In vitro maturation of bitch oocytes from advanced preantral follicles in synthetic oviduct fluid medium: serum is not essential

The ability of oocytes from preantral follicles to mature in vitro was assessed using a synthetic oviduct fluid (SOF) medium. Advanced preantral follicles (approximately 210 microm diameter) were isolated from the ovaries of domestic bitches and assigned to one of four treatment groups: (1) SOF (n = 230); (2) SOF + 3 mg/ml bovine serum albumin (+BSA, n = 220); (3) SOF + 20% fetal bovine serum (+FBS, n = 227); or (4) SOF + 3 mg/ml BSA + 20% FBS (+BSA+FBS, n = 232), then cultured for up to 72 h. A group of control follicles was not cultured (n = 103). The percentages of oocytes reaching metaphase I to metaphase II stages (MI to MII) did not differ between treatments at each culture period. Within treatments, the percentages of oocytes at MI to MII stages did not differ with duration of culture. However, when compared to the control group (0.97%) the percentages of oocytes at MI to MII increased (P < 0.05) in the SOF group after 48 h (10.0%) and 72 h (12.2%) of culture. In the +BSA (10.1%) and +FBS (9.7%) groups, the percentages of oocytes at MI to MII increased (P < 0.05) above control values only after 72 h of culture. The percentage of oocytes at MI to MII did not significantly increase in the +BSA+FBS group (3.9,6.6 and 7.6% at 24,48 and 72 h of culture, respectively) compared to the control group. These results indicate that under the described conditions supplementation of culture medium with BSA or FBS is not essential, and the simple medium SOF can support nuclear maturation of a small proportion of bitch oocytes in vitro.     

Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes

The process of oocyte maturation in the canine species is unique among mammals: oocytes are immature at ovulation and the resumption and progression of meiotic maturation occur in the oviduct. This study was performed to investigate (i) the effect of co-culture with infundibulum and ampullar oviductal epithelial cells on the in vitro maturation of canine oocytes and (ii) the culture time necessary to reach full meiotic maturation. For this purpose the oocytes, collected from the ovaries of bitches undergoing ovariectomies, were divided into three groups and cultured for 48 and 72 h with the following systems: (A) TCM 199 + 10% oestrus bitch serum + FSH (0.1 IU.mL(-1)), LH (0.1 IU.mL(-1)) + progesterone (1 microg.mL(-1)) + oestradiol (1 microg.mL(-1)) + cysteamine (100 microM); (B) medium A plus infundibulum cells; (C) medium A plus ampullar cells. Infundibulum and ampullar cells were recovered from the oviducts of bitches at the oestrus stage of their cycle. The results showed that after 48 h of incubation, a significantly higher meiotic resumption (P < 0.01) was observed in the oocytes cultured with infundibulum (59%) and ampullar cells (60.0%), than in the control group (40.0%). There was also a significantly (P < 0.01) higher meiotic progression to the MII in systems B and C (15.6% and 16.7%) than in system A (4.0%). After 72 h of culture, the percentages of meiotic resumption and progression were unchanged. These results showed that both the infundibulum and the ampullar oviductal epithelial cells positively influence the meiotic resumption and progression of canine oocytes and that 48 h are sufficient for the completion of nuclear maturation.     

Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa

The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity. The first objective of this study was to compare the effect of two different culture media and two different incubation times on in vitro maturation (IVM) of domestic cat oocytes. The second objective was to determine the developmental competence of in vitro matured cat oocytes after intracytoplasmic sperm injection (ICSI) with cat spermatozoa. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 medium or in synthetic oviductal fluid (SOF), both of which were supplemented with cysteamine, BSA, FSH, LH. Nuclear maturation was assessed after 24 h and 40 h of incubation. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 40 h of incubation were significantly higher (P<0.001) after culture with SOF (88/110, 80% and 159/192, 82.8%) than TCM 199 (86/129, 66.7% and 58/90, 64.4%). Oocytes (n = 231) matured in vitro in SOF for 24 h were fertilized by ICSI with frozen-thawed epididymal cat spermatozoa. After ICSI, one group of oocytes (n = 129) was activated with ethanol, and a second group (n = 102) was not activated. The developmental competence of all ICSI oocytes was examined after 7 days of in vitro culture. After 28 h of culture, the cleavage frequency of ICSI-activated oocytes was significantly higher (P<0.001) than that of IC     

