Fertilizability and developmental capacity of bovine oocytes cultured individually in a chemically defined maturation medium

This study investigated effects of adding hypotaurine (HT), beta-merocaptoethanol (beta-ME), or both into a chemically defined maturation medium (TCM-199 containing 0.1% polyvinyl alcohol: PVA) on maturation, fertilization and development of individually (single) cultured bovine oocytes. Mean GSH concentration in the oocytes cultured in the medium supplemented with either beta-ME (1.11 +/- 0.05 nM) or HT plus beta-ME (0.97 +/- 0.03 nM) was significantly (P < 0.05) higher than that in the medium containing PVA alone (0.75 +/- 0.03 nM). Adding beta-ME showed a significantly (P < 0.05) higher rate of the second metaphase stage (93.6 +/- 3.3%) than in the medium containing PVA alone (single-control) (65.2 +/- 7.9%). Adding both HT and beta-ME showed significantly (P < 0.05) higher rates (92.6 +/- 2.7%) of normal fertilization than did adding HT alone (63.5 +/- 4.6%). Also, adding both HT and beta-ME significantly (P < 0.05) lowered the polyspermy rate than did adding HT alone. Adding either beta-ME or both HT and beta-ME showed no significant difference in cleavage. Blastocyst development did not improve significantly adding either HT, beta-ME or both, although beta-ME alone or HT plus beta-ME tended to result in a higher rate of blastocysts (6.4 and 6.8%, respectively) than resulted without additives (1.6%). Our results show that adding beta-ME to a chemically defined maturation medium increased the intracellular GSH level of bovine oocytes cultured individually, and can improve the maturation rate leading to the blastocyst stage throughout in vitro production.     

Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium

The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.     

Differential effects of testosterone and dibutyryl cyclic AMP on the meiotic maturation of mouse oocytes in vitro

Fully grown, meiotically immature mouse oocytes were isolated and cultured under varying conditions with the aim of determining a) whether the inhibitory effects of testosterone on oocyte meiotic maturation require the synthesis of new oocyte proteins and b) if the meiosis-inhibiting effects of testosterone and dibutyryl cyclic AMP (dbcAMP) are distinct and can be differentiated. We found that the inclusion of puromycin in culture medium containing testosterone has no effect on the meiosis-inhibiting potency of testosterone or upon the reversibility of testosterone effects. We conclude that testosterone inhibits oocyte meiosis by a mechanism that is independent of protein synthesis. We also found that oocytes exposed to testosterone recover more rapidly, as evidenced by the timing of germinal vesicle breakdown (GVBD) following placement in a control medium, than do oocytes exposed to dbcAMP. Through further investigation of this phenomenon we have determined the sequence of testosterone and dbcAMP effects relative to the time course of GVBD. A testosterone-sensitive event occurs 20 min prior to GVBD, while the dbcAMP-sensitive event precedes GVBD by 41 min. The nature of this difference may involve the differential interaction of testosterone and dbcAMP with a set of puromycin-sensitive proteins that are required for GVBD. When oocytes were initially cultured in medium containing both puromycin and either testosterone or dbcAMP and then moved to medium containing puromycin alone the incidence of GVBD was reduced relative to oocytes never exposed to puromycin. This observation suggests that mouse oocytes contain proteins that are required for GVBD and that experience a high turnover rate. The degree of reduction in GVBD was a function of the length of puromycin exposure and was significantly greater in dbcAMP- than in testosterone-exposed oocytes. If oocytes were initially cultured in medium containing puromycin and dbcAMP, the rate of GVBD upon removal of dbcAMP was initially slow but increased with time. This observation is consistent with the hypothesis that dbcAMP inhibits oocytes at a point prior to the functioning of the puromycin-sensitive proteins. However, if oocytes were cultured in medium containing puromycin and testosterone the rate of GVBD following testosterone removal was not significantly reduced relative to oocytes that were not exposed to puromycin. This observation suggests that testosterone acts to inhibit meiosis at a site beyond the function of the puromycin-sensitive proteins or that testosterone causes a reduction in the turnover rate of these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Additional effect of epidermal growth factor during in vitro maturation for individual bovine oocytes using a chemically defined medium

