Oocytes In Medium Research Articles

CdSe/CdS/ZnS量子点对体外培养成熟卵母细胞的侵入性研究

The Kunming mice immatured oocytes in vitro maturation culture system are established to investigate the reproductive toxicity of CdSe/CdS/ZnS quantum dots (QDs). QDs stock solution is added into oocyte culture medium at a final concentration of 28.90 nmol/L. Then, QDs and oocytes are co-cultured at 37 °C, 5% CO2 for 4, 8 and 20 h, respectively. The morphological information of oocytes are observed and analyzed under phase-contrast fluorescence microscope. The results demonstrate that QDs enter cumulus cells and accumulate with co-culture time. QDs can not penetrate oocytes zona pellucida, which is confirmed by laser scanning confocal microscope with high spatial resolution. After being treated for 20 h and being rejected by oocytes, QDs decrease the ratio of oocyte in vitro maturation dramatically.

In-vitro Culture with a Tilting Device in Chemically Defined Media During Meiotic Maturation and Early Development Improves the Quality of Blastocysts Derived from In-vitro Matured and Fertilized Porcine Oocytes

Under physiological conditions, mammalian oocytes and embryos appear to be stimulated not only chemically but also mechanically, such as by compression, shear stress and/or friction force in the follicle and female reproductive tract. The present study was undertaken to examine the effects of kinetic culture with a tilting device in chemically defined media during in vitro maturation (IVM) of porcine oocytes and in vitro culture (IVC) following in vitro fertilization (IVF) on the early developmental competence and quality of blastocysts. After culture in a chemically defined IVM medium, modified porcine oocyte medium (mPOM) containing gonadotropins and dibutyryl cAMP for 20 h, the mean diameter of the cumulus-oocyte complexes (COCs) was larger in the tilting culture than in the static controls, whereas the diameter of the oocytes did not differ. When culture of the COCs was continued additionally in a fresh medium without gonadotropins and dibutyryl cAMP for 24 h, the incidences of oocytes completing GVBD and developing to the metaphase-II stage did not differ between the tilting and static culture systems. Furthermore, the sperm penetration after IVF and developmental competence of the oocytes to the blastocyst stage were not different between the tilting and static systems during IVM and IVC. However, tilting culture during both IVM and IVC had a significant positive effect on the number of cells per blastocyst (P<0.05). These observations indicate that tilting culture during IVM and IVC in chemically defined media improves the quality of blastocyst, as determined by the number of cells per blastocyst, without any effects on penetrability and developmental competence.

Addition of sphingosine-1-phosphate to human oocyte culture medium decreases embryo fragmentation

Apoptosis is implicated in the fragmentation of preimplantation mammalian embryos, yet the extent of this association remains controversial. The aim of this study was to assess the ability of sphingosine-1-phosphate (S1P), a known anti-apoptotic substance, to reduce the fragmentation rate of human preimplantation embryos when added to their culture microenvironment. Mature human oocytes were inseminated using intracytoplasmic sperm injection, incubated for 3 days and evaluated for embryo quality and fragmentation by the same embryologist. Oocytes in the study group were manipulated and cultured in culture medium supplemented with S1P to a 20 micromol/l concentration. A total of 46 patients donated 177 mature oocytes for the study group and 546 oocytes for the control group. The fertilization rate was significantly lower in the S1P-supplemented group (52.4% versus 67.3%; P=0.002) and the proportion of grade I embryos with less than 15% fragmentation was significantly higher in the same group (79.5% versus 53.9%; P<0.0001). Sphingosine-1-phosphate added to the culture medium of human preimplantation embryos is associated with a significantly lower fragmentation rate and hence better quality embryos. The clinical significance of these findings on reproductive outcome remains highly speculative awaiting further studies to translate this improvement in embryo quality into better pregnancy rates.

348 CO-CULTURE OF PORCINE OOCYTES - CUMULUS COMPLEXES DERIVED FROM SMALL FOLLICLE WITH CUMULUS-CELL MASSES FROM MIDDLE FOLLICLE IMPROVES MEIOTIC MATURATION OF THE OOCYTES