Nuclear maturation and development of IVM/IVF canine embryos in synthetic oviductal fluid or in co-culture with buffalo rat liver cells

In vitro embryo production in the domestic bitch can provide valuable insights for conservation of endangered canids. In the present study, canine oocytes underwent in vitro maturation (IVM) in simple or complex media, with production of in vitro matured and fertilized (IVM/IVF) canine embryos. Cumulus–oocyte complexes (COCs) were harvested from ovaries by slicing and subjected to IVM in four media (SOF, TCM 199, Ham-F10, and DMEM/F12). After culture for 48 h, oocytes were stained and examined for nuclear maturation. There were no significant differences in the mean (±S.D.) percentage of nuclear maturation (metaphase II) of oocytes cultured in SOF (18.6 ± 7.6%), TCM 199 (18.3 ± 4.5%), Ham-F10 (13.9 ± 8.2%), or DMEM/F12 (11.9 ± 4.2%). For assessment of embryo development, oocytes were matured for 48 h in synthetic oviductal fluid (SOF), fertilized with frozen-thawed sperm, and presumptive zygotes were cultured for 7 d, either in SOF or as co-cultures with BRL cells in TCM 199. Percentages of IVM/IVF oocytes that developed to the 2-cell, 3–4-cell, and 5–7-cell stages were higher (P < 0.05) following culture in SOF versus BRL cell co-cultures (33.6 ± 1.2% vs 13.7 ± 1.2%, 24.7 ± 0.5% vs 8.7 ± 1.1%, and 15.1 ± 2.2% vs 4.3 ± 1.3%, respectively). However, none of the embryos developed beyond the 8–16-cell stage. In conclusion, simple or complex media successfully induced resumption of meiosis and nuclear maturation of canine oocytes. Furthermore, SOF supported in vitro development of IVM/IVF canine embryos to the 8–16-cell stage.     

Effects of culture media and energy sources on the inhibition of nuclear maturation in bovine oocytes

The influence of the culture medium and energy sources on spontaneous nuclear maturation and inhibition of maturation in bovine cumulus-enclosed oocytes (CEO) was examined. CEO were cultured in Medium 199, minimum essential medium, M16, or synthetic oviduct fluid (SOF), all containing 3 mg/mL bovine serum albumin (BSA), and SOF without BSA, alone or supplemented with hypoxanthine (HYPO, 4 mM) or forskolin (FSK, 100 microM) for 21 h. More CEO remained at the GV stage in M16 compared to other media (P < 0.05). Supplementation with HYPO increased and FSK reduced the percentage of CEO remaining at the GV stage (P < 0.05) only in M16. The effects of energy sources, in the absence or presence of HYPO or FSK, were examined in CEO cultured in M16 salts+PVA. Glucose (0.5 and 5.5 mM), pyruvate (0.32 and 3.2 mM), lactate (3.3 mM) and glutamine (1.3 mM) significantly reduced the percentage of CEO remaining at the GV stage compared to M16 salts alone; only glutamine significantly increased the percentage of CEO at the MII stage compared to M16 salts. In M16 salts+HYPO, glucose (0.5 mM), pyruvate (0.32 mM), lactate (3.3 mM) and glutamine (1.3 mM) significantly reduced the percentage of GV and degenerate oocytes and increased the percentage of CEO at the MI stage. In M16 salts+FSK, the energy sources significantly decreased the percentage of oocytes with condensed chromosomes and increased the percentage of CEO reaching metaphase I. In conclusion, meiotic inhibitors had different effects in different culture media and glucose, pyruvate, lactate and glutamine were stimulatory to nuclear maturation. It was noteworthy that some of the results obtained were contrary to previous findings in mouse oocytes.     

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.     

