Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

Meiotic and developmental competence of prepubertal and adult swine oocytes

The present study was conducted to compare meiotic and cytoplasmic competence of prepubertal and adult porcine oocytes, and the effects of EGF (0 to 100 ng/mL), FSH (0 to 400 ng/mL) and prepubertal pFF (0 to 10%) on nuclear maturation. Prepubertal oocytes were less responsive to FSH and pFF than were adult oocytes in terms of stimulation of nuclear maturation. The best nuclear maturation rates for prepubertal oocytes were obtained with 10 ng/mL EGF and 400 ng/mL FSH, whereas for adult oocytes no additional effect of EGF was seen in the presence of 400 ng/mL FSH. Supplementation with pFF had no additional effect on MII yield over that obtained with EGF plus FSH. After maturation in the presence of EGF, FSH and cysteamine, fertilization rates were not different between adult and prepubertal oocytes, but polyspermy was more frequent in prepubertal oocytes (31 +/- 17% vs. 17 +/- 7% in prepubertal and adult oocytes, respectively, P < 0.05). The addition of pFF to maturation medium decreased oocyte fertilization of adult oocytes and polyspermic fertilization in prepubertal oocytes. Blastocyst yield and developmental competence were significantly reduced in prepubertal oocytes compared to adult oocytes. The mean cell numbers in blastocysts cultured for 7 days ranged from 61 to 74, and did not differ among groups. Finally, the viability of the 2- to 4-cell embryos and blastocysts produced was assessed by embryo transfer experiments. One offspring was obtained after transfer of 2- to 4-cell embryos, and one after transfer of in vitro-produced blastocysts. In conclusion, although prepubertal gilt oocytes appeared less meiotically and developmentally competent than their adult counterparts, they can be used to produce blastocysts able to develop to term.     

Developmental competence of porcine oocytes selected by brilliant cresyl blue and matured individually in a chemically defined culture medium

The objective of this study was to investigate the effects of oocyte selection using brilliant cresyl blue (BCB) and culture density during individual in vitro maturation (IVM) on porcine oocyte maturity and subsequent embryo development using a chemically defined medium. Cumulus-oocyte complexes (COCs) were classified as BCB-positive or BCB-negative after exposure to a BCB solution for 90 min. The classified COCs were matured in a group (15 COCs per 100-μL droplet) or individually (1 COC per 1-, 2.5-, 5-, or 10-μL droplet). Meiotic competence, intraoocyte glutathione concentration, and developmental competence after intracytoplasmic sperm injection were monitored. The BCB selected oocytes competent for nuclear and cytoplasmic maturation. Furthermore, meiotic competence for oocytes matured individually in a 5-μL droplet was superior (P < 0.05) to that of oocytes matured in a 1-μL droplet. Also, the culture density in a 5-μL droplet during IVM resulted in a higher (P < 0.05) rate of cleaved embryos than that in a 1-μL droplet and produced a similar rate of blastocysts compared with that of a group culture system. Conversely, BCB selection did not improve cleavage and blastocyst formation. In conclusion, it was possible to predict porcine oocytes competent for maturation using oocyte selection with BCB. Moreover, a 5-μL droplet during the individual IVM culture was most suitable for oocyte maturation and subsequent embryo development, although every culture density used in this study supported development up to the blastocyst stage.     

Regulation of oocyte mitochondrial DNA copy number by follicular fluid, EGF, and neuregulin 1 during in vitro maturation affects embryo development in pigs

Little is known about mitochondrial DNA (mtDNA) replication during oocyte maturation and its regulation by extracellular factors. The present study determined the effects of supplementation of maturation medium with porcine follicular fluid (pFF; 0, 10%, 20%, and 30%) on mtDNA copy number and oocyte maturation in experiment 1; the effects on epidermal growth factor (EGF; 10 ng/mL), neuregulin 1 (NRG1; 20 ng/mL), and NRG1 + insulin-like growth factor 1 (IGF1; 100 ng/mL + NRG1 20 ng/mL), on mtDNA copy number, oocyte maturation, and embryo development after parthenogenic activation in experiment 2; and effects on embryo development after in vitro fertilization in experiment 3. Overall, mtDNA copy number increased from germinal vesicle (GV) to metaphase II (MII) stage oocytes after in vitro maturation (GV: 167 634.6 ± 20 740.4 vs. MII: 275 131.9 ± 9 758.4 in experiment 1; P < 0.05; GV: 185 004.7 ± 20 089.3 vs. MII: 239 392.8 ± 10 345.3 in experiment 2; P < 0.05; Least Squares Means ± SEM). Supplementation of IVM medium with pFF inhibited mtDNA replication (266 789.9 ± 11 790.4 vs. 318 510.1 ± 20 377.4; P < 0.05) and oocyte meiotic maturation (67.3 ± 0.7% vs. 73.2 ± 1.2%, for the pFF supplemented and zero pFF control, respectively; P < 0.01). Compared with the control, addition of growth factors enhanced oocyte maturation. Furthermore, supplementation of NRG1 stimulated mitochondrial replication, increased mtDNA copies in MII oocytes than in GV oocytes, and increased percentage of blastocysts in both parthenogenetic and in vitro fertilized embryos. In this study, mitochondrial biogenesis in oocytes was stimulated during in vitro maturation. Oocyte mtDNA copy number was associated with developmental competence. Supplementation of maturation medium with NRG1 increased mtDNA copy number, and thus provides a means to improve oocyte quality and developmental competence in pigs.     

