Mokhamad Fahrudin

In vitro maturation, fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle.

This study was conducted to examine the effect of the donor cats reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.

Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions

BackgroundRecent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined.MethodsIn vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6.ResultsUnder 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts.ConclusionThese results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1–2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

Effect of protein supplementation on development to the hatching and hatched blastocyst stages of cat IVF embryos

The effects of protein supplementation in culture medium on development to the hatching and hatched blastocyst stages of cat in vitro-fertilized embryos were investigated. In the first experiment, presumptive zygotes derived from in vitro maturation and in vitro fertilization (IVF) were cultured in modified Earles balanced salt solution (MK-1) supplemented with 0.4% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS) for 9 days. There were no significant differences between the BSA and FBS groups with respect to the proportion of cleavage and development to the morula and blastocyst stages of zygotes. However, the presence of FBS in the medium enhanced development to the hatching blastocyst stage of zygotes compared with the BSA group (31.4% v. 7.8%). Moreover, 2.9% of zygotes cultured with FBS developed to the hatched blastocyst stage. The mean cell number of blastocysts derived from zygotes cultured with FBS was significantly higher (P<0.01) than that from zygotes cultured with BSA (136.6 v.101.5). In the second experiment, embryos at the morula orblastocyst stage, which were produced by culturing in MK-1 supplemented with 0.4% BSA after IVF, were subsequently cultured in MK-1 with 0.4% BSA or 5% FBS. Significantly more morulae developed to the blastocyst (P<0.05) and hatching blastocyst stages (P<0.01) in the FBS group than in the BSA group (71.5% and 53.6% v. 44.9% and 6.0%, respectively). Although none of the morulae cultured with BSA developed to the hatched blastocyst stage, 11.5% of morulae cultured with FBS developed to the hatched blastocyst stage. Moreover, the proportion of development to the hatching blastocyst stage of blastocysts was significantly higher (P<0.01) in the FBS group than in the BSA group (68.7% v. 9.8%). None of the blastocysts cultured with BSA developed to the hatched blastocyst stage, whereas 7.3% of blastocysts cultured with FBS developed to the hatched blastocyst stage. The results of the present study indicate that supplementation with FBS at different stages of early embryo development promotes development to the hatching and hatched blastocyst stages of cat IVF embryos.

Morphological classification of the ovaries in relation to the subsequent oocyte quality for IVF-produced bovine embryos

Although some inferences have been made regarding the morphological aspects of the ovaries in relation to the subsequent oocyte developmental competence in an in vitro system, the influence of ovarian morphology, taken as a pair, has yet to be demonstrated. The present study addresses this limitation. Forty pairs of ovaries from 5 morphological classes were examined to determine whether their characteristics could influence oocyte yield and developmental competence in vitro. An ovary was designated as bearing a corpus luteum (CL) with a dominant follicle (DF) a cyst (CY) or none of these structures (NO). Thus, the paired classes considered in this study consisted of 1) CL-NO 2) CL-DF 3) CL + DF-NO 4) NO-DF and 5) NO-NO. Comparisons were made among the members of 3 subgroups CL, NO and DF. Within the CL-subgroup, the pairs of CL-NO ovaries resulted in higher (P < 0.01) number of oocytes, cleavage rates and blastocyst formation per ovary than in the other categories (CL + DF-NO and CL-DF), with the latter being superior (P < 0.01) to that of CL + DF-NO in terms of cleavage rates. In the NO-subgroup, NO-CL pairs yielded higher (P < 0.01) rates of oocyte recovery and cleavage than the NO-DF pairs, and the latter was inferior (P < 0.05) to that of NO-NO ovaries for the 2 indices. Further, blastocyst rates from the NO-CL pairs was higher (P < 0.01) compared with those of NO-CL + DF, NO-DF, and NO-NO groups. And, in the DF-subgroup, the DF-CL pairs gave a higher (P < 0.05) oocyte yield and cleavage rate (P < 0.01) than the pairs of DF-NO ovaries but not significantly different in blastocyst formation. The overall oocyte recovery, cleavage and blastocyst rates for the 5 classes were, in a decreasing order CL-NO; NO-NO; CL-DF; CL + DF-NO; and DF-NO. Our results suggest that the morphological classification of ovarian pairs could be a useful means for predicting the developmental competence of oocytes in vitro, and that the presence of a dominant follicle in either one or both ovaries of a pair has a negative effect on the IVF-produced bovine embryos.