Effect of meiotic stages and maturation protocols on bovine oocyte's resistance to cryopreservation

We investigated the effect of meiotic stages and two maturation protocols on bovine oocyte's resistance to cryopreservation. Oocytes at germinal vesicle breakdown (GVBD) and metaphase II (MII) stage as well as oocytes matured for 22 h in media supplemented with FSH or LH were vitrified by the open pulled straw method. After warming, oocytes underwent additional 16 h (GVBD group) or 2 h (MII group) maturation. Then they were subjected to in vitro fertilization and culture. Some oocytes that matured in the medium supplemented with LH were subjected to parthenogenetic activation after vitrification to determine their developmental potential in absence of fertilization. Survival of oocytes after vitrifying/warming was determined after 22 h in fertilization medium. Cleavage and blastocyst formation rates were used to assess their developmental competence. In both experiments, a portion of unvitrified MII oocytes were subjected to in vitro fertilization and culture as control groups. In Experiment 1, similar cleavage rates were obtained for both GVBD and MII oocytes (53.56 versus 58.01%, P > 0.05). However, significantly higher proportion of cleaved embryos from vitrified MII oocytes developed into blastocysts than those from vitrified GVBD oocytes (1.06 versus 8.37%, respectively, P < 0.01). In Experiment 2, vitrified MII oocytes matured in medium supplemented with LH were superior to vitrified MII oocytes matured in FSH supplementation not only in cleavage rates (61.13 versus 50.33%), but in blastocyst formation rates (11.79 versus 5.19%, P < 0.01) as well. Cleavage and blastocyst formation rates of parthenogenetically activated oocytes were similar to those that were fertilized. Nevertheless, the vitrifying/ warming process significantly compromised the oocytes' developmental capacity since the vitrified oocytes showed significant reduction in both cleavage and blastocyst rates compared to those of not vitrified controls in both experiments (P < 0.01). We showed that oocytes at different maturation stages respond to cryopreservation differently and MII stage oocytes have better resistance to cryopreservation than GVBD stage oocytes. The maturation protocols also influence oocyte's ability to survive cryopreservation. Poor developmental potential after vitrification seem to have resulted from the cryodamage to the oocyte itself. These results suggested the importance of maturation on the developmental competence of cryopreserved oocytes.     

The effect of vitamins during maturation of caprine oocytes on subsequent developmental potential in vitro

Only a small proportion of goat oocytes selected for in vitro oocyte maturation (IVM) can successfully complete cytoplasmic maturation and support embryonic development. To produce goat blastocysts more efficiently in vitro, it is necessary to identify factors required during oocyte maturation. The objective of this study was to determine the role of vitamins during maturation of caprine oocytes in semi-defined medium on subsequent developmental capacity in vitro. Cumulus oocyte complexes (COCs) collected from a local abattoir were matured in synthetic oviductal fluid (SOF) medium supplemented with BSA, LH, FSH, and EGF in the presence or absence of MEM vitamins for 24 h. The COCs were co-incubated with frozen-thawed sperm in Bracket and Oliphant fertilization medium for 18-22 h. Embryos were cultured in G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 72 h. Addition of vitamins significantly increased (P<0.05) overall blastocyst development (16.4+/-1.2% versus 12.3+/-1.1%), and tended to increase (P<0.06) the percentage of cleaved embryos (61.4+/-3.0% versus 52.7+/-2.6%). Addition of MEM vitamins to SOF maturation medium significantly increased (P<0.05) mean blastocyst cell number compared with control medium (107.7+/-6.0 versus 85.1+/-6.3). Hatched blastocysts tended to have increased (P<0.06) cell numbers in the vitamin-treated group (150.5+/-8.4 versus 123.4+/-8.8). These results suggest that addition of vitamins during oocyte maturation is beneficial for subsequent blastocyst development and viability.     

Equine oocyte in vitro maturation: influences of sera, time, and hormones.

Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 micrograms/ml), equine luteinizing hormone (eLH; 100 micrograms/ml), bovine follicle-stimulating hormone (FSH; 5 micrograms/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P less than 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P less than 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.     

