Oocyte Maturation Medium Research Articles

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Adiponectin enhances in vitro development of swine embryos

Recent studies have shown that factors from adipose tissue influence and regulate the reproductive system. Hormones such as leptin and resistin are now known to regulate several reproductive processes. Adiponectin is the most abundant protein secreted by adipose tissue, and its circulating concentration is inversely related to adiposity and body mass index. Little is known about the involvement of adiponectin in reproduction. In the present study, the effect of recombinant adiponectin on the meiotic maturation and early embryo development in vitro was investigated, using porcine oocytes. Adiponectin receptors, AdipoR1 and AdipoR2, were found to be expressed in porcine oocytes and cumulus cells of both small and large follicles. Both AdipoR1 and AdipoR2 were immunolocalized to cumulus–oocyte complexes (COCs), oocytes, and early developing embryos. When included in oocyte maturation medium for 46 h, adiponectin significantly decreased the frequency of meiotic immature oocytes derived from large follicles (3–6 mm) but not from small follicles (<3 mm). From studies of oocytes matured in the presence of adiponectin and mitogen-activated protein kinase (MAPK) pathway inhibitors MEK1 (PD98059), MEK1/2 (U0126), and p38MAPK (SB203580) it was concluded that adiponectin enhances oocyte maturation thought the p38MAPK pathway. Finally, a superior rate of embryo development to the blastocyst stage was achieved by embryos cultured in the presence of adiponectin. These results indicate that adiponectin has a positive effect on the meiotic maturation and in vitro embryo development of porcine oocytes and suggests a physiological role for this adipokine in early development in mammals.

Effect of a phosphodiesterase type 3 inhibitor in oocyte maturation medium on subsequent mouse embryo development

To investigate the effect of a phosphodiesterase type 3 inhibitor in oocyte maturation medium on subsequent embryo development. Animal study. University laboratory. CD-1 mice. Immature oocytes were arrested meiotically in medium containing a specific phosphodiesterase type 3 inhibitor, cilostamide (1 micromol/L, 5 micromol/L, and 10 micromol/L), for 6 and 24 hours of prematuration culture, and then matured in vitro in inhibitor-free medium. Oocyte maturation, fertilization, and early embryonic development in vitro. After 6 and 24 hours of prematuration culture with 1 micromol/L, 5 micromol/L, and 10 micromol/L cilostamide, there were no differences in maturation, fertilization, and early embryonic development compared with non-arrested controls. Prematuration culture with cilostamide, for either 6 or 24 hours, may not be beneficial for subsequent embryonic development of immature mouse oocytes.

Effect of polyvinylpyrrolidone on bovine oocyte maturation in vitro and subsequent fertilization and embryonic development

The exact role of polyvinylpyrrolidone (PVP) in culture medium for oocyte maturation is still largely unknown. Bovine cumulus–oocyte complexes (COC) were cultured in in-vitro maturation (IVM) medium supplemented with 10% fetal bovine serum (FBS), 0.3% PVP (K-90) or 10% serum substitute supplement (SSS) respectively. The rates of oocyte maturation, fertilization and early embryonic development were evaluated. In addition, the status of DNA fragmentation in the oocytes was determined by comet assay, and the ratio of trophectoderm (TE) cells and inner cell mass (ICM) in blastocysts was determined by differential staining. Furthermore, the percentage of apoptotic cells in the blastocysts was examined by TUNEL assay. The results indicated that the effect of PVP in IVM medium was similar to FBS in terms of oocyte maturation and subsequent embryonic development. However, the addition of SSS in IVM medium retarded further embryonic development and resulted in more oocyte DNA fragmentation and a higher ratio of TE cells and ICM in the blastocysts. However, the number of apoptotic cells in blastocysts was similar among the three groups. These results suggest for the first time that the addition of PVP in oocyte maturation medium is not only a suitable substitute for serum but is also beneficial to in-vitro oocyte maturation.