It is known that maturation rate of oocytes derived from small follicles (SF) is lower than that of oocytes from middle follicles (MF). Since it has been reported that cumulus cells have important role during oocytes maturation, the ability of SF oocytes to complete the meiotic maturation may be affected by additional cumulus-cell mass. The present study was undertaken to examine the effects of co-culture of oocyte-cumulus complexes (OCCs) derived from SF with additional cumulus-cell masses on in vitro maturation and developmental competence of the oocytes. OCCs were aspirated from small (SF; 1-2 mm in diameter) or middle follicles (MF; 3-6 mm in diameter) of prepuberal ovaries. OCCs were cultured in porcine oocyte medium (POM; Research Institute for the Functional Peptide, Yamagata, Japan) supplemented with gonadotropins and dbcAMP for a first 20-h period and then in gonadotropin-free and dbcAMP-free POM for another 24 h. Culture medium was collected after the first 20-h culture and the end of IVM, and analyzed for the protein profiles. Following IVM, some oocytes were co-incubated with spermatozoa in a drop of modified Medium199 containing 0.4% BSA and 5 mM caffeine for 8 h and then incubated in PZM5 (Research Institute for the Functional Peptide, Yamagata, Japan) for 6 days. Sperm penetration, cleavage, and the early development of the oocytes were examined before culture in PZM5 or Day 2 and Day 6 of culture, respectively. OCCs derived from SF were co-cultured with cumulus-cell masses derived from SF or MF during IVM (SFO-SFC and SFO-MFC groups, respectively). Some OCCs derived from SF or MF were cultured for IVM without additional cumulus-cell masses (SFO and MFO, respectively). After culture, meiotic maturation of the oocytes was examined. To analyze the developmental competence of oocytes of SF, MF, and SFO-MFC groups, sperm penetration, pronuclear formation, cleavage, and blastocyst formation were examined. Protein profiles in the IVM media were examined by 10% SDS-PAGE. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P ≤ 0.05). After culture for IVM, the diameters of SFO and SFO-MFC were not different from that of MFO (113.3-114.5 μm). The maturation rate of SFO-MFC oocytes (75.5 ± 6.2%) was higher than SFO (52.2 ± 2.8%) and comparable with the rate of MFO oocytes (83.2 ± 6.3%), while there was not significant difference between the mature rate of SFO+SFC oocytes (63.6 ± 4.0%) and SFO oocytes. There were no significant differences between groups in sperm penetration, pronuclear formation, and cleavage. Blastocyst formation of SF oocytes was not improved by co-culture with MF cumulus-cell masses. Certain band was detected only in MF medium of collected at 20 h (24.5 kD). From these results, we conclude that secretions from cumulus-cell masses derived from MF well improve the meiotic progress of oocytes derived from SF, but not the early development following IVF.

Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization

A major challenge of assisted reproduction technologies (ARTs) is to mimic the natural environment required to sustain oocyte and embryo survival. Herein, we show that the ceramide-metabolizing enzyme, acid ceramidase (AC), is expressed in human cumulus cells and follicular fluid, essential components of this environment, and that the levels of this enzyme are positively correlated with the quality of human embryos formed in vitro. These observations led us to develop a new approach for oocyte and embryo culture that markedly improved the outcome of in vitro fertilization (IVF). The addition of recombinant AC (rAC) to human and mouse oocyte culture medium maintained their healthy morphology in vitro. Following fertilization, the number of mouse embryos formed in the presence of rAC also was improved (from approximately 40 to 88%), leading to approximately 5-fold more healthy births. To confirm these observations, immature bovine oocytes were matured in vitro and subjected to IVF in the presence of rAC. Significantly more high-grade blastocysts were formed, and the number of morphologically intact, hatched embryos was increased from approximately 24 to 70%. Overall, these data identify AC as an important component of the in vivo oocyte and embryo environment, and provide a novel technology for enhancing the outcome of assisted fertilization. Eliyahu, E., Shtraizent, N., Martinuzzi, K., Barritt, J., He, X., Wei, H., Chaubal, S., Copperman, A. B., Schuchman, E. H. Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization.

Transforming growth factor-α in a defined medium during in vitro maturation of porcine oocytes improves their developmental competence and intracellular ultrastructure

