Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes

The present study aimed to analyze detailed morphological and functional characteristics of mouse in vitro matured oocytes after a pre-maturation culture (PMC) by temporary nuclear arrest with the specific phosphodiesterase 3-inhibitor (PDE3-I) Cilostamide. In a first experiment the lowest effective dose of Cilostamide was determined. Cumulus-oocyte complexes (COCs), isolated from small antral follicles, were exposed to different concentrations of Cilostamide (ranging from 0 (control) to 10 microM) for 24 hr. Afterwards, oocytes were removed from PDE3-I-containing medium and underwent in vitro maturation (IVM) for 16-18 hr. A concentration of 1 microM Cilostamide was the lowest effective dose for maximum level of inhibition and reversibility of meiosis inhibition. This concentration was used in further experiments to evaluate oocyte quality following IVM in relation to different parameters: kinetics of meiotic progression, metaphase II (MII) spindle morphology, aneuploidy rate, fertilization, and embryonic developmental rates. The results were compared to nonarrested (in vitro control) and in vivo matured oocytes (in vivo control). Following withdrawal of the inhibitor, the progression of meiosis was more synchronous and accelerated in arrested when compared to nonarrested oocytes. A PMC resulted in a significant increase in the number of oocytes constituting a MII spindle of normal morphology. None of the oocytes exposed to PDE3-I showed numerical chromosome alterations. In addition, fertilization and embryonic developmental rates were higher in the PMC group compared to in vitro controls, but lower than in vivo controls. These results provide evidence that induced nuclear arrest by PDE3-I is a safe and reliable method to improve oocyte quality after IVM.     

In vitro maturation of bovine oocytes in the presence of growth hormone accelerates nuclear maturation and promotes subsequent embryonic development

Regulatory effect of GH on follicular growth and development in the cow is well documented. The aim of this study was to investigate the role of GH on in vitro bovine oocyte maturation. Therefore bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of 10, 100, or 1,000 ng/ml bovine GH (NIH-GH-B18). The COCs were incubated at 39°C in a humidified atmosphere with 5% CO₂ in air and nuclear stage was assessed after 2, 4, 8, 16, 22, and 24 hr of incubation using DAPI staining. To assess the effect of GH on developmental capacity of the oocytes, COCs were incubated in the presence of GH for 22 hr, followed by IVF and in vitro embryo culture. Cultures without GH served as controls. For subsequent development, the embryos were cultured in M199 supplemented with 10% FCS on a monolayer of BRL cells. Embryos were scored morphologically and the efficiency of the culture system was evaluated as (1) the percentage of cleaved embryos 4 days after IVF, (2) the percentage of blastocysts on day 9 expressed on the basis of the number of oocytes at the onset of culture, and (3) the percentage of hatched blastocysts on day 11 expressed on the basis of the total number of blastocysts present at day 9. GH (100 and 1,000 ng/ml) significantly accelerated nuclear maturation (P < 0.001). A 4 and 8 h the percentage of oocytes in GV stage after GH treatment (54% and 19%) was significantly lower than the control (64% and 41%). Similarly at 16 and 22 h the percentage of oocytes in MII stage was significantly higher in the GH-treated group; (58% and 77%) and (46% and 62%) for GH and control respectively. The number of oocytes in MII beyond 22 hr of culture did not differ; 100 and 1,000 ng/ml GH induced significant cumulus expansion (P < 0.05), which was not observed in the absence of GH. Addition of 100 and 1,000 ng/ml GH during maturation significantly (P < 0.01) enhanced subsequent cleavage rate from (64% and 67%) in control to (75% and 81%) in GH-treated group; embryonic development in terms of day 9 blastocyst formation was also significantly increased in the presence of GH (29% and 34%) compared to the control (18% and 24%). The hatchability of the blastocysts was not influenced by GH. From the present data, it can be concluded that GH present during IVM has a beneficial effect on subsequent development. © 1996 Wiley-Liss, Inc.     

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.     