Human Umbilical Cord Blood Serum in Culture Medium on Oocyte MaturationIn Vitro

In vitro maturation (IVM) of immature oocytes for infertile patients is an attractive treatment. It can avoid side effects of ovarian stimulation with gonadotropins. However, at the present the successful results of IVM treatment are lower than conventional in vitro fertilization (IVF) treatment. The key issue may be the IVM medium for immature oocyte maturation. In the present study, we compared 20% (v/v) human follicular fluid (hFF) and 20% (v/v) human umbilical cord serum (hUS) as a supplement to IVM medium. A total of 47 patients with polycystic ovary syndrome (PCOS) underwent 47 IVM treatment cycles. Immature oocytes (349) collected from 32 patients were cultured in IVM medium supplemented with hFF, and immature oocytes (160) collected from 15 patients were culture in IVM medium supplemented with hUS. The results indicate that the final maturation rate of oocytes cultured in IVM medium supplemented with hUS (93.8%) is significantly higher than those cultured in IVM medium supplemented with hFF (77.1%). The percentage of high-quality embryos produced from IVM medium supplemented with hUS (50.0%) is significantly higher than IVM medium supplemented with hFF (23.8%). In addition, the results also indicate that the final maturation rate of oocytes is higher in IVM medium supplemented with hUS and the time course of oocyte maturation is hastened. Following transfer 6 out of 15 patients (40.0%) become pregnant when IVM medium was supplemented with hUS, and 7 out of 31 patients (22.6%) were pregnant when IVM medium was supplemented with hFF. These results suggest that IVM medium containing hUS appears to be a more effective means to stimulate in vitro oocyte maturation and is capable of achieving a promising clinical outcome.

A Growth-Maturation System That Enhances the Meiotic and Developmental Competence of Porcine Oocytes Isolated from Small Follicles1

In livestock, most of the follicles on the ovarian surface are small follicles. A procedure that supports the in vitro growth and maturation of these small follicle-derived oocytes may offer a new source of useable oocytes for both biotechnological and fundamental research purposes. The objective of the current study was to test the hypothesis that providing a more growth-supporting and less maturation-promoting environment during the first phase of small follicle-derived oocyte maturation may improve oocyte competence for meiosis and embryo development upon activation. In our small follicle-derived oocyte growth-maturation system (SGM group), cumulus-oocyte complexes (COCs) from small follicles (1-3 mm) were first cultured in oocyte growth medium for 24 h, then in oocyte maturation medium for 20 h. As controls, COCs from small (SM group) and large (LM group) follicles were cultured using a conventional in vitro maturation (IVM) approach in which they were directly cultured in oocyte maturation medium. At 24 h of culture, the percentage of small follicle-derived oocytes that underwent germinal vesicle breakdown (GVBD) in the SGM group was comparable to that of large follicle-derived oocytes (LM group) but was significantly higher than that of the SM group (P < 0.05). At 44 h of culture, compared to 36% in the SM group, 55% of the SGM group oocytes reached metaphase II (MII; P < 0.05). In addition, the level of cyclin B in oocytes of the SGM group was comparable to that of oocytes from LM group and was significantly higher than that of oocytes from the SM group (P < 0.05). When activated and in vitro fertilized (IVF), 7.3 and 9.0 times more parthenogenetic and IVF embryos developed to blastocyst stage in the SGM group than in the SM group (P < 0.05). The mRNA expression levels of three developmentally important genes--DNA-methyltransferase 1, Pou domain class 5 transcription factor 1, and Fibroblast growth factor receptor 2--in embryos of the SGM group were comparable to those of embryos developed from the LM group, whereas they were significantly lower in those of the SM group (P < 0.05). Our data suggest that the oocyte growth-maturation system facilitates the final stage of oocyte growth and thus resulted in better oocyte nuclear, cytoplasmic maturation, and developmental competency compared with the conventional direct oocyte maturation system.