We examined the effects of transforming growth factor-α (TGF-α) to develop a defined medium for in vitro maturation (IVM) of porcine (Sus scrofa domesticus) oocytes. Cumulus-oocyte complexes (COCs) were matured in porcine oocyte medium containing 3 mg/mL polyvinyl alcohol (POM) and TGF-α (0, 1, 10, or 100 ng/mL) in the presence or absence of the gonadotropins equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In the absence of gonadotropins, adding 10 ng/mL TGF-α increased (P < 0.05) the percentage of oocytes that reached metaphase II (24.2%) compared with that of the control (no TGF-α addition; 5.6%). In the presence of gonadotropins, although maturation rate did not differ among TGF-α treatments (75.4% to 84.8%), the rate of blastocyst formation (28.1%) was higher (P < 0.05) in the TGF-α group (28.1%) than that in the control group (15.9%) after in vitro fertilization and embryo culture. An electron microscope study revealed that TGF-α–treated oocytes contained more homogenous lipid droplets than did control oocytes. Moreover, mitochondria surrounded by the endoplasmic reticulum were observed only in the TGF-α–treated oocytes. In blastocysts derived from the latter oocytes, mitochondria with numerous cristae were frequently observed compared with that in blastocysts from control oocytes. When the Day-5 blastocysts obtained from oocytes matured with TGF-α were surgically transferred into four recipients, a total of 29 piglets were farrowed. We concluded that the addition of TGF-α to the defined IVM medium of porcine oocytes improved the subsequent blastocyst formation and that the blastocysts produced by the defined in vitro production system have developmental competence to full term after embryo transfer.

Involvement of polyadenylation status on maternal gene expression during in vitro maturation of porcine oocytes

During mammalian oocyte maturation, protein synthesis is mainly controlled through cytoplasmic polyadenylation of stored maternal mRNAs. In this study, the role of polyadenylation modification of maternal transcripts in pig oocytes was investigated by adding cordycepin (3'-dA), a potent polyadenylation inhibitor, to the culture medium of porcine oocytes maturing in vitro. 3'-dA significantly prevented cumulus expansion regardless of the concentration used, and inhibited pig oocyte maturation in a dose-dependent manner. Further, 3'-dA 1 microg/ml-treated MII oocytes experienced significantly lower rates of cleavage (29%) and blastocyst formation (15.35%) compared to control MII oocytes (58.6% and 35.3%, respectively). Western blotting revealed that the activity of mitogen-activated protein kinase (MAPK) and p34(cdc2) was significantly decreased in oocytes and cumulus cells treated with 3'-dA at a concentration of 1 microg/ml or greater. To further explore the underlying molecular mechanisms, expression patterns and polyadenylation states of four important genes, C-mos, cyclin B, GDF9 and BMP15, were studied as representative maternal transcripts by real-time PCR and the PAT assay. 3'-dA at concentrations above 1 microg/ml significantly prevented polyadenylation and caused aberrant expression of C-mos and GDF9 during oocyte maturation. These results suggest that polyadenylation inhibitor blocked pig oocyte maturation in vitro by one or more of the following actions: (1) inactivation of MAPK and MPF in oocytes, especially at the late stages (MI and MII); (2) prevention of cumulus cell expansion through inactivation of cellular MAPK; and (3) inhibition of the maternal mRNA polyadenylation process, which in reverse, disrupted the maternal mRNA patterns in pig oocytes' maturation in vitro.

Metabolomic assessment of oocyte viability

The aim of the current study was to evaluate whether near-infrared (NIR) spectroscopy-generated metabolomic data obtained from oocyte culture samples would correlate with nuclear maturity status and derived embryo development. A total of 412 oocyte culture samples were collected from 43 patient cycles. Metabolomic profiles of metaphase I and II oocytes were obtained by NIR spectroscopy and were significantly different from each other and from profiles of prophase I (germinal vesicle) oocytes ( P < 0.001 at the 95% confidence interval). Additionally, NIR spectroscopic analysis of culture medium of oocytes that developed to grade A embryos on day 3 demonstrated significantly higher viability indices (0.62 ± 0.23) than those that developed to grades C/D (0.42 ± 0.26; P < 0.006); and on day 5 grade A (0.37 ± 0.20) was also higher than grades C/D (0.14 ± 0.21; P < 0.02). Metabolomic profiles of oocytes that resulted in pregnancy had higher viability indices (0.87 ± 0.27) than those that did not (0.44 ± 0.17; P < 0.0001). The results of the current study demonstrate that metabolomic profiling from spent culture medium of the oocyte is related to nuclear maturity, is able to predict embryo development at day 3 and day 5 stages, and relates to embryo viability.