Bovine Blastocysts Obtained from Reconstructed Cytoplast and Karyoplasts Using a Simple Portable CO2 Incubator

To enable both the multiplication of elite livestock and the engineering of transgenic animals for various agricultural and biochemical purposes, scientists around the world are intensively studying efficient ways of improving developmental competency of bovine embryos reconstructed by somatic cell nuclear transfer. Because it is widely accepted that culture conditions along with many other factors contribute to the developmental competency of reconstructed embryos, this preliminary study was designed to test whether or not bovine reconstructed embryos could develop in vitro using a simple portable CO(2) incubator. CO(2) and O(2) gas tensions and air pressure can be varied using this system. The parameters used in the five conducted trials were low CO(2) (2-5%) and O(2) (8-10%) gas tensions, and negative air pressure (of 300 mm Hg). Chamber temperature was maintained at 38.5 degrees C. Bovine fetal fibroblasts were used as donor karyoplasts and were fused into microsurgically enucleated M II oocytes followed by activation and culture. From the 250 enucleated oocytes, 217 (86.8%) fused, 183 (73.2%) cleaved, and 43 (17.2%) developed to the blastocyst stage. While relatively low developmental rates were achieved, technical proficiency may have been a contributing factor. Further studies using this system are needed to determine optimal levels of O(2), CO(2), and air pressure.

Handmade Somatic Cell Cloning and Related Studies in Farm Animals

ABSTRACT The method of enucleating recipient oocytes by bisection with a metal-blade has been adapted to somatic cell nuclear transfer (SCNT) using zona-free mammalian oocytes, and the process named handmade cloning (HMC). Besides the simplification of enucleation without using a manipulator, this technique also provides for a reduction in the amount or the elimination of expensive equipment that is usually indispensable for conventional SCNT. In this review, we will highlight some recent studies on HMC and related studies in farm animals, especially cloning using enucleation by the gradient centrifugation of zona-free oocytes to produce a large number of recipient cytoplasts at once.

Nuclear Replacement of In Vitro-Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method

The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.

44 EFFECT OF CYTOPLAST VOLUME ON FERTILIZATION AND EMBRYO DEVELOPMENT IN PORCINE M-II OOCYTES RECONSTRUCTED WITH KARYOPLASTS AND CYTOPLASTS OBTAINED BY THE 'CENTRI-FUSION' METHOD

Metaphase-II chromosome transfer (M-II transfer) of oocytes is considered to be one of the advanced procedures to improve fertilization and developmental abilities of oocytes with poor cytoplasmic maturation. The aim of this study was to investigate the developmental capacity after IVF and IVC of porcine oocytes reconstructed from karyoplasts and cytoplasts produced by centri-fusion (Fahrudin et al. 2007 Cloning Stem Cells 9, 216–228). In brief, IVM oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) with a visible first polar body were centrifuged at 13 000g for 9 min to stratify the cytoplasm. Then the zonae pellucidae were removed with pronase treatment. Zona-free oocytes were layered on a 300-µL discontinuous gradient of Percoll in TCM-HEPES with 5 µg mL–1 of cytochalasin B. After centrifugation at 6000g for 4 s, fragmented cytoplasms with approximately equal volumes were obtained, stained with Hoechst-33342, and classified into cytoplasm with (K; karyoplast) or without (C; cytoplast) chromosomes. One karyoplast was fused with 0, 1, 2, 3, and 4 cytoplasts (K, K + 1C, K + 2C, K + 3C, and K + 4C, respectively) by an electric stimulation with a single DC pulse (1.5 kV cm–1 for 20 µs) and cultured for 1 h. Zona-free oocytes without any reconstruction served as control oocytes. The diameters of the reconstructed and control oocytes were measured. All specimens were fertilized in vitro with frozen–thawed boar sperm, and cultured using the well of the well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Their fertilization status and developmental competence were examined. Data were analyzed by ANOVA followed by Duncans multiple range tests. The diameter differed significantly among K to K + 4C oocytes (75.0–127.1 µm; P < 0.05), whereas the diameter of K + 2C oocytes was similar to that of the control oocytes (110.5 µm). Regardless of the cytoplast volume, sperm penetration rates (73.1–93.8%) for K to K + 4C oocytes were not significantly different compared to control oocytes (78.0%). Male pronuclear formation rates of K to K + 4C oocytes (92.3–97.1%) were also not different significantly different compared to control oocytes (96.6%). However, monospermy rates of K oocytes was significantly higher (61.6%; P < 0.05) than those of the reconstructed (K + 1C to K + 4C; 18.2–34.9%) and control oocytes (32.9%). The blastocyst formation rates in K, K + 1C, K + 2C, and K + 3C groups (0.0–9.8%; P < 0.05) were significantly lower than those in the control and K + 4C groups (17.8% and 15.3%, respectively; P < 0.05). The total cell numbers per blastocyst in K + 1C and K + 2C groups (7.5 and 8.3 cells, respectively) were significantly lower than in the control, K + 3C, and K + 4C groups (15.3–26.2 cells; P < 0.05). These results suggest that the cytoplast volume of porcine M-II transferred oocytes, produced by reconstruction from a karyoplast and cytoplast(s) and centri-fusion, is important for their ability to develop to the blastocyst stage and influences cell number.