Tissue-Specific Effects of In Vitro Fertilization Procedures on Genomic Cytosine Methylation Levels in Overgrown and Normal Sized Bovine Fetuses1

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo-fertilized controls (n=14) indicated an apparently normal phenotype for IVF1 fetuses (n=5), but IVF2 fetuses (n=10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (-16.1% and -9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

Effects of hexoses on in vitro oocyte maturation and embryo development in pigs

The objective was to determine the effects of supplementing hexoses in oocyte maturation and embryo culture medium on in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes and in vitro development of in vitro produced (IVP) porcine embryos. In the first experiment, oocytes were matured in vitro in modified North Carolina State University (NCSU)-37 medium, supplemented with hexoses (glucose, fructose or galactose) at various concentrations: 0 (control), 2.5, 5.5 and 10 mM. Supplementing the maturation medium with either glucose or fructose (5.5 mM) increased the percentages of oocytes that matured to metaphase II (79.4 and 70.2%, respectively), as compared with the control group ( P < 0.05). However, supplementing galactose had no effects on meiotic maturation and fertilization. In the second experiment, cleaved embryos were collected 3 days after IVF of oocytes matured in the maturation medium supplemented with 5.5 mM of glucose; they were cultured for an additional 4 days in modified NCSU-37 medium, supplemented with 5.5 mM of glucose, fructose or galactose. The incidence of blastocyst formation was higher ( P < 0.05) in the glucose and fructose groups (18.6 and 18.2%, respectively) than in the galactose group and non-supplemented control group (12.9 and 9.2%). Moreover, fructose supplementation increased the total cell number/blastocyst (48.0 versus 37.6) and reduced the index of DNA-fragmented nucleus in the blastocysts (7.6% versus 11.8%), as compared with glucose supplementation ( P < 0.05). In conclusion, fructose was a practical alternative to glucose for supporting IVM of porcine oocytes and fructose was superior to glucose for producing high-quality porcine embryos in vitro.

Comparison of three in vitro culture systems for maturation of early preantral mouse ovarian follicles

The aim of this study was to compare three different culture systems for in vitro follicular growth and oocyte maturation in ovarian follicles of mice in order to assess the technique with the optimal growth and improved rate of meiotic maturation. The three systems tested were culture under oil, on a hydrophobic membrane and on agar respectively. Early preantral follicles were cultured for 12 days in alpha-MEM GlutaMAX medium. Follicular growth, oocyte meiotic maturation, oocyte extrusion, atresia and estradiol production were analysed. Follicular development showed two phases in the three systems, with slow growth before day 5 and subsequent acceleration. The percentage of follicles transferred into oocyte maturation medium was significantly higher after culture under oil. The proportion of oocytes that achieved nuclear maturation (metaphase II) was higher when follicles were cultured under oil or on a hydrophobic membrane than on agar. Our results support the use of culture under oil for in vitro follicular growth from the early preantral stage in order to obtain metaphase II oocytes. Fertilization ability of these oocytes and the capacity to obtain healthy mice in a reproducible manner warrants further investigation.

Addition of Recombinant Human Growth Hormone to In Vitro Maturation Medium of Bovine Oocytes

The aim of this study was to increase the bovine embryonic development rate, adding recombinant human growth hormone (rhGH) to maturation medium of bovine oocytes. Oocytes were matured for 24 h in TCM 199 Earle's salts and five treatments were developed: T1, 0.01 IU/ml of recombinant human follicle stimulating hormone (rhFSH); T2, 0.01 IU/ml of rhFSH + 100 ng/ml of rhGH; T3, 0.01 IU/ml of rhFSH + 1000 ng/ml of rhGH; T4, 100 ng/ml of rhGH; and T5, 1000 ng/ml of rhGH at 39 degrees C and 5% of CO(2) in air and saturated humidity. In vitro fertilization from cumulus-oocyte complexes was conducted in TALP-Fert medium (18-22 h) and spermatozoa were selected by Percoll gradient. Zygotes were incubated in SOFaaci medium in 5% of CO(2) in air, 5% of O(2) at 39 degrees C and saturated humidity for 11 days. There was no statistical difference in cleavage rate and embryo production on day 7 and day 9 among treatments. However, the hatching rate increased significantly in the T4 and T5 treatments (11.0 and 12.8%, respectively), compared with the T1 treatment (4.6%) (p < 0.05). Therefore, the rhGH addition to the oocyte maturation medium showed beneficial effects on the hatching rate of in vitro-produced bovine embryos.