Successful Piglet Production in a Chemically Defined System for In-vitro Production of Porcine Embryos: Dibutyryl Cyclic AMP and Epidermal Growth Factor-family Peptides Support In-vitro Maturation of Oocytes in the Absence of Gonadotropins

To induce meiotic resumption of porcine oocytes, it is thought to be necessary to expose the cumulus-oocyte complexes (COCs) to gonadotropins during in-vitro maturation (IVM). However, the detailed mechanism of meiotic resumption by gonadotropins is still unknown, and successful piglet production has not been reported by using oocytes matured in gonadotropin-free media and fertilized in vitro. The present study was undertaken to examine the combinational effects of epidermal growth factor (EGF)-family members and dibutyryl cyclic AMP (cAMP) in a chemically defined medium on IVM of porcine oocytes and the developmental competence following in vitro fertilization (IVF). The basic IVM medium was a chemically defined medium, modified porcine oocyte medium (mPOM). Supplementation of the IVM medium with 10 or 1000 ng/ml EGF, amphiregulin and betacellulin during the whole IVM period, except for 10 ng/ml amphiregulin, increased the percentage of oocytes maturing to the metaphase-II stage. When COCs were exposed to both dibutyryl cAMP and EGF-family members during the first 20-h of IVM and then culture was continued in the absence of EGF-family members and dibutyryl cAMP, the incidence of metaphase-II oocytes was significantly increased and was not different from that of oocytes cultured in a standard IVM system with gonadotropins. The developmental competence of the oocytes to the blastocyst stage following IVF was no different from that of control oocytes matured with gonadotropins. When these blastocysts were transferred into the uterine horn of three recipients, all of gilts became pregnant and delivered a total of 11 piglets. These observations indicate that supplementation of a chemically defined maturation medium with EGF-family members and dibutyryl cAMP during the first 20 h of IVM can support well the meiotic progress and developmental competence of porcine oocytes.

241 DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS

Exposure of porcine oocyte–cumulus complexes (OCC) to gonadotropins induces meiotic resumption, but the details of this mechanism are still unknown. The present study was undertaken to examine combinational effects of EGF-like factors and dibutyryl cyclic AMP (dbcAMP) in a chemically defined medium on in vitro maturation (IVM) of porcine oocytes. The OCC were aspirated from 3- to 6-mm-diameter follicles of prepuberal ovaries and used in the current study. The basic culture medium was a chemically defined medium, Porcine Oocyte Medium (POM; Research Institute for the Functional Peptides, Yamagata, Japan). In the first experiment, various concentrations (0, 10, and 1000 ng mL–1) of EGF-like factors (EGF, amphiregulin, and betacellulin) were added to POM during an entire IVM period (44 h). In the second experiment, to determine the additive effect of EGF-like factors, each EGF-like factor with an effective concentration was combined with the others. In the last experiment, to examine the combined effect with dbcAMP, OCC were exposed to EGF (10 ng mL–1), amphiregulin (1000 ng mL–1), and dbcAMP (1 mm) during the first 20 h of IVM and then the culture was continued in the absence of EGF-like factors and dbcAMP. After culture, in all experiments, meiotic resumption and the progress of oocytes were examined after denuding, fixing, and staining. Statistical analyses was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P &lt; 0.05). In the first experiment, all treatments without supplementation with 10 ng mL–1 amphiregulin increased the incidence of oocytes maturing to the MII phase, as compared with controls (29.1 to 39.3% v. 11.1%, P &lt; 0.05). In the second experiment, combinations with 2 kinds of EGF-like factor slightly (but not significantly) improved the percentage of oocytes at the MII stage (37.7 to 47.4%). In the last experiment, supplementation with 1 mm dbcAMP during the first 20 h of IVM, regardless of the presence of EGF-like factors, significantly increased the incidence of MII oocytes as compared with controls, whereas the incidence was the highest when 1 mm dbcAMP, 10 ng mL–1 EGF, and 1000 ng mL–1 amphiregulin were supplemented (75.5%). When those oocytes were cultured in a chemically defined medium after in vitro fertilization, the developmental competence of oocytes to the blastocyst stage (25.0%) was not different from oocytes matured in the presence of gonadotropins and dbcAMP during the first 20 h of IVM (17.3%). These observations indicate that supplementation of a chemically defined maturation medium with EGF-like factors and dbcAMP during the first 20 h of IVM can support the meiotic progress and developmental competence of porcine oocytes well. Currently, we are examining the developmental competence of those oocytes after embryo transfer. The results will be presented at the meeting. This study was supported by MAFF AgriBio1605.