This study was performed to establish an individual bovine oocyte-IVP system using a chemically defined simple medium (mSOFaa containing 1 mg/ml polyvinyl alcohol: PVA) and to investigate the effects of epidermal growth factor (EGF) during oocyte maturation on in vitro maturation, fertilization and embryonic development. Cumulus-oocyte complexes were collected from bovine ovaries and were matured in mSOFaa containing PVA (control medium) supplemented with 0, 1, 10 or 50 ng/ml of EGF. Two further groups (TCM199 and mSOFaa, supplemented with 10% fetal calf serum were also included. In this study, mSOFaa containing PVA were used as a basic medium for fertilization and embryo development in vitro. Experiments were conducted in both group- and individual-IVP systems. In the group-IVP system, the proportion of matured oocytes (MII) in the control medium (62.7% +/- 5.0%) was significantly (p < 0.05) lower than in all other treatments, and in the individual-IVP system, the addition of 1 ng/ml EGF significantly (p < 0.05) increased the maturation rate (1 ng/ml EGF vs control: 76.2% +/- 5.4% vs 57.1% +/- 14.4%). The addition of EGF did not affect the proportions of penetrated and normally fertilized oocytes in either individual- or group-culture systems. In the group-IVP system, no significant difference among treatments was found in the rate of blastocyst formation, whereas in the individual-IVP system the control medium supplemented with 10 ng/ml EGF resulted in a significantly (p < 0.05) higher the rate of blastocyst formation (20.0 +/- 5.2%) than that in the control medium (6.2% +/- 3.5%). These results indicate that bovine oocytes can successfully develop to blastocysts in an individual-IVP system using a single chemically defined medium, and that the group-IVP system also resulted in a similar level of blastocyst formation to that in a standard multiple-media system in our laboratory. The effect of EGF during oocyte maturation medium differed depending on whether embryos were cultured individually or in groups.     

Effects of different serum supplements in maturation medium on meiotic and cytoplasmic maturation of pig oocytes

The temporal progression of meiotic and cytoplasmic maturation of pig oocytes cultured in a medium supplemented with 0.4% polyvinylalcohol (PVA), 10% fetal calf serum (FCS), 10% newborn piglet serum (NPS), 10% porcine follicular fluid (PFF) or 10% porcine seminal fluid (PSF) was examined after 20, 30, 40 and 50 hours of culture. There were no differences in germinal vesicle breakdown (GVBD) among FCS and NPS supplements. After 20 hours of culture, the frequency for GVBD was higher (P < 0.05) in FCS and NPS (54% and 52%, respectively) than in PVA (32%) and PFF (33%) culture media but were not different at 40 and 50 hours of culture. Supplementation with PSF resulted in a rapid chromosome condensation of pig oocytes after 20 hours culture, but all GVBD oocytes stopped developing at the condensed germinal vesicle stage. Oocytes were not penetrated by spermatozoa when inseminated following 20 hours of culture, while high penetration (87 to 100%) and polyspermy rates (86 to 100%) were consistently obtained in all the supplement groups when inseminated after 30, 40 or 50 hours of culture. Male pronuclear formation rates at 10 to 12 hours after insemination, following a 50-hour culture period in FCS and NPS, were 28 and 28%, respectively, in comparison with 54% in PVA and 59% in PFF. The results indicate that supplementing maturation media with serum such as FCS and NPS reduced the ability of pig oocytes to form a male pronucleus, and further suggest that the detrimental effects may be due to accelerated progression of maturation events.     

Suppression of spontaneous maturation of isolated cumulus-free mouse oocytes by a calmodulin antagonist

Oocytes isolated from antral follicles undergo spontaneous maturation when cultured in vitro. W7, a calmodulin antagonist, at concentration of more than 50 microM blocked the occurrence of spontaneous germinal vesicle breakdown (GVBD) in isolated cumulus-free mouse oocytes. The inhibition of maturation was observed in more than 90% of oocytes when W7 was added within 15 min after the initiation of incubation of the oocytes. The block was partially reversible. Hypoxanthine, estradiol-17 beta, testosterone and progesterone did not influence the inhibition induced with W7. The present results suggest that calmodulin is involved in the early stage of mouse oocyte maturation.     