294 THE EFFECT OF PLASMIN ON THE IN VITRO FERTILIZATION ABILITY OF PORCINE GAMETES

Plasminogen activators (PAs) are specific proteolytic enzymes that convert the inactive proenzyme plasminogen to plasmin. The plasmin formed is a nonspecific, potent protease that cleaves blood fibrin clots and several other extracellular proteins. The purposes of the present study were (1) to assess the effect of plamin on sperm viability and acrosome reaction (AR), (2) to examine the effect of plasmin on zona pellucida (ZP) solubility and the binding of sperm to ZP, and (3) to evaluate the effect of plasmin on fertilization responses, including penetration and incidence of polyspermy during in vitro fertilization in the pig. Ejaculated semen was collected from three mature Duroc boars by artificial vagina. The same three boars were used for all experiments. The oocyte maturation medium used was North Carolina State University-23 (NCSU-23) medium supplemented with 10% (v/v) porcine follicular fluid (pFF), 0.6 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG; Sigma-Aldrich Corporation, St. Louis, MO, USA), and 10 IU/mL pregnant mare's serum gonadotropin (PMSG; Sigma). Porcine spermatozoa, which were washed in Dulbecco PBS (Sigma), were resuspended and incubated in fertilization medium (mTBM) containing 0, 0.1, 1.0, 10.0, or 100.0 ng/mL plasmin (Sigma). Data were analyzed by ANOVA and Duncan's multiple-range test using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The present study suggests that sperm viability was not affected by plasmin treatment. Also, addition of plasmin in doses ranging between 0.1 and 100.0 ng/mL for 2, 4, or 6 h to washed boar spermatozoa resulted in enhancement of acrosome reaction (AR), compared with untreated cells. Concentrations of 0 and 0.1 ng/mL plasmin (83 � 15 and 95 � 18 sperm/oocyte, respectively) had no effect on sperm binding, whereas 1.0 (123 � 21 sperm/oocyte), 10.0 (124 � 16 sperm/oocyte), and 100 ng/mL (124 � 15 sperm/oocyte) plasmin increased (P &lt; 0.05) sperm binding, compared with the control. The zona pellucida solubility (zona digestion time) was significantly (P &lt; 0.05) lower in medium with 1.0 (123 � 24 s), 10.0 (99 � 15 s), or 100.0 ng/mL (95 � 19 s) plasmin, compared with control (176 � 27 s). When porcine oocytes and spermatozoa were co-incubated in various concentrations of plasmin for 6 h, the penetration rate was significantly (P &lt; 0.05) higher in medium with 1.0 ng/mL plasmin (77.5 � 3.1%), compared with control. However, there were no significant differences in the polyspermic rates and mean numbers of sperm (MNS)/oocyte among the groups treated with plasmin and the control group. We found that addition of plasmin to fertilization medium increases the percentage of acrosome-reacted spermatozoa and the sperm-binding ability of the pig ZP. These results suggest that plasmin may play a role in events related to fertilization in the pig.

276 IN VITRO FERTILIZATION PROCEDURES AFFECT THE HEPATIC GENOMIC CYTOSINE METHYLATION LEVEL AND THE PHENOTYPE OF BOVINE FETUSES

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable bovine Day 80 fetuses to assess the effects of different in vitro fertilization (IVF) protocols on hepatic genomic cytosine methylation levels and on fetal phenotype. Two groups of IVF-fetuses (IVF1, n = 5 and IVF2, n = 10) were compared with control fetuses generated by artificial insemination (AI, n = 14). Both IVF protocols were previously employed to generate live offspring but differed with respect to gonadotropins in the oocyte maturation medium (0.01 units/mL b-FSH and b-LH for IVF1 versus 0.2 units/mL o-FSH for IVF2) and serum concentrations in the embryo culture media (5% estrous cow serum in IVF1 versus 10% estrous cow serum in IVF2). Analysis of variance (General Linear Model Procedure, SPSS for Windows version 12.0; SPSS GmbH Software, Munich, Germany) showed that fetus group significantly affected fetus weight and length (P &lt; 0.01), heart (P &lt; 0.05) and liver (P &lt; 0.01) weight, and 5-methylcytosine (5 mC) content of liver DNA (P &lt; 0.001). Comparison of group means (t-test) showed that methylation levels in both groups of IVF fetuses differed significantly from AI controls. We observed hepatic DNA hypomethylation (-15.4% vs. AI control, P &lt; 0.01) in IVF1 fetuses, and hypermethylation (+11.6% vs. AI control, P &lt; 0.001) in IVF2 fetuses, but only IVF2 fetuses showed phenotypic abnormalities. The IVF2 fetuses were significantly heavier (18.6%, P &lt; 0.01) and longer (4.3%, P &lt; 0.05) than AI fetuses, with increased heart (21.8%, P &lt; 0.05) and liver (25%, P &lt; 0.001) weights, and thus displayed an overgrowth phenotype. A clinical-chemical screen of 18 plasma parameters failed to detect abnormalities in IVF1 fetuses but revealed significantly increased levels of insulin-like growth factor 1 (40.8%, P &lt; 0.001) and creatinine (28.5%, P &lt; 0.05) in IVF2 fetuses. Our data indicate that bovine IVF-procedures can induce protocol-specific and persistent changes in hepatic cytosine methylation level with or without obvious concomitant changes in fetal phenotype. This suggests that epigenetic change after bovine IVF could be more widespread than previously thought and highlights the value of epigenetic diagnostic screening.

295 RELATIONSHIP BETWEEN PLASMINOGEN ACTIVATOR/PLASMIN SYSTEM AND IN VITRO FERTILIZATION ABILITY IN THE PIG

Plasminogen activators (PAs) are serine proteases, known to be secreted by a large number of cell types, that convert plasminogen into plasmin. The present study was undertaken to identify PAs in porcine gametes and to investigate a possible role of plasminogen in fertilization in vitro in the pig. The oocyte maturation medium used was North Carolina State University-23 (NCSU-23) medium supplemented with 10% (v/v) porcine follicular fluid (pFF), 0.6 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG), and 10 IU/mL pregnant mare's serum gonadotropin (PMSG). To determine the changes of PA activities in porcine oocytes during maturation, the cumulus-oocyte complexes (COCs) were incubated in NCSU-33 in an atmosphere of 5% CO2 in air at 39�C for 0, 24, or 48 h. On the other hand, to investigate the release of PAs by boar spermatozoa, fresh spermatozoa were pre-incubated in fertilization medium (mTBM) for 0, 2, 4, or 6 h. After culture, 40 COCs, 40 cumulus-free oocytes, and 40 � 106 spermatozoa were separately put into microtubes containing 20 �L of sample buffer [5.0% (w:v) sodium dodecyl sulfate (SDS), 20% (v:v) glycerol, and 0.0025% (w:v) bromophenol blue in 0.125 M Tris-HCl buffer] and frozen at -80�C until used for analysis. PA activities in porcine oocytes and spermatozoa were quantified using SDS-PAGE, casein-agar zymography, and densitometry. Data were analyzed by ANOVA and Duncan's multiple-range test using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). In the COCs cultured for 24-18 h, tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed. Also, PA activities increased as duration of culture increased. However, no uPA activity was detected in cumulus-free oocytes. In procine fresh spermatozoa, tPA, uPA, and tPA-PAI were observed. When spermatozoa were incubated for 2, 4, or 6 h in fertilization medium, the rate of acrosome reaction (AR) in spermatozoa increased as the duration of culture increased, but PA activities decreased gradually. However, PA activities in sperm-conditioned medium increased as duration of culture increased. On the other hand, to determine the effect of plasminogen on fertilization ability of porcine oocyte and spermatozoa, plasminogen (50 �g/mL) was added in fertilization medium. Addition of plasminogen to fertilization medium increased (P &lt; 0.05) AR in spermatozoa and sperm binding to the zona pellucida (ZP), compared with control group. The ZP solubility (zona digestion time) was higher in medium with than that without plasminogen. When porcine oocytes and spermatozoa were co-incubated in fertilization medium with plasminogen, the polyspermic rate was lower in medium with than that without plasminogen. Also, plasminogen significantly (P &lt; 0.05) increased formation rate of the male pronucleus in oocytes penetrated by spermatozoa. These results suggest that supplementing of plasminogen in fertilization medium may play a positive role in improving of fertilization ability in vitro in the pig.

Leptin enhances oocyte nuclear and cytoplasmic maturation via the mitogen-activated protein kinase pathway.

Recent studies have suggested that leptin has a central role in female reproduction, including ovarian function. The leptin receptor (Ob-R) has six isoforms and can signal through either the MAPK or the Janus-activated kinase/signal transducer and activator of transcription signal-transduction pathway, depending on the isoform. Expression of Ob-R has been reported in human and mouse oocytes; however, the physiological role of leptin during follicular development and oocyte maturation is largely unknown. In the current study, expression of Ob-R during oocyte growth and maturation was investigated in porcine oocytes from small, medium, and large follicles and in oocytes in the germinal vesicle (GV), GV breakdown, and metaphase II (MII) stages at both the mRNA and protein levels. The proportion of oocytes expressing Ob-R was maximal in oocytes from medium follicles and at the GV breakdown stage (P < 0.05), whereas the proportion of oocytes expressing the long isoform, Ob-Rb, was found to be consistently low throughout growth and maturation. When included in oocyte maturation medium, leptin significantly increased the proportion of oocytes reaching MII (P < 0.01), elevated cyclin B1 protein content in MII-stage oocytes (P < 0.05), and enhanced embryo developmental potential (P < 0.05), suggesting that leptin plays a role in both nuclear and cytoplasmic maturation. During oocyte maturation, leptin increased phosphorylated MAPK content by 2.8-fold (P < 0.05), and leptin-stimulated oocyte maturation was blocked when leptin-induced MAPK phosphorylation was suppressed by a specific MAPK activation inhibitor, U0126 (P < 0.01), demonstrating that leptin enhances nuclear maturation via activation of the MAPK pathway.

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.

In vitro development of buffalo oocytes in media-containing fluids from different size class follicles.

Studies were conducted to investigate the effect of supplementation of fluid from different sized class [small (SFF, < 3 mm), medium (MFF, 3-8 mm) and large (LFF, > 8 mm)] of normal and cystic (CFF) ovarian follicles in oocyte culture media on oocyte maturation rate and embryo development in vitro and to test the efficacy of follicular fluid (FF) from different size classes as a whole oocyte maturation medium. Results suggested that FF were capable of developing buffalo oocytes to embryonic stage in vitro although its efficacy was lower than that of serum. Regardless of high maturation rates after in vitro maturation (IVM) in media containing FF or IVM in whole FF, low blastocyst rates were obtained after in vitro fertilization (IVF) and culture of embryos. Follicular fluid from small follicles had significantly (p < 0.05) higher potential of developing buffalo oocytes to embryonic stage in vitro than that from medium and large follicles. Cystic FF was not capable of supporting development of buffalo oocytes in vitro.