Effect of transport and storage temperature of ovaries on in vitro maturation of bitch oocytes

In this study, the effects of ovary transport and storage temperature on in vitro maturation of bitch oocytes were investigated. Ovaries were collected from 23 mature bitches and one randomly selected ovary of each pair (n=23 pairs) was transported in physiologic saline at 4 degrees C, while the other one at 35-38 degrees C for 2-4h. A total of 316 cumulus oocyte complexes (COCs) were obtained from the 4 degrees C group and 301 COCs from the 35-38 degrees C group. All COCs were matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), essential and non-essential amino acids at 38 degrees C in a humidified 5% CO2, 5% O2, and 90% N2 atmosphere for 72 h. At the end of the in vitro maturation period, nuclear maturation of oocytes was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), undetermined nuclear maturation (UDNM), and MI+MII. The nuclear maturation rates to MI, MII, and MI+MII stages were 60.44%, 10.75%, and 71.20% in the 4 degrees C group and 37.20%, 7.64%, and 45.85% in the 35-38 degrees C group, respectively. The data demonstrated that oocytes obtained from ovaries transported at 4 degrees C had higher maturation rates than from the ones transported at 35-38 degrees C (p<0.001).     

Influence of oviductal cells and conditioned medium on porcine gametes

The aim of this study was to optimise porcine in vitro fertilisation (IVF) with cryopreserved semen with the exploitation of the oviduct secretion. The oocytes were cultured in NCSU37 supplemented with db-cAMP (1 mM), porcine follicular fluid (pFF; 10%), cysteine (0.1 mg/ml) and beta-mercaptoethanol (25 microM) for 44 h (the first 20 h with 10 IU/ml hCG and PMSG). The oviductal epithelial cells (OEC) were cultured in TCM-199 medium (with 10% FCS, 0.2 mM pyruvate and 50 microg/ml gentamicin) for 48 h. To determine the effects of OEC and conditioned medium, oocytes were separated into five groups for the last 3 h of maturation and placed in: fresh maturation medium (controls), OEC-cNCSU with OEC in the maturation medium for 24 h; OEC-fNUSU with fresh OEC in maturation medium; cTCM with TCM-199 conditioned with OEC for 48 h; or fTCM with fresh TCM-199. Results indicate that OEC-cNCSU and OEC-fNCSU increase the number of oocytes reaching the two pronucleus (2PN) stage (p < 0.01) and decrease the polyspermy rate (p < 0.01) compared with controls. The rates are significantly lower than controls when cTCM and fTCM were used (p < 0.01). As regards blastocyst rates, an increase was observed in the OEC-cNCSU and cTCM groups (p < 0.05). For the second experiment, spermatozoa were incubated with OEC in IVF medium (mTBM medium supplemented with 0.1% BSA) without caffeine for 4 h prior to IVF. Results indicate that sperm treatment with OEC increases the 2PN rate (p < 0.01) compared with controls and reduces the polyspermy rate (p < 0.01). In conclusion, our study shows that co-incubation of OEC with both oocytes and sperm before IVF reduces polyspermy rates and improves embryo development.     

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.     

In vitro canine oocyte nuclear maturation in homologous oviductal cell co-culture with hormone-supplemented media

The aim of the present research was to verify the influence of oviductal cell co-culture previously supplemented with steroids (estrogen, progesterone, or both) on IVM rates for oocytes from anestrous bitches that were cultured in vitro for 48, 72 and 96 h. Oocytes harvested from anestrous bitches were selected and allocated into four groups: Group 1 (co-culture in oviductal epithelial cells without hormonal supplementation-control); Group 2 (estrogen supplementation); Group 3 (progesterone supplementation); Group 4 (estrogen+progesterone supplementation). The oviductal epithelial cell culture was established 72 h prior to oocyte co-culture. After periods of 48, 72 and 96 h, the degree of oocyte nuclear maturation was assessed. Co-culture in oviductal epithelial cells with estrogen was not as beneficial for canine IVM as supplementation with progesterone and estrogen, or progesterone supplementation alone. Therefore, it was feasible to use co-culture with oviductal epithelial cells obtained from anestrous bitches for IVM (monolayer culture with oviduct cells previously supplemented with progesterone). Final stages of oocyte maturation were achieved at 72 and 96 h of culture; therefore, the duration of maturation for oocytes obtained from bitches in different stages of the estrous cycle should be taken into account.     