Meiotic competence of bovine fetal oocytes following in vitro maturation

The in vitro ability between fetal and cow oocytes to resume meiosis and progression to metaphase-II (M-II) was compared. Cumulus oocyte complexes (COCs) were harvested from 2 to 6 mm follicles from ovaries of 7.5 month to term fetuses and adult cows. Cumulus cells were removed using 3 mg/ml hyaluronidase and repeated pipetting. Denuded oocytes were fixed in 3% glutaraldehyde, stained with DAPI and evaluated under fluorescent microscopy for nuclear status before in vitro maturation (IVM). COCs from fetal and adult ovaries were also matured in 200 microl droplets of medium 199 supplemented with 10 microg/ml FSH, 10/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM hepes and 10% FBS for 24 h at 39 degrees C and 5% CO(2). Matured oocytes were fixed, stained and evaluated as explained above for nuclear status namely stage of germinal vesicle (GV) development and subsequent meiotic competence. Data were analyzed using chi-square analysis. The majority of fetal oocytes (P<0.05) before IVM were at GV stages GV-I (27.7%), GV-II (37.6%) and GV-V (22.8%) compared to cow oocytes, which were at GV stages IV (28.3%) and V (46.7%). After IVM, fewer fetal oocytes were at earlier stages of GV development and majority (P<0.05) were at GV-V (24.0%), premetaphase (17.4%) and metaphase-I (M-I: 7.2%) stages. However, after IVM, more cow oocytes matured to M-II than did fetal oocytes (93.7% versus 26.9%; P<0.05). In conclusion, fetal oocytes do not mature in vitro as well as cow oocytes. Our findings suggest that the low meiotic competence of fetal oocytes can be attributed to their being at earlier stages of GV development before IVM.     

Effects of glucocorticoids on maturation of pig oocytes and their subsequent fertilizing capacity in vitro

The aim of this study was to assess the possible role of glucocorticoids in the maturation of pig oocytes and their subsequent fertilizing capacity in vitro. Pig cumulus-enclosed oocytes collected from prepubertal gilts were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microg/ml), LH (0.5 microg/ml), FSH (0.5 microg/ml), and estradiol-17beta (1 microg/ml) in the presence or absence of cortisol or dexamethasone (DEX) for 24 h; they then were cultured without hormonal supplements in the presence or absence of cortisol or DEX for an additional 16-24 h. Treatment of cumulus-enclosed or denuded oocytes with increasing concentrations of cortisol or DEX for 48 h resulted in a dose-response inhibition of germinal vesicle breakdown (GVB). Increasing duration (12-48 h) of treatment with DEX (10 microg/ml) led to a time-dependent inhibition of GVB, which achieved statistical significance by 12 h. The addition of DEX (10 microg/ml) to maturation medium immediately after culture or at 12 h, 24 h, or 36 h after culture also decreased the percentage of oocytes with GVB. When oocytes were exposed to DEX for 48 h, the maturation rate was reduced. The degree of this reduction was dependent on DEX, and a concentration of DEX higher than 0.1 microg/ml was needed. The inhibitory effect of DEX on the maturation of oocytes was prevented by the glucocorticoid receptor antagonist RU-486. Exposure of oocytes to DEX for 40 h did not prevent sperm penetration, affect the incidence of polyspermy, or decrease the ability of oocytes to form a male pronucleus. The intracellular concentration of glutathione (GSH) in cumulus-enclosed oocytes was 4.4 mM per oocyte. Exposure of oocytes to DEX (0.01-10 microg/ml) had no effect on GSH concentration. These results demonstrate that glucocorticoids directly inhibit the meiotic but not cytoplasmic maturation of pig oocytes in vitro. This inhibitory effect is not mediated through a decrease in the level of intracellular GSH.     

In vitro maturation and fertilization of swamp buffalo oocytes and their subsequent development

The present experiment was carried out to evaluate the maturation, fertilization and subsequent embryo culture of swamp buffalo oocytes in vitro. The oocytes (n=273) were collected and morphologically graded based on the structure of cumulus-oocyte complexes as Grade 1 (compact, n=81), Grade 2 (expanded, n=70), Grade 3 (partially denuded, n=65) or Grade 4 (completely denuded, n=57). More than 60% of the in vitro matured oocytes co-cultured with capacitated spermatozoa demonstrated evidence of fertilization or cleavage to the 2-cell stage when either Grade 1 or 2 oocytes were used. The percentage of fertilized oocytes undergoing 2-cell stage cleavages from Grade 3 (53%) and Grade 4 (46%) groups was significantly lower (P<0.01) than that observed in the Grade 1 (64%) and Grade 2 (68%) groups. Development to the 6 to 8 cell stage substantiated fertilization of Grade 1 and 2 oocytes. These results demonstrated that swamp buffalo oocytes are capable of maturing in vitro, forming embryos, and developing at least to the 8-cell stage in culture medium alone.     