315THE REDUCTION OF MATURATIONAL COMPETENCE BY STREPTOMYCIN DURING IN VITRO MATURATION OF GOAT FOLLICULAR OOCYTES

Antibiotics are commonly added to mammalian oocyte maturation media, but their effects on oocytes maturation have not been examined thoroughly. Goat follicular oocytes were used to investigate whether penicillin, streptomycin or gentamycin affect maturational competence of oocytes and subsequent parthenogenetic activation potential in vitro. Cumulus-oocyte complexes collected from a local abattoir were matured for 24h in five treatments, and matured oocytes were cultured for 48h in five treatments after parthenogenetic activation by treatment with ionomycin, followed by immediate exposure to 6-diethlaminopurine; (1) Control: TCM-199 medium with no antibiotics, (2) TCM-199 with 100IU/mL−1 penicillin (P-4687, Sigma, St. Louis, MO, USA), (3) TCM-199 with 50μgmL−1 streptomycin (S-1277, Sigma), (4) TCM-199 with 50μgmL−1 gentamycin (G-1264, Sigma) and (5) TCM-199 with both 100IUmL−1 penicillin and 50μgmL−1 streptomycin. Maturation rates at 24h post-in vitro maturation and parthenogenetic cleavage development at 48h post-activation were evaluated. Data were analyzed by ANOVA and Student’s t-test. Penicillin and gentamicin treatment groups did not affect maturation rates and percentages of cleavage to 2–4 cell stage at 48h post-chemical oocyte activation. However, when streptomycin was present in the maturation medium, the percentages of matured oocytes at 24h post-in vitro maturation of immature goat oocytes were significantly lower than those from the other groups. However, among the five treatments, there was no significant difference in cleavage rates of matured oocytes at 48h post-activation (Table 1). Therefore, streptomycin did interfere with the maturation of immature goat oocytes, but did not affect the subsequent development of matured goat oocytes. The mechanism by which streptomycin affects the maturation of goat follicular oocytes needs to be investigated further. We conclude that streptomycin in oocyte maturation medium can be detrimental during in vitro maturation of goat follicular oocytes. Table 1 Effect of antibiotics on maturational competence of goat follicular oocytes and subsequent parthenogenetic activation potential in vitro

319IN VITRO MATURATION OF EQUINE OOCYTES IN A COMPLETELY DEFINED MEDIUM SUPPLEMENTED WITH PROGESTERONE

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500ngmL−1) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1IUmL−1 (Pluset, Laboratorios Calier, Barcelona, Spain), EGF* 50ngmL−1, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytesmL−1): (1) MSOF+FCS 10% (MSOF-FCS), (2) MSOF+progesterone 100ngmL−1 (MSOF-P4), (3) MSOF. After 24h of culture in 5% CO2 in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10μgmL−1 in PBS) for 30min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P&amp;lt;0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P&amp;lt;0.05) increases the nuclear maturation rate at 24h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4±19.3ngmL−1, healthy preovulatory follicle 1094.3±170.9ngmL−1; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system.*All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated. Table 1 Maturation of equine oocytes in different media