Angiotensin II reverses the inhibitory action produced by theca cells on bovine oocyte nuclear maturation

The presence of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin II (Ang II) and Ang II receptors in the ovary is suggestive of a functional ovarian renin-angiotensin system (RAS). In cattle, the expression of Ang II is greatest in large follicles, suggesting that it is important during follicular growth and maturation. The present study was designed to investigate the role of Ang II in bovine oocyte nuclear maturation. Bovine cumulus-oocyte complexes (COCs) were cultured with or without follicular cells and Ang II or saralasin (Ang II antagonist). In the absence of follicular cells, Ang II at 0, 10(-11), 10(-9) and 10(-7) M did not affect the percentage of oocytes reaching the germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII) stage after 7-h (41.3 +/- 4.3, 35.3 +/- 4.0, 31.3 +/- 9.7, 38.7 +/- 8.6), 12-h (31.6 +/- 7.0, 34.7 +/- 6.1, 31.7 +/- 5.3, 28.9 +/- 9.1; mean +/- S.E.M.) and 18-h (44.9 +/- 7.3, 58.4 +/- 8.4, 53.1 +/- 7.4, 44.9 +/- 7.3) of culture, respectively. Similarly, saralasin at 0, 10(-11), 10(-9) and 10(-7) M did not affect the percentage of oocytes reaching MII stage after 18-h of culture (37.6 +/- 7.4, 34.4 +/- 7.7, 30.0 +/- 10.8 and 31.2 +/- 5.1, respectively). The theca cells (MII = 22.9%) or medium conditioned with follicular cells (GV = 65.5%, MI = 23.6%) inhibited oocyte maturation; however, theca cells (MII = 35.5 +/- 4.9; P < 0.05) or medium conditioned with follicular cells (GV = 34.6%, MI = 52.7%; P < 0.01) were not able to inhibit nuclear maturation when Ang II (10(-11) M) was present in the culture system. Theca cells remained viable during the culture period when Ang II was present. Therefore, results supported the idea of a role of Ang II in blocking the inhibitory effect of theca cells on nuclear maturation of bovine oocytes.     

Changes in global histone acetylation pattern in somatic cell nuclei after their transfer into oocytes at different stages of maturation

In our study, we have examined the pattern of global histone modification changes in somatic cell nuclei after their transfer into mouse oocytes at different stages of maturation or after their parthenogenetic activation. While germinal vesicle (GV) staged immature oocytes are strongly labeled with anti-acetylated histone H3 and H4 antibodies, the signal is absent in both metaphase I and metaphase II oocytes (MI, MII). In contrast, the oocytes of all maturation stages show a presence of trimethylated H3/K4 in their chromatin. When somatic cells were fused to intact or enucleated GV oocytes, both the GV and the somatic cell nucleus showed a very strong signal for all the antibodies used. On the other hand, when somatic cells nuclei that are AcH3 and AcH4 positive before fusion are introduced into either intact or enucleated MI or MII oocytes, their acetylation signal decreased rapidly and was totally absent after a prolonged culture. This was not the case when anti-trimethyl H3/K4 antibody was used. The somatic cell chromatin showed only a slight decrease in the intensity of labeling after its transfer into MI or MII oocytes. This decrease was, however, evident only after a prolonged culture. These results suggest not only a relatively higher stability of the methylation modification but also some difference between the oocyte and somatic chromatin. The ability to deacetylate the chromatin of transferred somatic nuclei disappears rapidly after the oocyte activation. Our results indicate that at least some reprogramming activity appears in the oocyte cytoplasm almost immediately after GV breakdown (GVBD), and that this activity rapidly disappears after the oocyte activation.     

Mitogen-activated protein kinase activity during goat oocyte maturation and the acquisition of meiotic competence