Non-invasive assessment of embryo viability by metabolomic profiling of culture media (‘metabolomics’)

Increasing the efficiency of the IVF procedure by improving pregnancy/implantation rates and at the same time lowering (or avoiding) the risks of multiple gestations are the primary goals of the current assisted reproductive technology. These aims require a much improved gamete/embryo testing and selection procedure, which, using the current approach of microscopy-based morphology evaluation is unlikely to be achieved. Therefore, alternative or additional, non-invasive techniques have been proposed which may be able to detect alterations of the culture environment surrounding gametes/embryos reflective of the (patho-)physiological processes. One of the most recently applied approaches is to measure metabolomic changes in the culture medium of embryos and oocytes (‘exometabolomics’). Initial studies have demonstrated that different types of spectrophotometric tests, including Raman and near-infrared (NIR) techniques, are similarly well capable of detecting specific changes of the ‘secretome’ (exometabolome). These studies have also demonstrated that metabolomic measurements correlate well with embryo development and morphology assessment. Furthermore, viability index on oocytes/embryos established by metabolomic tests may be a stronger predictor for implantation potential than traditional morphological assessment. Although the results of these initial investigations are promising, further prospective studies are required to define clearly the potential benefits and most relevant applications of this novel non-invasive technology in the field of assisted reproduction.

Defined System for In Vitro Production of Porcine Embryos Using a Single Basic Medium

We have previously indicated that porcine blastocysts can be produced by in vitro fertilization (IVF) and culture (IVC) in chemically defined porcine gamete medium (PGM) and porcine zygote medium (PZM)-5, respectively, In the present study, the effects of basic media and macromolecular components on in vitro maturation (IVM) were investigated to develop a defined system for in vitro embryo production using a single basic medium through IVM, IVF and IVC. Porcine immature oocytes were matured in porcine oocyte medium (POM) or modified North Carolina State University (mNCSU) 37, which were supplemented with either 10% (v/v) porcine follicular fluid (pFF) or 3 mg/ml polyvinyl alcohol (PVA) as a macromolecular component (designated POM+pFF, POM+PVA, mNCSU37+pFF and mNCSU37+PVA). In the maturation with mNCSU37+PVA, the percentages of oocytes that reached the metaphase II stages were significantly lower than those in the other treatments. Following IVM with the above media, oocytes were treated with an electrical stimulus and cycloheximide for parthenogenetic activation and were cultured in PZM-5 for 5 days. The rates of cleavage and blastocyst formation of parthenogenetic oocytes were significantly lowered for maturation with mNCSU37+PVA compared with the other treatments, while there were no significant differences in the total numbers of cells in blastocysts among the treatments. Following IVF and IVC, the rates of penetration, male pronucleus formation, cleavage and blastocyst formation were significantly lower when oocytes were matured in mNCSU37+PVA than in other maturation media. The normal fertilization rate was significantly higher in POM+PVA compared with the other treatments, although the total number of cells in blastocysts was reduced with the addition of PVA to both POM and mNCSU37 compared with pFF supplementation. These results demonstrate that porcine blastocysts can be produced by the defined system using a single basic medium.

Physiological temperature variants and culture media modify meiotic progression and developmental potential of pig oocytes in vitro

Ovarian follicles in vivo are cooler than surrounding abdominal and ovarian tissues. This study investigated whether typical follicular temperatures influence the maturation and developmental potential of pig oocytes in vitro. Oocytes were synchronised at the germinal vesicle (GV) stage and incubated at 39, 37 or 35.5 degrees C. When compared with 39 degrees C, which is often used for in vitro studies, lower temperatures delayed spontaneous progression to the metaphase I and II (MI and MII) stages of meiosis. The MII was delayed by about 12 h per degrees C. All oocytes had normal morphology. Oocytes reaching GV breakdown (GVBD) at 39 degrees C were subsequently unaffected by cooling, demonstrating thermal sensitivity during the pre-GVBD stage only. Simultaneous assay of maturation-controlling kinases (maturation promoting factor (MPF) and MAPK) showed that cooling delayed kinase activation, provided it was applied prior to GVBD. Activity profiles remained coupled to the stage of meiosis. Neither enzyme was directly thermally sensitive over this temperature range. Following in vitro fertilisation, fewer blastocysts developed from embryos derived from 35.5 or 37 degrees C oocytes as compared with those from 39 degrees C oocytes. Manipulation of fertilisation timings to allow for delayed maturation showed that over-maturing or aging at lower temperatures compromises subsequent embryo development, despite normal nuclear maturation; the GV stage was again the thermally sensitive period. Cleavage rates were improved by the culture of oocytes with follicle-stimulating hormone (FSH) at 37 but not at 35.5 degrees C. Inclusion of 20% follicular fluid in the oocyte medium restored the blastocyst rate to that seen at higher temperatures. Thus, FSH and follicular fluid may allow oocytes to achieve normal developmental potential at in vivo temperatures.