41EFFECTS OF MATURATION PERIOD OF PORCINE OOCYTES ON DEVELOPMENT FOLLOWING SOMATIC CELL NUCLEAR TRANSFER

Despite intensive research, porcine nuclear transfer is still characterized by low success rates. To determine the effect of maturation period of porcine oocytes on subsequent development following nuclear transfer, we investigated fusion rate, induction of activation and development to blastocyst stage of somatic cells. For this we used MII-oocytes after 38, 40, and 42h of maturation culture as recipients. Oocytes surrounded by a compact cumulus mass were selected and placed into North Carolina State University (NCSU) 37 oocyte maturation medium supplemented with 0.1mgmL−1 cysteine, 10ngmL−1 epidermal growth factor, 10% porcine follicular fluid, 50μm 2-mercaptoethanol, 0.5mgmL−1 cAMP, 10 IU each of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) for 22h in humidified air with 5% CO2 at 38.5°C. Subsequently the oocytes were moved to fresh NCSU37 maturation medium without cAMP, eCG and hCG and incubated for an additional 16, 18, and 20h. In the first experiment, a total of 878 MII-arrested oocytes were enucleated, fused with pig fetal fibroblasts in calcium-free medium and activated approximately 3h later with an electrical stimulus. This was followed by incubation in 6-dimethylaminopurine for 3h and subsequent analysis of development in vitro. Maturation period had no effect on the frequencies of fusion (87% v. 75% v. 84%, respectively), and cleavage (82% v. 81% v. 87%, respectively), but when MII-oocytes recovered at 40h of maturation were used as recipients, 41/279 (14,8%) the numbers of cloned embryos developing to the blastocyst stage on Day 7 of culture was significantly (ANOVA followed by multiple pairwise comparisons using Tukey test, 6 replicates, P&amp;lt;0,05) higher than those of embryos reconstituted with oocytes collected at 38h (27/285, 9.6%) and 42h (16/314, 4.9%). In the second experiment, reconstructed embryos derived from oocytes matured for 40h were surgically transferred to the oviducts of synchronized German Landrace gilts. Transfers were made on the first day of standing oestrus within 3h of activation to assess their development in vivo. Synchronization was achieved by injections of 1500IU eCG followed by 500IU hCG 3 days later. Of 4 recipients receiving an average of 150 zygotes (range, 136 to 163), 2 became pregnant as determined by ultrasound between Days 25 and 35 of gestation. Of the two pregnant recipients, one subsequently farrowed 4 piglets on Day 115 of pregnancy. These results indicate that the maturation period is critical and affects development of porcine nuclear transfer embryos. This study was funded by the Deutsche Forschungsgemeinschaft (DFG; SFB265).

252.Oocyte-secreted factor(s) regulate apoptosis of bovine cumulus cells

Paracrine factors secreted by the oocyte affect cumulus cell proliferation and differentiation. These factors may also act in an anti-apoptotic manner, maintaining the low incidence of cellular apoptosis within cumulus cells. The purpose of this study was to determine whether the incidence of apoptosis within cumulus cells is regulated by oocyte-secreted factors (OSF). Bovine cumulus-oocyte complexes (COC) aspirated from abattoir-derived ovaries were randomly allocated to 3 treatments: (1) groups of 5 intact COC; (2) groups of 5 oocytectomised complexes (OOX), where oocytes were removed microsurgically; and (3) groups of 5 OOX co-cultured with 25 denuded oocytes (DO). To examine a dose effect of OSF, OOX were also cultured with increasing numbers of DO (OOX+5DO, OOX+25DO, OOX+50DO). In both experiments, complexes were cultured in their respective treatments for 24 h in 50 �l of oocyte maturation medium (+/– rFSH; 0.1 IU/mL). Apoptosis was assessed using TUNEL, with all nuclei counterstained with propidum iodide (PI). Nikon TE2000 laser confocal scanning microscopy was used to visualise and quantify the incidence of apoptosis (TUNEL/PI). The proportion of apoptotic cells was determined by image analysis. Oocyte removal lead to a significant increase in cumulus cell apoptosis (OOX, 67% apoptotic; COC, 15%; P�&lt;�0.001). However, the incidence of apoptosis in OOX was restored to COC levels when co-cultured with DO (18%; P�&gt;�0.05, COC v. OOX+DO). FSH prevented apoptosis in all treatment groups (P�&lt;�0.05) decreasing the incidence by 27% in OOX and by 12% in COC. Cumulus cell apoptosis within OOX (+/– FSH) was reduced in a dose dependent manner by treating with increasing numbers of DO. These results indicate for the first time that oocyte-secreted factor(s) regulate the incidence of apoptosis within cumulus cells.