Meiotic arrest in human oocytes is maintained by a Gs signaling pathway

In mammalian oocytes, the maintenance of meiotic prophase I arrest prior to the surge of LH that stimulates meiotic maturation depends on a high level of cAMP within the oocyte. In mouse and rat, the cAMP is generated in the oocyte, and this requires the activity of a constitutively active, Gs-linked receptor, GPR3 or GPR12, respectively. To examine if human oocyte meiotic arrest depends on a similar pathway, we used RT-PCR and Western blotting to look at whether human oocytes express the same components for maintaining arrest as rodent oocytes. RNA encoding GPR3, but not GPR12, was expressed. RNA encoding adenylate cyclase type 3, which is the major adenylate cyclase required for maintaining meiotic arrest in the mouse oocyte, was also expressed, as was Galphas protein. To determine if this pathway is functional in the human oocyte, we examined the effect of injecting a function-blocking antibody against Galphas on meiotic resumption. This antibody stimulated meiotic resumption of human oocytes that were maintained at the prophase I stage using a phosphodiesterase inhibitor. These results demonstrate that human oocytes maintain meiotic arrest prior to the LH surge using a signaling pathway similar to that of rodent oocytes.     

Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs

The effects of ammonium in a chemically defined maturation medium on oocyte nuclear maturation and subsequent embryonic development of pigs after in vitro fertilization (IVF) and parthenogenetic activation (PA) were examined. Cumulus–oocyte complexes were matured in Purdue Porcine Medium (PPM) supplemented with 0 mM, 0.02 mM, 0.2 mM, 2 mM, or 20 mM ammonium chloride, or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) at 38.7 °C in 7% CO₂ in air for 40–44 h. No significant difference (P > 0.05) in nuclear maturation was found between oocytes matured in TCM + pFF or PPM with 0 mM, 0.02 mM and 0.2 mM ammonium chloride. However, nuclear maturation was decreased (P < 0.05) in oocytes matured in PPM with 2 mM or 20 mM ammonium. After IVF, oocytes matured in PPM with 20 mM ammonium resulted in embryos with reduced (P < 0.05) embryonic cleavage and blastocyst development than all other treatment groups. After PA, oocytes matured in PPM with 20 mM ammonium resulted in embryos with lesser (P < 0.05) embryonic cleavage compared to TCM + pFF. However, PA embryos derived from oocytes matured in PPM with both 2 mM and 20 mM ammonium had reduced (P < 0.05) blastocyst development compared with TCM + pFF. These results demonstrate the detrimental effect of ammonium during in vitro oocyte maturation on nuclear progression to metaphase II. Additionally, the presence of ammonium during in vitro maturation negatively influences subsequent embryonic development, although PA embryos appear to be more sensitive to the negative effects of ammonium during oocyte maturation than do IVF embryos.     

Meiotic arrest and aneuploidy induced by vinblastine in mouse oocytes

Young superovulated female mice were injected i.p. with single doses of vinblastine sulfate just before the onset of the first meiotic division. Secondary oocytes, fixed one by one on a slide, were cytogenetically scored. Evidence of the meiotic arresting activity of vinblastine was produced by the observation of increasing frequencies of M1-arrested oocytes and by the presence of undegenerated chromosome sets of first polar bodies. When the first meiotic division could be undertaken chromosome malsegregation occurred with high frequency, both in terms of aneuploidy and polyploidy. M1-blocked and polyploid oocytes have been interpreted as the consequence of irreversible damage to the spindle induced by vinblastine through its binding on tubulin low-affinity sites; this reaction, in fact, causes microtubule crystallization. According to this mechanism, dose-effect relationships of both phenomena show a threshold at 0.45 mg/kg. On the other hand, the incidence of aneuploid oocytes is correlated with meiotic delay, as detected by the delayed degeneration of polar bodies, and increases linearly with dose. Both phenomena are, therefore, stochastic and can be referred to the binding of the chemical on tubulin high-affinity sites, which is known to cause tubulin depolymerization in a colchicine-like way.     

Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 microM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P < 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P < 0.05) increased embryo development to the blastocyst stage by 33%-39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P < 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.     

Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 microg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.     