Effects of modification of in vitro fertilization techniques on the sex ratio of the resultant bovine embryos

The duration of sperm–oocyte co-incubation has been observed to affect the sex ratio of in vitro produced bovine embryos. The purpose of this study was to investigate some factors that may be responsible for the skewed sex ratio. The factors studied were selected combinations of the duration of co-incubation, the presence or absence of cumulus cells, and the level of hyaluronic acid (HA) in the culture medium. Experiment 1 examined the effect of selected combinations of different factors during the fertilization phase of in vitro oocyte culture. The factors were the nature of the sperm or its treatment, the duration of the sperm–oocyte co-incubation, and the level of hyaluronic acid in the culture medium. In experiment 2, the capacitation of frozen-thawed-Percoll-washed sperm (control), pre-incubated, and non-binding sperm was evaluated by the zona pellucida (ZP) binding assay and the hypo-osmotic swelling test (HOST). The purpose of experiment 3 was to determine the oocyte cleavage rate and sex ratio of the embryos (>5 cells) produced as a consequence of the 10 treatments used in experiment 1. In treatments 1–3 (experiments 1 and 3) COC were co-cultured with sperm for 1, 5 or 18 h. Polyspermic fertilization rose as the co-incubation period increased (1 h 6.5%, 5 h 15.9%, 18 h 41.8%; P < 0.05), and the highest rate of normal fertilization was observed for 5 h culture (73.4%; P < 0.05). The sex ratio was significantly ( P < 0.05) skewed from the expected 50:50 towards males following 1 h (64.4%) and 5 h (67.3%) co-incubation, but was not affected by 18 h incubation (52.3%). In treatment 4, sperm was pre-incubated for 1 h and cultured with COC for 5 h. Relative to control sperm, pre-incubation of sperm increased ZP binding (116 versus 180 per ZP; P < 0.05) and decreased the proportion of HOST positive sperm (65.8–48.6%; P < 0.05; experiment 2). Pre-incubation did not affect the rates of polyspermy, normal fertilization or the sex ratio of the embryos (experiments 1 and 3). The oocytes used in treatments 5–10 of experiments 1 and 3 were denuded prior to fertilization. Co-incubation of denuded oocytes for 1 h (treatment 5) or 5 h (treatment 6) resulted in levels of polyspermic fertilization similar to that for treatment 2 with significantly lower levels of normal fertilization (41.7% and 52.6%, respectively; P < 0.05), and the 1 h co-incubation significantly skewed ( P < 0.05) the proportion of male embryos to 70.0%. Denuded oocytes were fertilized for 5 h with sperm unable to bind to cumulus cells (NB sperm) in treatment 7 or those that bound to cumulus cells (B) in treatment 8. These two treatments had similar rates of polyspermic, normal and non-fertilization. However, the B sperm caused the sex ratio of the embryos to be significantly skewed to males (63.9%; P < 0.05). Fertilization of denuded oocytes in medium containing hyaluronic acid (0.1 mg/ml, treatment 9; 1.0 mg/ml treatment 10) significantly ( P < 0.05) reduced the incidence of polyspermic fertilization relative to treatments 2 and 6, and normal fertilization relative to treatment 2, but did not affect the sex ratio of the embryos. It was concluded that exposure of sperm to cumulus cells, either before fertilization of denuded oocytes or during the process of fertilization of complete COC, increased the proportion of male embryos produced by in vitro culture. It was hypothesized that this may be due to the capacitation state of the sperm, the cumulus–sperm interaction, and/or the ability of the sperm to bind to cumulus cells or oocytes.

In vitro Maturation and Fertilization of Riverine Buffalo Follicular Oocytes in Media Supplemented with Oestrus Buffalo Serum and Hormones

Effects of two maturation media (TCM-199 and Ham's F-12) with and without the addition of oestrus buffalo serum (OBS) and hormones (FSH, LH, E2) on the maturation rate of buffalo follicular oocytes were evaluated. The results revealed a significant (P &lt; 0.05) increase in the maturation rate when the OBS and hormones were added to TCM-199 than in Ham's F-12 medium. The percentage of maturation rates in TCM-199 + hormones + OBS, TCM-199 + hormones, TCM-199 + OBS and TCM-199 were 77.44, 55.17, 62.28 and 26.62 percent, respectively. While in Ham's F-12 + OBS + hormone, Ham'F-12 + hormone, Ham's F-12 + OBS and Ham's F-12 were 32.85, 27.52, 31.38 and 13.46 percent, respectively. A significantly higher (P &lt; 0.05) fertilization rate was recorded for modified Ca2+ free Tyrode's medium (63.72%) than in TALP (10.9%) and IVF-TL (32.18%). Thus, TCM-199 containing hormones and OBS appeared better for in vitro maturation, whereas modified Ca2+ free tyrode's medium was found to be more suitable for in vitro fertilization of buffalo follicular oocytes.