A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles

Growing porcine oocytes from early antral follicles (1.2-1.5 mm in diameter) do not mature to metaphase II (MII, 4%) under culture conditions which supported maturation (MII, 95%) of fully grown oocytes from large (4-6 mm) antral follicles. We hypothesized that FSH and dbcAMP supported growth and acquisition of meiotic competence. Growing oocytes (113.0 ± 0.4 μm, mean ± SEM) were cultured for 5 d in medium supplemented with 1 mM dbcAMP, 0.01 IU/mL FSH or both; in these media, oocytes reached, 120.5 ± 0.4, 123.5 ± 0.4 and 125.7 ± 0.2 μm, respectively, after 5 d, and then were matured in vitro for 48 h. Oocytes remained enclosed by cumulus cells when cultured with FSH (82%) or both FSH and dbcAMP (80%), but not with dbcAMP alone (0%). Furthermore, oocytes cultured with FSH maintained trans-zonal projections of cumulus cells. Oocytes remained at the GV stage at higher rates when cultured with dbcAMP and FSH (99%), or dbcAMP (97%), than with FSH (64%), or without either (75%). Following in vitro maturation, oocytes reached MII after in vitro growth with dbcAMP (19%), FSH (11%), or both (68%). When oocytes were cultured with both FSH and dbcAMP, activation of Cdc2 and MAP kinases in growing oocytes was similar to fully grown oocytes. In conclusion, growing porcine oocytes grew and acquired meiotic competence in medium supplemented with dbcAMP and FSH; the former maintained oocytes in meiotic arrest, whereas the latter maintained trans-zonal projections of cumulus cells to oocytes during in vitro growth culture.     

Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization

The present study examined the effect of epidermal growth factor (EGF) during in vitro maturation (IVM) and embryo culture on blastocyst development in the pig. In experiment 1, cumulus oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, hormonal supplements, and with or without EGF (0–40 ng/ml) for 20–22 hr. They then were cultured for an additional 20–22 hr without hormones. After maturation, cumulus-free oocytes were co-incubated with frozen-thawed spermatozoa for 5–6 hr. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 hr. In experiment 2, oocytes were matured in medium containing 10 ng/ml EGF, inseminated, and putative embryos were cultured in the presence of 0–40 ng/ml EGF. In experiment 3, oocytes were cultured in the presence of 0, 10 and 40 ng/ml EGF to examine the kinetics of meiotic maturation. In experiment 4, 2- to 4-cell and 8-cell to morula stage embryos derived from oocytes matured with 10 ng/ml EGF were transferred to the oviduct and uterus, respectively, of each of three recipient gilts (3 and 4 days post-estrus, respectively). The presence or absence of EGF during IVM did not affect cumulus expansion, nuclear maturation, fertilization parameters, or cleavage rate. However, compared to no addition (21%), presence of 1 (33%) and 10 ng/ml EGF (42%) during IVM increased (P < 0.01) the rate of blastocyst development in a concentration-dependent manner. Compared to 10 ng/ml EGF, higher concentrations (20 and 40 ng/ml) reduced (P < 0.01) blastocyst development in a concentration-dependent manner (35% and 24%, respectively). No difference was observed between no addition and 40 ng/ml EGF (22%). Compared to no addition and 10 ng/ml EGF, a significantly (P < 0.001) higher proportion (25% vs. 55%) of oocytes reached metaphase II stage 33 hr after IVM with 40 ng/ml EGF. However, no difference was observed at 44 hr. Transfer of embryos to six recipient gilts resulted in three pregnancies and birth of 18 piglets. The results show that EGF at certain concentrations in IVM medium can influence the developmental competence of oocytes. However, addition of EGF during the culture of pig embryos derived from oocytes matured in the presence of EGF is without effect. Birth of piglets provides evidence that embryos derived from oocytes matured in a medium containing EGF are viable. Mol. Reprod. Dev. 51:395–401, 1998. © 1998 Wiley-Liss, Inc.     

