Effect of cumulus and granulosa cells on meiosis resumption in murine oocytes in vitro

Effects of different follicular cell types on resumption of meiosis were studied. Cumulus enclosed oocytes (CEO), denuded oocytes (DO), cumulus cells (CCs) and mural granulosa cells (GCs) were used. Oocytes were obtained from mature gonadotrophin-stimulated and unstimulated mice. The resumption of meiosis was assessed by the germinal vesicle breakdown (GVBD) at the end of cultivation. It has been shown that GCs produced a meiosis activating substance due to gonadotrophin stimulation; for meiosis resumption connections between CCs and the oocyte were not necessary, but the very production of the meiosis activating substance, was, however, dependent on the initial connection between CCs and the oocyte. The presence of oocyte was necessary for stimulating CCs to produce a diffusible heat stable meiosis activating substance; gonadotrophins induced CCs to produce a diffusible thermostable meiosis activating substance. This substance induced, in a paracrine fashion, resumption of meiosis directly. It is proposed that the heat stable meiosis activating component of the used media from gonadotrophins-stimulated CEO may belong to a kind of meiosis activating sterols, previously isolated from human follicular fluid and from adult bull testes.     

Development of rabbit parthenogenetic oocytes and nuclear-transferred oocytes receiving cultured cumulus cells

The present study determined a suitable parthenogenetic activation procedure for rabbit oocytes and examined the developmental potential of enucleated oocytes receiving cultured cumulus cells. Unfertilized oocytes recovered from superovulated rabbits were activated with one or two sets of electrical pulses, with or without subsequent administration of 6-dimethylaminopurine (6-DMAP). The proportion of oocytes treated with one or two sets of electrical pulses and 6-DMAP that cleaved (87% and 98%, respectively) and developed into blastocysts (77% and 85%, respectively) was significantly higher (P < 0.05) than those activated with electrical pulses alone (30% and 42% for cleavage, 7% and 17% for blastocysts). Cumulus cells separated from ovulated oocytes obtained from mature rabbits were cultured for three to five passages and then induced to quiescence by serum starvation before nuclear transfer. The enucleated oocytes receiving cumulus cells were activated with electrical pulses followed by the addition of 6-DMAP, and cultured in vitro for 5 to 6 d or transferred to pseudopregnant recipient females 1 d after activation. Of 186 nuclear-transferred oocytes, 123 (66%) cleaved and 42 (23%) developed into blastocysts. After transfer of 174 nuclear-transferred oocytes to 8 recipient females, a total of 3 implantation sites were observed in 3 recipient females but no fetuses were obtained.     

Resumption of meiosis in pig oocytes cultured with cumulus and parietal granulosa cells: the effect of protein synthesis inhibition

In denuded and cumulus-enclosed pig oocytes, puromycin at concentrations 5, 10, and 25 micrograms/ml did not lower the rate of germinal vesicle breakdown (GVBD) after 24 h of culture. GVBD was prevented in 50, 75, and 100 micrograms/ml of puromycin. After 40 h of culture, 5 and 10 micrograms puromycin/ml impaired significantly incidence of metaphase II (42 and 30%), respectively. Concentrations of 25 and 50 micrograms puromycin/ml absolutely prevented the first polar body (I PB) expulsion. The results indicated that GVBD in pig oocytes is far less sensitive to puromycin than I PB expulsion. Culture of cumulus-enclosed pig oocytes isolated with a piece of membrana granulosa (C + P oocytes) did not allow GVBD after 24 and 32 h in control medium. After 24 h of culture, GVBD occurred in 43 and 56% of C + P oocytes in the medium supplemented with 17 and 25 micrograms puromycin/ml. GV was broken down in 80 and 68% of C + P oocytes cultured in 17 and 25 micrograms puromycin/ml for 32 h. It is concluded that inhibition of protein synthesis by puromycin released pig oocytes from the block exerted by granulosa cells.     