Effect of glucose and pyruvate on nuclear and cytoplasmic maturation of porcine oocytes in a chemically defined medium

The objective was to examine potential roles of glucose and pyruvate in nuclear and cytoplasmic maturation of porcine oocytes. Oocyte-cumulus complexes (OCC), derived from 3 to 6mm follicles, were cultured in a chemically defined medium (pyruvate-free mNCSU37-PVA), with or without 5.55mM glucose, during in vitro maturation (IVM); in the absence of glucose, germinal vesicle breakdown (GVBD) and nuclear maturation were prevented (P<0.05). Subsequently, OCC were cultured for IVM in glucose-containing mNCSU37-PVA supplemented with 6-amononicotinamide (6-AN) and diphenyleneiodonium (DPI), inhibitors of the pentose phosphate pathway (PPP); both compounds (>/=10muM 6-AN and >/=10nM DPI) inhibited resumption of meiosis (P<0.05). Supplementation of glucose-free maturation medium with increasing concentrations of pyruvate induced resumption of meiosis and increased the incidence of oocytes reaching metaphase-II in a concentration-dependent manner (P<0.05). More mature oocytes were obtained in the presence of pyruvate+glucose (P<0.05). After culture to allow maturation, glutathione content was higher in oocytes cultured in the presence of pyruvate alone than in those cultured in glucose alone; inclusion of 6-AN abolished responses to pyruvate (P<0.05). In conclusion, both glucose and pyruvate played a critical role in the release of porcine oocytes from arrest at the GV-I stage, probably through the PPP, whereas supplementation with pyruvate improved cytoplasmic maturation, as determined by oocyte glutathione content.     

Effects of phosphodiesterase inhibitors on spontaneous nuclear maturation and cAMP concentrations in bovine oocytes

It was previously demonstrated that inhibition of cAMP degradation with phosphodiesterase type 3 (PDE3) inhibitors resulted in the maintenance of bovine cumulus–oocyte complexes (COC) and denuded oocytes (DO) in meiotic arrest, while a PDE4 inhibitor was without effect. In this study, different inhibitors of PDE3 and PDE4 were tested for their effects on bovine oocyte nuclear maturation. Bovine COC and DO were cultured in TCM-199+10% fetal bovine serum (FBS) with or without different concentrations of the PDE inhibitors. The PDE3 inhibitor trequinsin significantly increased the percentage of COC remaining at the germinal vesicle (GV) stage after 7 h of culture (19.3, 60.3, and 67.8% GV for control and trequinsin 10 and 50 nM, respectively) while Ro 20-1724 (a PDE4 inhibitor) was without effect. In DO, only trequinsin at 10 nM had a significant effect after 7 h of culture (51.3 and 86.1% GV for control and trequinsin 10 nM, respectively). Trequinsin reduced the percentage of COC reaching the mature phase after 22 h, but was without effect on DO. The protein kinase A (PKA) inhibitor H-89 reversed the inhibitory effect of trequinsin in COC and DO, indicating that inhibition of nuclear maturation by trequinsin involves activation of PKA. Trequinsin increased cAMP concentrations in COC but not in DO, suggesting that cumulus cells may also contain a PDE3 isoenzyme.     

Effects of cooling germinal vesicle-stage bovine oocytes on meiotic spindle formation following in vitro maturation.

Attempts to cryopreserve bovine oocytes result in low survival because of their sensitivity to temperatures near 0 degrees C. This study evaluates the effects of chilling germinal vesicle-stage (GV) oocytes on their formation of microtubules and the meiotic spindle. In experiment 1, five groups of GV-stage oocytes, each consisting of approximately 90 oocytes, were held at 39 degrees C as controls, or at 31 degrees C, or cooled to 24, 4 or 0 degrees C for 10 min. After being treated, all oocytes were cultured at 39 degrees C for 24 hr. Compared to the controls, holding oocytes for 10 min at 31 or 24 degrees C did not significantly alter the formation of normal spindles, but chilling them to 4 or 0 degrees C did. After 24 hr of maturation, the respective percentages of oocytes containing normal meiotic spindles observed in the controls or those held at 31 or 24 degrees C were 69.8%, 71.9%, or 69.4% (P > 0.05). In contrast, the percentages of oocytes with normal spindles after they had been cooled to 4 or 0 degrees C were 44.0% or 29.1%, respectively. In experiment 2, approximately 90 oocytes/group were cooled to 4 degrees C for various times before being warmed and cultured. Regardless of the time of exposure, cooling oocytes to 4 degrees C reduced the formation of normal spindles. The percentages of oocytes cooled to 4 degrees C for 10, 20, 30, 45, or 60 min with normal spindles were 44.0%, 38.4%, 37.5%, 34.5% and 30.9%, respectively. In experiment 3, approximately 60 oocytes per group that had been held at 31 degrees C or cooled to 24, 4 or 0 degrees C for 10 min were allowed to mature for 24 hr before being subjected to in vitro fertilization. The cleavage rates of oocytes subjected to various chilling treatments exhibited the same pattern as that of oocytes with normal spindles. That is, there were no significant differences in cleavage rates among the control oocytes and those held at 31 or 24 degrees C (70.4%, 71.8%, and 72.4%; P > 0.05). However, only 37. 0% and 30.4% of oocytes chilled to 4 or 0 degrees C cleaved after fertilization. These results suggest that: (1) chilling bovine oocytes no lower than 24 degrees C does not reduce formation of normal meiotic spindles; (2) however, chilling oocytes to 4 degrees C or lower for as little as 10 min drastically reduces the formation of normal meiotic spindles and of fertilization; (3) the rates of fertilization and cleavage of resultant zygotes mimic that of formation of normal spindles.     