Nuclear maturation and embryo development of porcine oocytes vitrified by cryotop: Effect of different stages of in vitro maturation

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus–oocyte complexes (COCs) were cultured in IVM medium supplemented with 1 mM dibutyryl cAMP (dbcAMP) for 22 h and then for an additional 22 h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0–22, 26, 32, 38 and 44 h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44 h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44 h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2 h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44 h, but significantly decreased survival rate of oocytes matured for 0 or 22 h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P < 0.05) than that in the control. At 2 h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44 h of IVM were similar but were higher (P < 0.05) than those of oocytes vitrified at 0 or 22 h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38 h of IVM did not differ from the control and were higher (P < 0.05) than those of oocytes vitrified at 22, 26 or 32 h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44 h did not differ, but all were lower (P < 0.05) than those matured 0 h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.     

A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate-dependent meiotic arrest in murine oocytes

The effects of the putative maturation inhibitor in porcine follicular fluid on gonadotropinstimulated reversal of cyclic adenosine monophosphate (cAMP)-maintained meiotic arrest in mouse oocytes in vitro were assessed in this study. When cumulus cell-enclosed oocytes were cultured in a suboptimal inhibitory concentration of dibutyryl cAMP (dbcAMP), the effect of follicle-stimulating hormone (FSH) on oocyte maturation was initially inhibitory at 3 hr, but stimulatory at 6 hr. Supplementation of the medium with an ultrafiltrate of porcine follicuiar fluid (PM10-filtrate) completely suppressed FSH-promoted reversal of inhibition at 6 hr. Charcoal extraction eliminated this effect of the PM10-filtrate. FSH reversed the inhibition of maturation of cumulus cell-enclosed oocytes maintained by a high concentration of dbcAMP and suboptimal concentrations of the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX), during a 21–22-hr culture period. However, the effect of a completely inhibitory concentration of IBMX was not reversed by gonadotropin. A component of serum was also found to inhibit FSH reversal of dbcAMP-maintained meiotic arrest, and this activity was removed by charcoal extraction. In addition, when oocytes were cultured in medium containing a suboptimal concentration of dbcAMP plus a low molecular weight fraction (< 1,000) of porcine follicular fluid, porcine serum, or fetal bovine serum, a synergistic inhibition of maturation was observed. Experiments with highly purified gonadotropins revealed that reversal of dbcAMP-maintained meiotic arrest occurred only in response to FSH; neither highly purified luteinizing hormone nor human chorionic gonadotropin could mimic this action of FSH. Also, this effect was mediated by the cumulus cells, since FSH could not reverse dbcAMP-maintained meiotic arrest in denuded oocytes. Furthermore, elevating cAMP levels in denuded oocytes augmented, rather than reversed, the inhibitory action of dbcAMP on oocyte maturation. These data therefore suggest that dbcAMP- or IBMX-maintained meiotic arrest in vitro is reversed by an FSH-stimulated, cAMP-dependent process mediated by the cumulus cells and demonstrate that a factor present both in follicular fluid and serum prevents this action of the gonadotropin.     

Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.     

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10⁶ cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O₂. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O₂ using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.     

Protection of porcine oocytes against cell damage caused by oxidative stress during in vitro maturation: role of superoxide dismutase activity in porcine follicular fluid