350 EFFECT OF INDIRECT MAINTENANCE OF INTRACELLULAR cAMP IN CUMULUS–OOCYTE COMPLEXES USING 3-ISOBUTYL-1-METHYLXANTHINE ON GAP JUNCTIONAL COMMUNICATIONS AND OOCYTE MEIOTIC MATURATION

Oocyte and cumulus cells communicate through an extensive network of gap junctions (GJs), which permit the transfer of small molecules such as cAMP. Gonadotropin strongly enhances the intracellular cAMP concentration in cumulus cells, and induces oocyte meiotic resumption. Enhanced cAMP also triggers a reduction of GJ communications (GJCs) in cumulus-oocyte complexes (COCs), accompanied by cumulus expansion. Intracellular cAMP is modulated by both adenylate cyclase (AC) for synthesizing and phosphodiesterase (PDE) for degrading. Addition of AC to gonadotropin-free medium induces meiotic resumption of bovine oocytes without cumulus expansion, suggesting that maintenance of cAMP at a certain level in COCs may be crucial for either prolonged maintenance of GJCs or the timing of oocyte meiotic resumption. In the present study, we investigated the intracellular cAMP concentrations in porcine COCs or oocytes, and GJCs during in vitro oocyte maturation culture using PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Porcine COCs obtained from prepubertal gilts were cultured for 20 h (1st culture) using M199 containing 10% FCS (basic medium, BM group) with FSH (FSH group) or IBMX (IBMX group). Following this, the COCs were transferred into the basic medium containing FSH and LH, and cultured for another 24 h (2nd culture). At 6, 12, and 20 h of the 1st culture, intracellular cAMP in COCs or oocytes was measured. To determine GJCs in each COC, Lucifer Yellow fluorescent dye was microinjected into cumulus-enclosed oocytes at 6 or 12 h of the 1st culture, and the ability of dye transfer, which is related to the GJCs, from the oocyte to the surrounding cumulus cells was observed. At the end of the 1st culture, 30.8 ± 6.0% of the oocytes in the FSH group underwent germinal vesicle breakdown (GVBD), whereas only a few oocytes in the BM group (8.6 ± 2.4%) and the IBMX group (5.8 ± 3.0%) achieved GVBD (P < 0.05). In contrast, ratios of metaphase-II (M-II) stage oocytes at the end of the 2nd culture did not differ between the FSH group (75.7 ± 3.9%) and the IBMX group (68.2 ± 6.8%), although a few oocytes in BM group (10.1 ± 3.7%) reached the M-II stage (P < 0.05). Concentrations of cAMP in COCs and oocytes increased drastically in the FSH group compared to those of the BM and IBMX groups (P < 0.05). In addition, the concentration of cAMP in IBMX group oocytes was also higher than that in the BM group, with a significant difference detected at 20 h (P < 0.05). The GJCs in the FSH group were gradually closed, depending on the length of time in culture (54.9 ± 3.7% of COCs closed their GCJs at 12 h of the 1st culture). In contrast, in the IBMX group, only 23.0 ± 3.7% of COCs closed their GJCs at 12 h of the 1st culture, which was significantly different from that of the other two groups (P < 0.05). These results suggest that treatment with IBMX during the first half of IVM culture can induce subsequent meiotic resumption of porcine oocytes, and that a moderate increase of cAMP concentration in COCs or oocytes prolongs GJCs during the treatment.

183 ADDITION OF DIPHENYLENEIODONIUM OR DEHYDROEPIANDROSTERONE TO CULTURE MEDIA INHIBITS REACTIVE OXYGEN SPECIES PRODUCTION DURING THE EARLY CLEAVAGE STAGES OF IN VITRO-PRODUCED PORCINE EMBRYOS

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 µM (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 ± 2.3, 39.3 ± 1.7, and 42.5 ± 2.7, respectively) was significantly higher (P < 0.01) than those in the control (33.4 ± 1.6) and DPI-0.5 (32.7 ± 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P < 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 µM significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA.