinizing hormone (LH). Beneficial effects of undisturbed cumulus cells were demonstrated. Improved IVF followed insemination of cumulus-surrounded oocytes, but not denuded oocytes, after maturation with high LH (100 micrograms/ml) versus low LH (10 micrograms/ml), implicating cumulus cells in mediating hormonal enhancement. Oocytes matured with high LH resulted in embryos of superior viability, as reflected by cleavage to 4- to 8-cell stages. Pregnancy resulting from transcervical embryo transfer further documented embryonic viability. These findings should be useful in further development and implementation of reproductive and genetic technologies.

Animals↗
PubMed1983

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.

Animals↗