To elucidate the beneficial effects of porcine follicular fluid (pFF) added to maturation medium on the sustenance of cytoplasmic maturation responsible for the subsequent developmental competence after in vitro fertilization (IVF) of porcine oocytes, we focused on the antioxidative role of pFF in its function of protecting oocytes from reactive oxygen species (ROS)-induced cell damage. Porcine follicular fluid collected from small (2-6 mm) follicles had about 7.2-fold higher levels of superoxide dismutase (SOD) activity than that of fetal bovine serum (FBS), and this activity was markedly blocked by the CuZn-SOD inhibitor, diethyldithiocarbamate (DETC). The interruption of meiotic progression and the increasing intracellular glutathione (GSH) content throughout the maturation period, as well as an outbreak of DNA damage in oocytes and cumulus cells were difficult to detect in oocytes cultured in a medium supplemented with 10% pFF, even in the presence of ROS generated by the hypoxanthine-xanthine oxidase system, whereas cell damage encompassed by ROS was prominent in oocytes cultured with 10% FBS and 10% pFF plus 100 microM DETC. Similarly, significant enhancement to the degree of transformation of the sperm nucleus into the male pronucleus (MPN) after in vitro fertilization was shown by the addition of pFF to the maturation medium. The presence of DETC during in vitro maturation reduced the ability of oocytes to promote MPN formation to the same extent as oocytes matured with FBS. The proportion developing to the blastocyst stage was increased in oocytes that matured with pFF, but this developmental competence was significantly lowered by treatment with DETC (P < 0.05). These findings suggest that pFF plays a critical role in protecting oocytes from oxidative stress through a higher level of radical scavenging activity elicited from SOD isoenzymes, resulting in the enhancement of cytoplasmic maturation responsible for developmental competence postfertilization.     

Influence of caffeine pretreatment on biphasic in vitro maturation of dog oocytes

Phosphodiesterase (PDE) inhibitors have been utilized for in vitro maturation (IVM) of oocytes to manipulate the meiotic resumption and progression. Premature chromatin condensation and DNA replication of the oocytes, immediately after the decrease in the cAMP level, are the difficulties in canine IVM. Caffeine, a nonselective competitive PDE inhibitor, due to its structural similarity to adenosine molecule maintains the cAMP level by occupying PDE enzymes such as PDE-3A inside the oocyte and PDE-4 and PDE-5 in the cumulus cells. In this study, the effects of 12-hour caffeine pretreatment in a biphasic IVM protocol were assessed on maturation rates of canine oocytes. Sixty hours of culture after a 12-hour of 10 mM caffeine pretreatment resulted in 16.9% ± 2.4 of the oocytes reaching metaphase II stage (MII) and 25.9% ± 5.2 degeneration rate compared with the control group with 2.2% ± 2.2 MII and 37.6% ± 4.3 degeneration rates (P < 0.05). Caffeine pretreatment induced higher mitogen-activated protein kinases (MAPK1 and MAPK3) phosphorylation and maturation-promoting factor activity at 12 hours and activated MAPK1 and maturation-promoting factor at 48 hours after culture in cumulus-oocyte complexes (COCs) compared with the control group (P < 0.05). Fresh canine COCs were also analyzed before IVM using brilliant cresyl blue (BCB) staining. Oocytes showed difference in meiotic resumption (MI-MII) (BCB+ = 16.11% ± 5.5, BCB− = 9.86% ± 5.0; P < 0.05) after 60 hours of culture following 12-hour caffeine pretreatment. The BCB+ canine oocytes had higher MII rate than the BCB− group under caffeine pretreatment (10.2% ± 2.9 vs. 1.1% ± 1.1, respectively; P < 0.05). Results indicated that 12-hour caffeine pretreatment of canine COCs improves the MII maturation rates at 72 hours and BCB+ oocytes have higher competency in vitro for nuclear maturation.     

Expression of growth differentiation factor 9 (GDF-9) during in vitro maturation in canine oocytes

The aim of this study was to characterize in canine oocytes and cumulus cells the dynamic expression of growth differentiation factor 9 (GDF-9) in relation to meiotic development and cumulus expansion throughout in vitro maturation (IVM). Cumulus oocytes complexes (COCs) from ovaries of adult bitches were cultured intact for IVM during 0, 48, 72, and 96 hours. At 0 hours or after IVM, COCs were divided into two groups: one group remained with their cumulus cells and in the other group the cumulus cells were extracted. The expression levels of GDF-9 were determined in both groups using indirect immunofluorescence and Western blot analysis. For immunofluorescence assay, in vivo-matured oocytes collected from oviducts were also used as a positive control. The nuclear stage was analyzed in parallel with 4′-6-diamidino-2-phenylindole staining in denuded oocytes from all maturing groups. The intensity of fluorescence, indicative of GDF-9 expression level, decreased with time (P < 0.05). High expression was observed only in germinal vesicle nonmature oocytes; in contrast, second metaphase oocytes showed only low expression. Western blot analysis showed bands of approximately 56 kd and a split band of approximately 20 kd representing the proprotein and possibly two mature protein forms of GDF-9, respectively. The proprotein was detected in all samples, and it was highly expressed before IVM and in a lesser degree, during the first 48 hours, declining thereafter in coincidence with the expansion of the cumulus cell (P < 0.05). There was a negative correlation (r = −0.97; P < 0.05) between the expression level of GDF-9 and mucification. Mature forms were evident only in COCs, before culture and up to 48 hours of IVM. It was concluded that GDF-9 is expressed in canine oocytes and cumulus cells, mainly in the early developmental states, with low levels in mature oocytes in vitro and in vivo, representing the first approach of GDF-9 dynamic in dog oocyte maturation.     