Manipulation of chromosome condensation by protein synthesis inhibitors and cyclic AMP during maturation of bovine oocytes

We investigated the effects of cycloheximide on bovine oocyte chromosomes during meiotic maturation in vitro. Bovine oocytes at Metaphase I (MI) of the meiotic maturation were treated with 10 mug/ml cycloheximide alone or in addition to 5 mM dibutyrylcAMP (dbcAMP) plus 1 mM isobutylmetylxantine (IBMX). A maturation period of 15 to 18 h followed by 12-h treatment with cycloheximide appeared to be most efficient to induce interphase (86% with 16 h maturation). About 60% of oocytes returned to a metaphase state 12 h after the oocytes were transferred to cycloheximide-free medium. In contrast, up to 73% of cycloheximide-treated oocytes at 17 h of maturation remained in interphase if dbcAMP plus IBMX was included in the cycloheximide-free medium. This shows that dbcAMP plus IBMX can inhibit the development of conditions in the oocytes that are required for the transition to metaphase. The chromosome decondensation induced by protein synthesis inhibition at Metaphase I is reversible. This study shows that transition to interphase in bovine oocyte depends on the stage of maturation of oocytes and is sensitive to cAMP levels.     

Effects of different protein supplements in fertilization medium on in vitro penetration of cumulus-intact and cumulus-free bovine oocytes matured in culture

Bovine oocytes matured in culture were inseminated with frozen-thawed spermatozoa in BO medium containing 5 mM-caffeine, 10 mug/ml of heparin and different protein supplements at various concentrations. When cumulus-enclosed oocytes were inseminated, no significant differences were observed in the penetration rates (89 to 100%) between media with and without protein supplements and among the different concentrations of each protein supplement, except for 20% calf serum (CS), in which the penetration rate decreased drastically (43%). Notably higher incidences of polyspermy were obtained in medium with FCS (75 to 86%) than with either no supplement (25%) or with BSA (20 to 24%) and CS (13 to 49%). On the other hand, there was almost no penetration of cumulus-free oocytes in the nonsupplemented control medium. Concentration-dependent increases in penetration and polyspermy occurred with BSA, FCS and CS supplementation. A high concentration (5%) of FCS yielded a high incidence (97%) of polyspermy. A decrease in the penetration of cumulus-enclosed oocytes was observed when spermatozoa were capacitated with a high concentration (20%) of CS; difficulty of sperm penetration of cumulus-free oocytes occurred when the capacitation medium lacked protein supplementation; and an increased rate of polyspermy was observed following supplementation with FCS in both cumulus-enclosed and cumulus-free oocytes after insemination with spermatozoa from 5 different bulls.     

Follicular wall maintains meiotic arrest in bovine oocytes cultured in vitro

Cumulus oocyte complexes (COCs) were cocultured with parts of the follicular wall. Coculture conditions were such that the COCs were 1) in continuous contact with the follicular wall (FWC), 2) separated from the follicular wall at collection but in contact with it during culture (FWR), and 3) separated from the follicular wall, but cultured in its vicinity (FWNR). Oocytes cultured for 24 hr under FWC conditions maintained the germinal vesicle stage. Under FWR conditions the germinal vesicle stage was not maintained, but an arrest at metaphase I of meiosis occurred in mostof the oocytes. When COCs were cultured in the vicinity of the follicular wall (FWNR), meiosis was resumed and similar numbers of oocytes progressed to metaphase II of meiosis as compared to cultures of COCs without coculture with parts of the follicular wall. When COCs were isolated from the follicular wall after 24 hr of culture and additionally cultured for another 24 hr, the oocytes showed the same capability of resuming meiosis as fresh, isolated cumulus oocyte complexes. It is concluded that maintenance of contact with the follicular wall is necessary to maintain meiotic arrest. When COCs restore a physical contact with the follicular wall during culture, an arrest at metaphase I occurs. © 1994 Wiley-Liss, Inc.     

Effects of bovine follicular fluid on maturation of bovine oocytes

Three experiments were conducted to determine the effects of follicular fluid and media on bovine oocyte maturation. Experiments 1 and 3 test the effects of follicular fluid obtained at different times after the LH surge on bovine oocyte maturation in vitro, while Experiment 2 was designed to compare TALP and Medium 199 as serum-free maturation media. Bovine follicular fluid (BFF) was obtained from preovulatory follicles either before (0 h BFF) or at 4, 8, 12 or 20 h after a GnRH-induced LH surge. Oocytes were obtained from follicles 1 to 6 mm in diameter from ovaries retrieved from a slaughterhouse. In Experiment 1, both 0 h and 4 h BFF inhibited resumption of meiosis, whereas BFF collected at 8, 12 and 20 h did not. When oocytes were cultured in media that contained equal portions of 0 and 8 h BFF, meiosis was not inhibited. In Experiment 2, Medium 199 supplemented with bovine serum albumin (BSA) was superior to Tyrode's medium with albumin, lactate and pyruvate for oocyte maturation. In Experiment 3, a higher percentage (P<0.05) of oocytes cultured for 18 h in 40% 20 h BFF in Medium 199 reached Metaphase-II (64%) than those cultured in 0 h BFF (41%) or control medium (39%). There was a transient meiotic arrest due to 0 h BFF as evidenced by the higher percentage of oocytes with germinal vesicles at 8 h of incubation (35% with 0 h vs 20% with 20 h; P<0.05). Furthermore, expansion of cumulus cells was induced in 8 and 20 h BFF, but not 0 h BFF.     

Effect of follicle-stimulating hormone on cyclic adenosine monophosphate level and on meiotic maturation in mouse cumulus cell-enclosed oocytes cultured in vitro

We have reported that in vitro treatment with follicle-stimulating hormone (FSH) delays by about 3 h spontaneous meiotic resumption in cumulus cell-enclosed mouse oocytes. In the present paper we show that the temporary meiotic block is accompanied by a transient increase of cAMP concentration in the oocyte. In cumulus cell-oocyte complexes stimulated with 1 microgram/ml FSH, cAMP significantly increases within 1 h both in the whole complex (from a basal value of 1.9 +/- 0.2 to 169 +/- 13 fmol) and in the enclosed oocyte (from 0.9 +/- 0.2 to 2.4 +/- 0.2 fmol), then progressively decreases to basal values. Stimulation by FSH does not cause any cAMP increase in denuded oocytes. As the concentration of cAMP in the cells decreases, the percentage of oocytes escaping the meiotic block imposed by FSH increases. If the complexes are cultured in the presence of 1 microgram/ml FSH plus 1 mM isobutyl-1-methylxanthine (1BMX), cAMP concentration increases approximately 250-fold in the complex, and 10-fold in the enclosed oocyte; the level of cAMP in the oocyte drops very rapidly (50% degradation in less than 2 min) if the oocyte is then transferred to IBMX-free medium. The data are discussed in terms of the possible role of cAMP transfer from cumulus cells to the oocyte in the regulation of meiotic progression in mouse oocytes.     