Developmental potential of bovine oocytes cultured in different maturation and culture conditions

Diverse groups of chemicals in culture media are needed for successful bovine oocyte maturation and embryo development during which dramatic cytoplasmic and nuclear reprogramming events take place. In vitro embryo production (IVP) procedures frequently include supplements such as serum and/or co-culture with various types of somatic cells. However, the presence of undefined serum in culture media introduces a variation from batch to batch, increases viral or prion contamination risk, and leads to problems during fetal development. The aim of the present study was to investigate the possibility of using chemically defined-synthetic serum substitute (SSS) in place of fetal calf serum (FCS) during maturation and long-term culture to stimulate in vitro maturation (IVM), fertilization (IVF) and subsequent embryo development. In Experiment I, the effect of the protein source on in vitro maturation was tested by maturing oocytes in culture media supplemented with 10% FCS (Control Group), 10% SSS (Group I) and 10% SSS + 10 ng/ml epidermal growth factor (EGF) (Group II). In Experiment II, effects of SSS on both oocyte maturation and embryo development during in vitro culture (IVC) were tested by maturing oocytes in media supplemented with 10% FCS (FCS Group) or 10% SSS + 10 ng/ml EGF (SSS Group), followed by IVF and IVC in SOF media supplemented with 10% FCS and 10% SSS on day 4 for FCS and SSS Groups, respectively. Even though rates for cleavage and development to blastocyst stage were not different, blastocyst cell numbers were higher in Group II containing SSS and EGF. The SSS supplementation group had higher apoptotic nuclei as compared to the FCS Group in Experiment II. Transcripts for heat shock protein 70 (Hsp70), interferon tau (IF-τ), DNA methyltransferase 3a (Dnmt3a), desmosomal glycoprotein desmocollin III (DcIII) and insulin-like growth factor II receptor (Igf-2r) were altered in different culture conditions in Experiment I. However, only glucose transporter-1 (Glut-1) mRNA was different in the SSS and FCS Groups in the second experiment. In summary, SSS and EGF in maturation medium and replacement of FCS with SSS alone in culture medium on day 4 of IVC support oocyte maturation and embryo development in vitro. However, significance of culture condition induced changes on the genome-wide abundance of messenger ribonucleic acid and the significance of the apoptotic nuclei during fetal development still remain to be determined.

Ovine cumulus cells estradiol-17ß production in the presence or absence of oocyte

The objective of the present study was to compare the in vitro production of estradiol-17ß (E 2) by cumulus cells in the presence or absence of ovine oocyte. Moreover, the relationship between the concentration of produced estradiol-17ß and oocyte nuclear maturation was assessed. Ovaries collected from the local abattoir were transported to the laboratory in saline at 30–35 °C within 1–3 h after collection. The oocytes of follicles, 2–6 mm in diameter, were recovered by aspiration. The oocytes with evenly granulated cytoplasm and which were surrounded with at least three layers of cumulus cells were selected and subjected to culture in pre-incubated oocyte culture medium (OCM). Before culturing, the selected oocytes were randomly divided into five treatment groups: Group 1, cumulus enclosed oocytes cultured in OCM (Group COCs); Group 2, denuded oocytes cultured in OCM (Group D); Group 3, denuded oocytes co-cultured with a cumulus cell-monolayer in OCM (Group D + M); Group 4, denuded oocytes co-cultured with previously cultured (for 26 h) cumulus cell-monolayer (10 5 cells/ml) in refreshed OCM (Group D + M 26); Group 5, cumulus cell-monolayer (10 5 cells/ml) cultured in OCM (Group M). After an incubation period (26 h at 38.6 °C, 5% CO 2 and 100% humidity), the media were collected and kept at −20 °C until hormonal assay. The concentration of E 2 was determined by RIA method. For assessment of nuclear status, the completely denuded oocytes were subjected to DAPI staining. The highest percentage of metaphase II (MII) stage oocytes was observed in Group N (91%) and the lowest percentage was observed in Group D (6%) and Group D + M 26 (6%). The mean production of E 2 was highest and lowest in Group D + M (378.69 ± 54.34 pg/ml) and Group D + M 26 (109.15 ± 8.24 pg/ml), respectively. The production of E 2 was significantly ( P < 0.01) higher in Group D ± M when compared with Groups M and D ± M 26. Regarding the nuclear maturation, the percentage of MII stage oocytes was significantly ( P < 0.001) higher in Group COCs compared to the other groups. The results suggest that steroidogenic activity of cumulus cells in in vitro condition can be influenced by the pattern of connection between cumulus cells and the oocyte. Moreover, the nuclear maturation of oocytes is not influenced by the different production levels of E 2.