Detection of ovine lentivirus in the cumulus cells, but not in the oocytes or follicular fluid, of naturally infected sheep

The aim of this study was to examine the Maedi-Visna virus (MVV) infection status of oocytes, cumulus cells, and follicular fluid taken from 140 ewes from breeding flocks. MVV proviral-DNA and MVV RNA were detected using nested-PCR and RT-PCR MVV gene amplification, respectively in the gag gene. Nested-PCR analysis for MVV proviral-DNA was positive in peripheral blood mononuclear cells in 37.1% (52/140) of ewes and in 44.6% (125/280) of ovarian cortex samples. The examination of samples taken from ovarian follicles demonstrated that 8/280 batches of cumulus cells contained MVV proviral-DNA, whereas none of the 280 batches of oocytes taken from the same ovaries and whose cumulus cells has been removed, was found to be PCR positive. This was confirmed by RT-PCR analysis showing no MVV-viral RNA detection in all batches of oocytes without cumulus cells (0/280) and follicular fluid samples taken from the last 88 ovaries (0/88). The purity of the oocyte fraction and the efficacy of cumulus cell removal from oocytes was proved by absence of granulosa cell-specific mRNA in all batches of oocytes lacking the cumulus cells, using RT-PCR. This is the first demonstration that ewe cumulus cells harbor MVV genome and despite being in contact with these infected-cumulus cells, the oocytes and follicular fluid remain free from infection. In addition, the enzymatic and mechanical procedures we used to remove infected-cumulus cells surrounding the oocytes, are effective to generate MVV free-oocytes from MVV-infected ewes.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

The presence of cumulus cells on nuclear maturation of sheep oocytes during in vitro maturation

This study was carried out to investigate the role of maturation by cumulus cells and the initial bond between the cumulus cells and the oocyte on nuclear maturation of sheep oocytes. Ovaries collected from the local abattoir were transported to the laboratory in saline at 30–35 °C within 1–3 h after collection. The oocytes of follicles, 2–6 mm in diameter, were recovered by aspiration and collected in a pre-incubated (at 38.6 °C, 5% CO₂, and 100% humidity) Hepes-modified TCM 199 medium. After preliminary evaluation, the oocytes with evenly granulated cytoplasm and which were surrounded with at least two layers of cumulus cells (good quality oocytes) were selected and subjected to culture in pre-incubated bicarbonate-buffered TCM 199 supplemented with 0.05 IU/ml recombinant human follicle stimulating hormone (rhFSH), 1 IU/ml human chorionic gonadotropin (hCG), and 1 μg/ml estradiol (OCM: oocyte culture medium). Before culturing, the selected oocytes were randomly divided into four treatment groups: Group 1, cumulus enclosed oocytes cultured in OCM; Group 2, denuded oocytes cultured in OCM; Group 3, denuded oocytes co-cultured with a cumulus cell monolayer in OCM; Group 4, denuded oocytes cultured in OCM in the presence of cumulus cells-conditioned medium. After an incubation period (26–27 h), the nuclear status of the oocytes in each treatment group was assessed using a 2% orcein stain. The rate of oocytes reaching the metaphase II (MII) stage (metaphase of second stage of meiosis division) was 82%, 5%, 11%, and 47% for Groups 1, 2, 3, and 4, respectively. The differences between groups were significantly (P < 0.05) different. The percentage of MII oocytes in Group 4 (47%) was higher than that obtained in Group 3 (11%), indicating a higher efficiency in a cumulus cell-conditioned medium, compared to the cumulus cells monolayer in providing the proper condition for sheep oocyte nuclear maturation. The results suggest the ability of sheep oocytes to resume meiosis in the absence of gap junctional communication (GJC) between the cumulus cells and oocyte being drastically interrupted while for optimum oocyte nuclear maturation, the intact physical contact between the oocyte and cumulus cells is essential.     

Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.     

Studies on the effects of lactate transport inhibition, pyruvate, glucose and glutamine on amino acid, lactate and glucose release from the ischemic rat cerebral cortex