Melatonin supplementation during in vitro maturation of oocyte enhances subsequent development of bovine cloned embryos

Oocyte quality, which is directly related to reprogramming competence, is a major important limiting factor in animal cloning efficiency. Compared with oocytes matured in vivo, in vitro matured oocytes exhibit lower oocyte quality and reprogramming competence primarily because of their higher levels of reactive oxygen species. In this study, we investigate whether supplementing the oocyte maturation medium with melatonin, a free radical scavenger, could improve oocyte quality and reprogramming competence. We found that 10⁻⁹ M melatonin effectively alleviated oxidative stress, markedly decreased early apoptosis levels, recovered the integrity of mitochondria, ameliorated the spindle assembly and chromosome alignment in oocytes, and significantly promoted subsequent cloned embryo development in vitro. We also analyzed the effects of melatonin on epigenetic modifications in bovine oocytes. Melatonin increased the global H3K9 acetylation levels, reduced the H3K9 methylation levels, and minimally affected DNA methylation and hydroxymethylation. Genome-wide expression analysis of genes in melatonin-treated and nontreated oocytes was also conducted by high-throughput RNA sequencing. Our results indicated that melatonin ameliorates oocyte oxidative stress and improves subsequent in vitro development of bovine cloned embryos.     

Comparison of various maturation treatments on in vitro maturation of goat oocytes and their early embryonic development and cell numbers

In the present study, comparison of 2 different culture media (Ham's F-12 and M-199) for supporting in vitro maturation of goat oocytes, and their subsequent embryonic development was evaluated in the presence or absence of sera (estrous goat serum, EGS and fetal calf serum, FCS) and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol, 1 ug/ml). Neither medium (Ham's F-12 or M-199) when supplemented with EGS and hormones showed any notable changes in the maturation rate nor in cleavage and blastocyst development. The mean cell number for blastocysts was also significantly low (P < 0.05). However, Ham's F-12 medium supplemented with FCS and hormones showed a considerable increase in the maturation rate, but subsequent embryonic development was not appreciably increased. However, maturation, cleavage and blastocyst development rates of oocytes matured in M-199 medium in combination with 10% FCS and hormones were significantly higher (P < 0.05). Mean cell number per blastocyst was also significantly increased in this latter treatment compared with that of the other groups (P < 0.05). The results thus indicated that both the culture medium and serum have a marked effect on maturation and subsequent embryonic development. Further, the results also showed that the combination of M-199 with FSH, LH and E2 supplemented with 10% FCS was the most efficacious medium for in vitro maturation and subsequent embryonic development of the media, sera and hormone combinations studied.     

Low concentrations of MEM vitamins during in vitro maturation of porcine oocytes improves subsequent parthenogenetic development

To investigate the effects of water-soluble vitamin supplementation for IVM/IVC of porcine oocytes and evaluate maturation and developmental capacity in vitro, porcine cumulus oocyte complexes (COCs) was matured in NCSU-23-based medium with water-soluble vitamins for 44 h and then cultured in PZM-3 for 7 days following activation. The COCs were allocated into five treatment groups and matured in various concentrations of MEM vitamins (control, 0.05, 0.1, 0.2, 0.4, and 1x). Metaphase II plates of the cumulus-free oocytes were observed following Hoechest 33258 staining. The COCs were allocated into four treatment groups, matured in various concentrations of MEM vitamins (control, 0.05, 0.1, 0.2, and 0.4x) and cultured in PZM-3 following activation. Also, COCS were matured without MEM vitamins and cultured in PZM-3 with various concentrations (control, 0.1, 0.4, 1.0, and 2.0 x) of MEM vitamins. Furthermore, 2 x 2 factorial (IVM/IVC) experiments were performed in IVM medium with or without 0.05 x MEM vitamins and IVC medium with or without 0.4x MEM vitamins to examine the in vitro development of parthenogenetic embryos. Maturation rates of COCs treated with MEM vitamins did not differ significantly among groups. However, compared to the control group, oocytes matured with the addition of 0.05 x MEM vitamins developed to blastocysts at a higher percentage (P<0.05) following activation and culture in PZM-3 without MEM vitamins. Total cell number of blastocysts was significantly higher in the 0.05 x group. Addition of 0.4x MEM vitamins decreased (P<0.05) cleavage and blastocyst developmental rates compared with 0.05 x MEM vitamins-treated group. In contrast, addition of vitamins to PZM-3 medium for in vitro culture of activated porcine oocytes did not affect development. In conclusion, addition of a low concentration of MEM vitamins to IVM medium for porcine oocytes enhanced subsequent development and improved embryo quality.     