Changes in MPF and MAPK activities during meiotic maturation of goat oocytes were investigated. Detection of MPF activity occurred concomitantly with GVBD, increased at MI, decreased during anaphase-telophase I transition, and increased thereafter in MII oocytes. The appearance of MAPK activity was delayed compared to MPF activity. MAPK activity increased after GVBD and persisted during the MI-MII transition. Whether MAPK was implicated in goat oocyte meiotic competence was also investigated by using oocytes from different follicle size categories that arrest at specific stages of the maturation process (GV, GVBD, MI, and MII). Results indicate that the ability of goat oocytes to resume meiosis is not directly related to the presence of Erk2. The ability to phosphorylate MAPK is acquired by the oocyte during follicular growth after the ability to resume meiosis. GVBD-arrested oocytes exhibited a high level of MPF activity after 27 hr of culture. However, 28% of oocytes from this group contained inactive MAPK, and 72% exhibited high MAPK activity. In addition, 29% of GVBD-arrested oocytes contained a residual interphasic network without recruitment of microtubules around the condensed chromosomes; 71% of GVBD-arrested oocytes displayed recruitment of microtubules near the condensed chromosomes and contained asters of microtubules distributed throughout the cytoplasm. These results indicate that oocytes arrested at GVBD were not exactly at the same point in the meiotic cell cycle progression, and suggest that MAPK could be implicated in the regulation of microtubule organization. The data presented here suggest that in goat oocytes, MAPK is not implicated in the early events of meiosis resumption, but rather in post-GVBD events such as spindle formation and MII arrest. © 1996 Wiley-Liss Inc.     

Effects of wortmannin on the kinetics of GVBD and the activities of the maturation-promoting factor and mitogen-activated protein kinase during bovine oocyte maturation in vitro

The present study was conducted with the objective of examining the effect of wortmanin, a specific PI 3-kinase inhibitor, on the kinetic of GVBD, and on the activities of the maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase during bovine oocyte maturation. The time sequence for GVBD was not different between oocytes cultured with or without wortmannin. Most of the cultured oocytes were at the filamentous bivalents stage after 4 h of culture. Six hours after the start of culture, most of the oocytes possessed germinal vesicles with condensed bivalent, and by 10 h of culture nearly all of the cultured oocytes underwent GVBD. A gradual increase in MPF activity until 12 h of culture was observed in the presence and absence of wortmannin. A sharp decrease in MPF activity in oocytes cultured without wortmannin treatment was recorded at 14 h of culture. Thereafter, MPF regained activity, reaching a maximum level at 20 to 24 h of culture. For oocytes cultured with wortmannin, no decline in the activity of MPF was observed during the interval from 12 to 24 h of culture. For these oocytes the MPF activity remained nearly stable during this transition until the end of incubation. The presence of wortmannin in the maturation medium did not alter MAP kinase activity. Taken together, these observations indicate that inhibition of PI 3-kinase does not modulate the time sequence of GVBD or the pattern of MAP kinase activity in bovine oocytes. However, PI 3-kinase might be one of the molecules that regulate the sharp reduction in the activity of MPF during the MI/MII transition.     

The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology,fertilization and embryo development

In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal epithelial cells (pOECs) during maturation increases cytoplasmic maturation and blastocyst development. These aspects, as well as blastocyst quality, may be improved when adult sow oocytes are matured with pOEC. Therefore, the effect of the presence of pOEC on sow oocyte morphology, fertilization and the progression of embryo development was evaluated. The pOEC were cultured in M199 for 18 h, then cultured in NCSU23 for 4 h before the oocytes were added. Oocytes from 2 to 6 mm follicles were matured in 500 microl NCSU23, with eCG and hCG, for 24 h, and then cultured with or without pOEC, in NCSU23 without hormones, for 18 h. In vitro fertilization took place in modified Tris-buffered medium, for 6 h, and the presumptive zygotes were then cultured for 162 h in NCSU23. Morphology of the IVM oocytes was compared to that of immature oocytes and in vivo matured MII oocytes from slaughtered sows in estrus. The in vitro matured oocytes had a greater diameter and a wider perivitelline space than the immature and in vivo matured MII oocytes (P < 0.01). Penetration, polyspermy and pronucleus formation did not differ between the pOEC and Control groups, although the total penetration rate was higher for the Control oocytes (26% versus 39%; P < 0.01). Fewer blastocysts developed in the pOEC group than in the Control group (19% versus 27%; P < 0.01), but blastocyst growth was accelerated, leading to a higher percentage of hatched blastocysts (3% versus 10%; P < 0.01). Finally, the average blastocyst cell number was higher in the pOEC group (47 versus 40; P < 0.05) and a greater percentage of blastocysts contained a superior number of nuclei. In conclusion, the addition of pOEC during the second half of in vitro maturation resulted in fewer blastocysts formed, but of those blastocysts that did form the quality was improved.