A rat four vessel occlusion model was utilized to examine the effects of ischemia/reperfusion on cortical window superfusate levels of amino acids, glucose, and lactate. Superfusate aspartate, glutamate, phosphoethanolamine, taurine, and GABA were significantly elevated by cerebral ischemia, then declined during reperfusion. Other amino acids were affected to a lesser degree. Superfusate lactate rose slightly during the initial ischemic period, declined during continued cerebral ischemia and then was greatly elevated during reperfusion. Superfusate glucose levels declined to near zero levels during ischemia and then rebounded beyond basal levels during the reperfusion period. Inhibition of neuronal lactate uptake with alpha-cyano-4-hydroxycinnamate dramatically elevated superfusate lactate levels, enhanced the ischemia/reperfusion evoked release of aspartate but reduced glutamine levels. Topical application of an alternative metabolic fuel, glutamine, had a dose dependent effect. Glutamine (1 mM) elevated basal superfusate glucose levels, diminished the decline in glucose during ischemia, and accelerated its recovery during reperfusion. Lactate levels were elevated during ischemia and reperfusion. These effects were not evident at 5 mM glutamine. At both concentrations, glutamine significantly elevated the superfusate levels of glutamate. Topical application of sodium pyruvate (20 mM) significantly attenuated the decline in superfusate glucose during ischemia and enhanced the levels of both glucose and lactate during reperfusion. However, it had little effect on the ischemia-evoked accumulation of amino acids. Topical application of glucose (450 mg/dL) significantly elevated basal superfusate levels of lactate, which continued to be elevated during both ischemia and reperfusion. The ischemia-evoked accumulations of aspartate, glutamate, taurine and GABA were all significantly depressed by glucose, while phosphoethanolamine levels were elevated. These results support the role of lactate in neuronal metabolism during ischemia/reperfusion. Both glucose and glutamine were also used as energy substrates. In contrast, sodium pyruvate does not appear to be as effectively utilized by the ischemic/reperfused rat brain since it did not reduce ischemia-evoked amino acid efflux.     

Inhibitory effects of the porcine follicular fluid on estradiol and progesterone secretion by cultured rat granulosa cells

The effect of porcine follicular fluid on estradiol and progesterone secretion was examined using a rat granulosa cell culture with FSH and testosterone in the medium. Follicular fluids from small (less than 5 mm) and large (greater than 6 mm) follicles (SFFI, LFF1) were treated with charcoal, and then fractionated by filtration through an Amicon XM-50 and an PM-10 membrane. The addition of 25% SFF1 and LFF1 into the culture system significantly inhibited estradiol and progesterone secretion (P less than 0.005). These inhibitory activities were observed in PM-10 retentates (10,000-50,000 MW) and filtrates (less than 10,000 MW) of SFF1 and LFF1. The addition of XM-50 filtrates (less than 50,000 MW) of SFF1 and LFF1 caused a dose-dependent inhibition of estradiol and progesterone secretion. The dose-response relationship between the filtrates and estradiol secretion was linear with a significant correlation coefficient. The addition of the filtrates exerted no inhibitory effect on the growth of the cells cultured. XM-50 filtrate of LFF1 from a batch with a low ratio of small/large follicles showed a lower inhibitory activity on estradiol secretion than that of LFF1, while the inhibitory activities in both filtrates on progesterone secretion were almost equivalent. These results suggest that the follicular fluid of small porcine follicle contains nonsteroidal regulators capable of inhibiting estradiol and progesterone secretion by cultured rat granulosa cells, and that the estradiol secretion inhibitor activity decreases in the fluid of large follicle while the progesterone secretion inhibitor activity does not decrease in it.     

Meiotic state of bovine oocytes is regulated by interactions between cAMP,cumulus, and granulosa

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.     

Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes

Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the first polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 microl droplets of three different culture systems: (1) TCM-199 + steer serum (10%): (2) TCM-199 + steer serum (10%) + PMSG (40 IU/ml); and (3) TCM-199 + steer serum (10%) + PMSG (40 IU/ml) + estradiol 17beta (1 microg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 degrees C) mineral oil and incubated in a CO2 incubator (39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 16-18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying first polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9-10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of first polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22-24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded first polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies.     

Chromosome configuration during in vitro maturation of goat, sheep and buffalo oocytes

The competence of meiotic chromosome configuration at the time of co-culture of oocytes with spermatozoa is an essential prerequisite for successful in vitro fertilization (IVF). Although this technology has been used in several livestock species, various intrinsic and extrinsic factors affecting the high repeatablity of IVF have yet to be understood. The present study was conducted to determine the appropriate time for coculture of oocytes and spermatozoa in order to optimize the fertilization rate in sheep, goats and buffalo. Oocytes were collected from the ovaries of slaughtered animals. The oocytes were divided into 10 groups and cultured for maturation in TCM-199 supplemented with estrous cow serum for different durations at 38.5 x 0.5/C in a CO(2) incubator. Sheep and goat oocytes were removed from culture medium after 0,6,12,22,24,26,28,30,32 and 36 and buffalo oocytes after 0,6,12,16,20,22,24,26,28, and 36 h. The oocytes were treated with hypotonic solution (0.75 M KCl) and fixed in Carony's fixative on glass slides. The fixed oocytes were stained with Giemsa solution, and the meiotic chromosomes were evaluated under a compound microscope at x 1000 magnification. Observations were recorded on a total of 1328 oocytes (sheep, 409; goat, 727 and buffalo, 192). The sequential configurations of diffused chromatin, pachytene, diplotene (along with nucleoli), diakinesis and metaphase II (MII) were analyzed at different durations of culture. Control oocytes (fixed at 0 h without incubation) were mostly at the pachytene stage, and as the duration of culture increased the instances of diplotene, diakinesis and finally MII increased. Oocytes at the MII stage of meiosis are known to be at the optimal stage of development for co-culture with spermatozoa and successful in vitro fertilization. On the basis of sequential configuration of chromosomes, it was found that the optimal duration of in vitro maturation of oocytes is 32, 30 and 24 h for sheep, goats and buffalo, respectively.     