P-202: Oocyte cryopreservation: An analysis of cyclin B and map kinase as an indicator for oocyte activation following cyropreservation in calcium containing and calcuim free medium

P-202: Oocyte cryopreservation: An analysis of cyclin B and map kinase as an indicator for oocyte activation following cyropreservation in calcium containing and calcuim free medium

A unique preovulatory expression pattern plays a key role in the physiological functions of BMP-15 in the mouse

Mutations in the bone morphogenetic protein 15 (BMP-15) gene cause female infertility in the monoovulatory human and sheep; however, in the polyovulatory mouse, loss-of-function of BMP-15 results only in reduced ovulation rate. To elucidate the cause of these species-specific differences, we investigated the functional role of BMP-15 in the mouse ovary. Here, we found that the functional mature form of BMP-15 is barely detectable in the mouse oocytes until just before ovulation, when it is markedly increased. Further, we found that BMP-15 induces cumulus expansion in mouse cumulus-oocyte complexes. The oocyte culture medium from immature mice primed with pregnant mare serum gonadotropin followed by human chorionic gonadotropin also stimulated cumulus expansion, and this activity was attenuated by BMP-15 antibody. Interestingly, the oocyte culture medium from mice treated with pregnant mare serum gonadotropin alone had no effect. Moreover, BMP-15 stimulated the expression of EGF-like growth factors in cumulus cells as well as a series of molecules downstream of EGF-like growth factor signaling, including cyclooxygenase 2, hyaluronan synthase 2, tumor necrosis factor-stimulated gene 6, and pentraxin 3, all of which are necessary for normal cumulus expansion. An antagonist of the EGF receptor completely abolished the effect of BMP-15 in inducing cumulus expansion. These results are consistent with the phenotype of BMP-15-null mice, which exhibit normal folliculogenesis but have defects in the ovulation process. The species-specific differences in the phenotypes caused by BMP-15 mutations may thus be attributed to the temporal variations in the production of the mature form of BMP-15.

The effect of ovine oocyte diameter on nuclear maturation

The study was carried out to investigate the relationship between the development potential and the diameter of ovine oocytes. Ovaries collected from a local abattoir were transported to the laboratory within 1–3 h of slaughter. The oocytes from follicles (2–6 mm in diameter) were recovered by aspiration and stored in a preincubated (at 38.6 °C, 5% CO 2 and maximum humidity) hepes-modified TCM 199 solution. Good quality oocytes (evenly granulated cytoplasm with at least two layers of surrounding cumulus cells) were selected and subjected to culture in TCM 199 supplemented with 0.05 IU/ml rhFSH, 1 IU/ml hCG and 1 μg/ml E 2 (OCM; oocyte culture medium). Before culturing, the selected oocytes ( n = 1495) were divided into three categories based on diameter: (i) <110 μm; (ii) 110–150 μm; and (iii) >150 μm. In each category half of the oocytes were denuded of the cumulus cells and the rest remained intact (cumulus enclosed oocytes). All of the oocytes, denuded and cumulus enclosed oocytes in each category were cultured in OCM for a period of 26–27 h. After the incubation period, the nuclear status of the oocytes in each experimental group was assessed using a 2% orcein staining technique. The percentage of oocytes that reached the MII stage was 81, 82, and 84% for the cumulus enclosed oocytes with diameters of <110, 110–150, >150 μm, respectively. The corresponding values for the denuded oocytes were 4, 5, and 6%, respectively. The oocytes displayed no size-related ability to undergo meiotic maturation in both cumulus enclosed and denuded oocytes. However, as expected the rate of nuclear maturation in cumulus enclosed oocytes was higher than that of denuded oocytes in all the size categories. The results suggest that in sheep the antral follicles ranging from 2 to 6 mm contain fully grown oocytes which, despite their varaiabilty in diameter, show good competence for in vitro nuclear maturation.