Effect of cytoplasmic lipid content on in vitro developmental efficiency of bovine IVP embryos

The objective was to evaluate the effect of cytoplasmic lipid content on the embryonic developmental efficiency of bovine in vitro embryo production (IVP) embryos. Ovaries from Korean native cows (Bos taurus coreanae) were collected from a local abattoir, and cumulus-oocyte complexes (COCs) were recovered from follicles 2 to 8 mm in diameter. The oocytes were divided into three groups, dependent on their cytoplasm color: pale color (PC), brown color (BC), and dark color (DC). The COCs were fertilized using frozen-thawed semen from a single Hanwoo bull. Based on measurement of the cytoplasmic color intensity of oocytes after 22 h of in vitro maturation (IVM), the DC group had lower (P < 0.05) color intensity than that in the BC and PC groups (56.3 ± 2.7, 93.3 ± 5.1, and 123.9 ± 12.0, respectively). Based on MitoTracker Green FM staining, the number of mitochondria in the DC (170.1 ± 31.2) group was significantly higher than that in the BC (137.5 ± 30.8) and PC (105.5 ± 25.3) groups. The cleavage rate in the DC (81.5%) group was also higher than that in the PC (50.4%) group (P < 0.05), as was the development rate to blastocyst stage (18.9% vs. 9.8%). Finally, cell numbers of blastocysts in the DC (150.8 ± 28.0) group were higher (P < 0.05) than that in the BC (107.6 ± 17.8) and PC (80.5 ± 12.3) groups. In conclusion, cytoplasm color was a useful selection parameter for abattoir-derived oocytes destined for IVP.     

Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor-b?₁ (TGF-b?₁), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A₄), and estradiol-17b? (E₂) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte-cumulus cell complexes (OCC) were cultured in the media containing the abovementioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 μg/ml) together and LH (2 μg/ml) and FSH (1.5 μg/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E₂ (1 μg/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A₄ (1 μg/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF-b?₁ had no effect on GVBD or cumulus expansion. These studies indicate that these hormones may have differing roles in oocyte maturation and that their interactions may be part of an intricate system regulating the maturation of oocytes during follicular development in vivo. © 1993 Wiley-Liss, Inc.     

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

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

Effect of sericin supplementation in maturation medium on cumulus cell expansion,oocyte nuclear maturation,and subsequent embryo development in Sanjabi ewes during the breeding season

The purpose of this study was to evaluate the effect of sericin with different concentrations (0% [control], 0.1%, 0.5%, 1.0%, and 2.5%) added to the IVM medium on cumulus cell expansion, oocyte nuclear maturation, and subsequent embryo development in Sanjabi ewes during the breeding season. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body extrusion. After IVF with fresh ram semen, presumptive zygotes were cultured 8 days in potassium simplex optimization medium supplemented by amino acids, and the percentages developing to the two-cell and blastocyst stages were measured as the indicators of early embryonic developmental competence. More cumulus–oocyte complexes matured with 0.5% sericin underwent germinal vesicle breakdown and reached metaphase II stage compared with the control cumulus–oocyte complexes matured without sericin (P ≤ 0.05). The present findings indicated that supplementation with 0.5% sericin during the maturation culture may improve the nuclear maturation and the cumulus cell expansion. Furthermore, the percentage of blastocysts obtained from 0.5% and 0.1% sericin (37.8 ± 1.76% and 34.8 ± 1.09%, respectively) was higher (P ≤ 0.05) than that of the control medium (29.60 ± 1.67%). However, addition of 1% and 2.5% of sericin to the IVM medium oocytes had a negative effect on nuclear maturation and cumulus cell expansion. Furthermore, the percentage of cleavage and blastocyst rate was significantly lower in the 1% and 2.5% sericin groups than in the control group. These findings showed that supplementation of IVM medium with 0.5% sericin may improve the meiotic competence of oocytes and early embryonic development in Sanjabi ewes during the breeding season.     

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 μg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ±13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 μg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 μg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10⁶ cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 μg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10⁶ cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 μg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 μg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 μg/mL Zn relative to 0 Zn (IVM alone) and 0.7 μg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.