Effects of fructose supplementation in chemically defined protein-free medium on development of bovine in vitro fertilized embryos

The present study evaluated the possible embryotrophic role of fructose supplementation in potassium simplex optimization medium (KSOM) on preimplantation development of bovine in vitro matured and fertilized (IVF) embryos under chemically defined conditions. In Experiment 1, the rates of cleavage (74.0-75.5%) and blastocyst formation (21.0-24.5%) were not affected by the supplementation of fructose in KSOM in absence or presence of glucose. In Experiment 2, the rates of cleavage (71.7-77.3%) and blastocyst formation (19.9-26.3%) did not differ significantly among the concentrations (0.0, 0.2, 1.5, 3.0, 5.6mM) of fructose supplementations in KSOM in presence of glucose. Moreover, the number of total ICM and TE cells, and percentage of ICM to total cell in blastocysts did not differ significantly among the concentrations of fructose supplementations in presence of glucose. In Experiment 3, the rates of cleavage (67.3-74.7%) and blastocyst formation (14.4-19.3%) did not differ significantly among the concentrations (0.0, 0.2, 1.5, 3.0, 5.6mM) of fructose supplementations in KSOM in absence of glucose. Although the number of total and ICM cells, and percentage of ICM to total cells in blastocysts did not differ significantly among the concentrations of fructose supplementations, 1.5mM fructose supplementation in absence of glucose had significantly (P<0.05) higher number of TE cells (106.2) than that of 5.6mM (84.0) supplementation. The study indicates that, fructose up to 5.6mM concentration can be used as an alternative for energy substrate in culture media without any detrimental effect on pre-implantation development in bovine IVF embryos.     

Sperm penetration in vitro of human oocytes matured in a chemically defined medium

Human oocytes were obtained randomly from the ovaries excised from 28 patients at different stages of the menstrual cycle. When 114 oocytes were cultured for 40-40.5 h in a chemically defined medium 80 showed degeneration. When 16 of the remaining 64 oocytes were examined for their maturation, germinal vesicle breakdown (GVBD) had occurred in 11 oocytes. Another 48 oocytes were inseminated by ejaculated spermatozoa which were washed twice and preincubated for 3 h. When examined 10 h later, 19 (39.6%) were penetrated with an enlarged sperm head or male pronucleus(ei) and its corresponding sperm tail(s).     

The importance of NaCl concentration in a chemically defined medium for the development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined medium (modified Tyrode's solution) without glucose. When different concentrations of NaCl were added to the medium, the proportions of embryos developed to the >/=8-cell, morula and blastocyst stages 96, 144 and 192 h post insemination, respectively, were significantly higher at 89 to 114 mM than 64 to 76 and 126 to 139 mM NaCl. A high proportion (28%) of blastocyst-stage embryos 192 h post insemination was obtained at 89 mM NaCl. When calculated osmolarity in the medium with 64 mM NaCl was varied by adding D-sorbitol, significantly higher proportions of morula-stage embryos were obtained at 265 to 315 mOsm (27 to 38%) than 215 (9%) and 365 (2%) mOsm, but the development to the blastocyst stage was difficult at any osmolarities (215 to 365 mOsm) tested. In the medium with a fixed osmolarity (315 mOsm) but with different concentrations (64 to 114 mM) of NaCl, there were no differences in the proportions (29 to 33%) of morula-stage embryos among different NaCl concentrations. However, significantly higher proportions of embryos developed to the blastocyst stage at 89 to 101 mM (22 to 23%) than 64 to 76 (0 to 9%) and 114 (11%) mM NaCl. When Cl- concentration in the medium with 64 mM NaCl was adjusted by adding choline chloride, significantly higher proportions of embryos developed to the morula stage at 97 to 122 mM (32 to 40%) than 72 (6%) and 147 (2%) mM Cl-, but few embryos developed to the blastocyst stage at any Cl- concentrations (72 to 147 mM) tested. In the medium with 64 or 114 mM NaCl and each with 2 different Na (+)K (+) ratios, there were no differences in the proportions of morula- and blastocyst-stage embryos between different Na+ K+ ratios (31 and 39 at 64 mM NaCl, and 39 and 47 at 114 mM NaCl) at each NaCl concentration. When glucose was added to the medium with 89 mM NaCl 120 h postinsemination, there were no significant differences in the proportions (40 to 48%) of morula-stage embryos 144 h post insemination among different concentrations (0 to 6.95 mM) of glucose. The proportion (33%) of blastocysts 192 h post insemination at 2.78 mM glucose was significantly higher than the values at 0 (22%), 5.56 (19%) and 6.95 (15%) mM but not different compared with the values at 1.39 (23%) and 4.17 (28%) mM. In conclusion, NaCl concentration in a defined medium is one of the most important factors for the development of bovine embryo to the blastocyst stage, but the development of embryos up to the morula stage is also regulated by osmolarity and/or Cl-concentration.     

Inhibition of LH-induced and spontaneous meiotic maturation in mouse oocytes by N alpha-tosyl-L-lysine chloromethylketone

The effect of N alpha-tosyl-L-lysine chloromethylketone (TLCK), an inhibitor of trypsin-type proteases, on luteinizing hormone (LH)-induced and spontaneous meiotic maturation and follicular production of cAMP in mice was determined. When follicle-enclosed mouse oocytes were incubated with LH (1 micron/ml), they underwent the breakdown of the germinal vesicle (GVBD). TLCK (0.02-0.5 mM) inhibited LH-induced GVBD in folliculated oocytes. The concentration (0.5 mM) of TLCK that inhibited LH-induced GVBD did not significantly suppress LH-induced cAMP production by follicle cells. The effect of TLCK on spontaneous maturation in cumulus cell-enclosed and denuded oocytes was also determined. TLCK strongly inhibited spontaneous maturation in denuded oocytes only if it was added to the incubation medium for 1-3 h before oocytes were liberated from the follicular tissue. The inhibition of oocyte maturation by TLCK was significantly greater in cumulus cell-enclosed oocytes than in denuded oocytes, either with or without preincubation with TLCK. These results suggest that trypsin-type protease in oocytes participates in the process of meiotic maturation in mouse oocytes.     

Effects of leptin supplementation in in vitro maturation medium on meiotic maturation of oocytes and preimplantation development of parthenogenetic and cloned embryos in pigs

The objective of the present study was to investigate the effects of leptin addition in in vitro maturation (IVM) medium on meiotic maturation of oocytes and preimplantation development of parthenogenetic and cloned embryos in pigs. In experiment 1, oocytes were matured in North Carolina State University 23 (NCSU-23) medium supplemented with various concentrations of leptin: 0, 1, 10 and 100 ng/ml. IVM medium added with 10 or 100 ng/ml leptin significantly increased the rate of oocytes reaching metaphase II compared to the control (76.8% and 73.8% versus 61.7%). In experiment 2, the influence of the timing of leptin addition in IVM medium on meiotic maturation of porcine oocytes was assessed, and maximum maturation rate of oocytes developing to metaphase II was achieved when supplemented during the first half (0-22 h), the latter half (22-44 h) or the entire maturation period (0-44 h) compared to the control (80.5%, 84.7% and 78.1% versus 70.4%). In experiment 3, leptin strikingly increased the blastocyst rate of parthenogenetic embryos at the concentration of 10 ng/ml (37.5% versus 21.7%) and this increase was independent of the addition timing (0-44, 0-22, 22-44 h) compared to the control (32.5%, 34.6% and 31.5% versus 16.2%). Moreover, total cell number per blastocyst of parthenogenetic embryos was obviously increased in the 10 and 100 ng/ml leptin treatments as compared with the control (36, 38 versus 28). In experiment 4, 10 ng/ml leptin treatment significantly increased the rate of cleavage (72% versus 56%) of cloned embryos. Meanwhile, the rate of blastocyst formation was also improved although no significant difference was found (12.8% versus 7.1%). Collectively, our results indicate that leptin supplementation in IVM medium may be beneficial not only for developmental potential of oocytes but for subsequent developmental competence of embryos produced by parthenogenetic activation and the cleavage of embryos derived by somatic cell nuclear transfer (SCNT).     

The influence of different types of media supplement on the meiotic maturation of bovine oocytes in vitro

This work was designed to evaluate the influence of different types of media supplements in the maturation of preovulatory oocytes in cattle. We studied 4 different supplements: serum from estrous cattle (ECS), serum from fetal calves (FCS), amniotic fluid from cattle (bAF) and follicular fluid from cattle (bFF). Preovulatory bovine oocytes recovered from ovaries of mature cows after slaughter were cultured for 24 h in TCM-199 medium containing 20% of one of these protein supplements. The percentages of oocyte maturation were significantly higher (P < 0.001) in ECS (78%), FCS (79%) and bAF (77%) than in bFF (52%). These data indicate that supplementation with ECS, FCS or bFF is adequate for maturation of bovine oocytes without addition of other compounds.