The movement of pyruvate, lactate and lactate dehydrogenase into rabbit oviductal fluid.

Pyruvate, lactate and lactate dehydrogenase appeared linearly in 2 ml 0.9% NaCl recirculated through the rabbit oviduct for 4 h in vivo. In oviducts from rabbits injected 3 days previously with 100 i.u. hCG, the rate of appearance of all three constituents was considerably reduced. It is considered unlikely that the lactate dehydrogenase secreted brings about the interconversion of pyruvate and lactate in the oviduct lumen.     

Effect of intraovarian factors on porcine follicular cells: cumulus expansion, granulosa and cumulus cell progesterone production

The role of granulosa cell conditioned media (CM) containing luteinization stimulator (LS), and the role of EGF in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral follicles was investigated. The CM were prepared by incubation of granulosa cells isolated from large antral follicles. After 24h incubation, more than 61 or 64% of OCC expanded to the +3 and +4 stage in the presence of CM (50%) or EGF (10ng/ml), respectively. The stimulatory effect of LS and EGF on the cumulus expansion was accompanied by the enhanced hyaluronic acid synthesis. Complete suppression of cumulus expansion stimulated by LS and EGF was observed in the presence of 10 micromol/l genistein (tyrosine kinase inhibitor), in the presence of 10mmol/l LiCl (the inhibitor of inositol 1,4,5-trisphosphate metabolism), and 100 micromol/l gallopamil, verapamil and norverapamil (calcium channel blockers). Stimulatory effect of EGF on the cumulus expansion of OCC isolated from large follicles was accompanied by the increased cumulus cell progesterone production. However, EGF did not affect the progesterone production by OCC isolated from small follicles. To determine whether EGF could modulate the granulosa cell steroidogenesis also, the effect of EGF on granulosa cells isolated from large (LGC) and small (SGC) follicles was compared. EGF (10ng/ml) failed to affect the progesterone synthesis during 72h culture of SGC but significantly enhanced the LGC progesterone production. Our results indicate that luteinization factor stimulates the cumulus expansion and hyaluronic acid synthesis by the OCC isolated from large antral follicles. The mechanism of LS- and EGF-induced cumulus expansion may involve tyrosine kinase activation and calcium mobilization. In addition, these results indicate the different response of porcine cumulus and granulosa cells originating from small and large follicles on the stimulatory effect of EGF.     

Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells

The present study was conducted to determine the effects of cumulus cells and sodium pyruvate during in vitro maturation of bovine oocytes on maturation, fertilization, and subsequent development. Cumulus-enclosed oocytes (CEOs) and cumulus-denuded oocytes (CDOs) were cultured for 24 h in polyvinylpyrrolidone-Hepes-tissue culture medium 199 with or without sodium pyruvate. Oocytes were fertilized in vitro and then cultured in CR1aa for 10 days. Before in vitro fertilization, the glutathione (GSH) content of some oocytes was measured. Maturation and normal fertilization rates of CDOs cultured with sodium pyruvate and CEOs were higher than that of CDOs cultured without sodium pyruvate. The CEOs showed significantly higher rates of development to the blastocyst stage than CDOs. The GSH contents of oocytes significantly decreased in CDOs after maturation culture, but the GSH contents of oocytes in CEOs remained at the same level as oocytes before culture. These results indicate that sodium pyruvate promotes nuclear maturation of bovine CDOs and that a continuing presence of cumulus cells during maturation is important for subsequent development of zygotes to the blastocyst stage. However, blastocysts produced from CDOs in the presence of sodium pyruvate showed a developmental competence to be normal calves, but it is not known if CDOs cultured without sodium pyruvate also were capable of developing into calves.