Effect of protein synthesis inhibition before or during in vitro maturation on subsequent development of bovine oocytes

The overall objective of this study was to assess the effect of maintaining meiotic arrest in bovine oocytes in vitro on developmental competence. In Experiment 1 the effect of inhibition of meiotic resumption using cycloheximide (CX), on subsequent was examined. Immature cumulus oocyte complexes (COCs, n = 804) were cultured in the absence (24 h) or presence of CX for 6, 12, 18 or 24 h. The control was inseminated 24 h later, while CX-treated oocytes were cultured for a further 24 h before insemination. In Experiment 2 the effect of exposing the oocyte (n = 1239) during meiotic arrest to putative stimulatory substances (pFSH and FCS) was examined. In Experiment 3, to study the importance of protein synthesis during maturation, synthesis was blocked for a 6-h period at various times (6, 12, 18 h) after start of culture (n = 1117). In Experiment 1, there was no difference in cleavage rate between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower after CX treatment (64 vs 42 to 51%; P < 0.05). This was reflected in a lower rate of blastocysts at Day 6 (9 to 15 vs 31%, P < 0.002). While the blastocyst rate at Day 8 was lower in CX-treated oocytes, the effect was only significant when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with the control (17 to 19 vs 40%; P < 0.0002). In Experiment 2, addition of either FSH or FCS to oocytes in the presence of CX had no effect on any of the parameters studied, even though there was a positive effect in control oocytes. In Experiment 3, treatment with CX after the oocytes had matured for varying periods resulted in decreased blastocyst rates at Days 6 and 8 of culture. The most significant drop in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P < 0.0001). In conclusion, CX-blocked oocytes retained their developmental competence, although final blastocyst yields were reduced.     

Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression.

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.     

In vitro maturation of ovarian oocytes from unstimulated rhesus monkeys: assessment of cytoplasmic maturity by embryonic development after in vitro fertilization

One-hundred and sixty-six cumulus-enclosed oocytes, obtained from ovaries of unstimulated rhesus monkeys, were subjected to six different treatments in vitro--two types of media (simple = TALP; complex = CMRL) x three levels of gonadotropins (none, FSH, FSH + hCG)--to assess their ability to undergo maturation, fertilization, and embryo development. A summary of development in culture for all experimental treatments is as follows: 58% of oocytes underwent germinal vesicle breakdown; 37% extruded a first polar body; 17% had more than one pronucleus and/or two polar bodies after insemination (i.e., were activated/fertilized); and 12% cleaved (i.e., developed) to at least the 2-cell stage in vitro. Of 45 oocytes incubated only in medium (either simple or complex) without gonadotropins, only 3 were activated/fertilized (6.7%), and only one embryo developed to at least the 2-4-cell stage (2.2%). There were no differences between oocytes incubated with only FSH and oocytes incubated with FSH + hCG. Activation/fertilization (20.7% vs. 6.7%) and embryo development (greater than or equal to 2 cells; 15.7% vs. 2.2%) were significantly higher in treatments with than without gonadotropin supplementation. There were no statistically significant differences attributable to incubation in different media during oocyte maturation. Cumulus-enclosed oocytes recovered from unstimulated ovaries of rhesus monkeys can resume maturation during culture in vitro, as shown by their ability to be fertilized and by the cleavage in vitro of the resultant zygotes.     

Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro

The survival and developmental capacity of cumulus cell-enclosed oocytes frozen (1) at the germinal vesicle (GV) stage, after maturation in vitro with (2) and without (3) FSH, and (4) after gonadotrophin-stimulated ovulation were assessed. Survival, defined as the number of morphologically normal oocytes, after freeze-thaw at the GV stage (69%), was lower than for oocytes frozen after ovulation (84%), and after maturation in vitro with FSH (88%) and without FSH (81%). Treatment with DMSO without freezing had no effect on survival when compared with untreated controls except in immature GV-stage oocytes for which there was a slight reduction. After insemination in vitro, 9% of frozen-thawed GV-stage oocytes cleaved to two equal blastomeres, but none developed to blastocysts. Of oocytes matured in vitro before freezing, 17% cleaved to the 2-cell stage and 18% of these developed to blastocysts. When oocytes were matured in vitro in the presence of FSH, however, the percentage cleaving to the 2-cell stage after freeze-thaw was improved to 55%, and 77% of 2-cell stage embryos developed to blastocysts. When ovulated cumulus cell-enclosed oocytes were frozen, 88% cleaved and 67% of the cleaved embryos developed to blastocysts. When 158 two-cell embryos resulting from oocytes matured in vitro with FSH were transferred to the oviducts of pseudopregnant foster mothers, 41 genetically marked live young were produced (26%).(ABSTRACT TRUNCATED AT 250 WORDS)