Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells

This paper describes the use of the protein synthesis inhibitor cycloheximide (CHX) to synchronize nuclear progression during meiotic maturation in porcine oocytes, and also the time-dependence of nuclear maturation on exposure of the oocyte to cumulus cells. Prior to culture, the majority of oocytes were at the germinal vesicle (GV) stage (95-100%), but distributed from GVI to GVIV (GVI 56.1 +/- 9.1%, GVII 15.3 +/- 1.4%, GVIII 21.5 +/- 7.1%, GVIV 7.1 +/- 3.5%). During culture of cumulus-enclosed oocytes (COCs) from 12 h to 48 h in a conventional culture system, all meiotic stages were represented at any time point examined, with 63.6 +/- 4.2% of oocytes maturing to metaphase II (MII). Cycloheximide blocked the progression of nuclear development in a dose-dependent manner. Treatment for 12 h with CHX at 1-25 microg mL(-1) resulted in 95-100% oocytes being arrested and synchronized at GVII. With >5 microg mL(-1) CHX, all oocytes were arrested before germinal vesicle breakdown (GVBD) (mostly at GVIII) by 24 h. A 12 h preincubation with 5 microg mL(-1) CHX followed by 24 h of further culture without CHX resulted in >80% of oocytes maturing to MII. The profile of nuclear progression during maturation revealed discrete peaks of occurrence of different meiotic stages, with GVBD at 6-12 h, metaphase I (MI) at 10-18 h and anaphase I/telophase I at 16-20 h. After 12 h preincubation with 5 microg mL(-1) CHX, denuded oocytes (DOs) matured to MI as COCs. However, DOs matured to MII as normal when denuded at MI. In conclusion, CHX not only efficiently blocks and synchronizes the meiotic progression of porcine oocytes at a specific GV stage, but it also effectively synchronizes subsequent meiotic progression to MII, resulting in discrete peaks of occurrence of different meiotic stages. Using this technique, the study showed that cumulus cells are essential for oocytes to mature from MI to MII but exposure to cumulus cells must occur before MI.     

OOCYTE MEMBRANE MATURATION AND OOCYTE-SPERM RELATIONSHIP: TRANSMISSION ELECTRON MICROSCOPY STUDY

The success of in vitro maturation (IVM) depends greatly on the acquisition of immature oocytes. Immature oocytes in prophase I (PI) and metaphase I (MI), aspirated after controlled ovarian hyperstimulation, were incapable of fertilization, leading to a lower fertilization rate. Therefore, they must be evaluated on a fine structure level for their in vitro maturation (IVM) processes and their relationship with sperm. Oocyte membrane maturation and oocyte-sperm relationship were studied using transmission electron microscopy. A total of 55 human oocytes obtained from 20 patients at various times and 83 oocytes obtained from the dissected ovarians of female Wistar rats were used for transmission electron microscopy (TEM) evaluation. Despite being in either prophase I and metaphase I or in metaphase II, the oocytes were not fertilized after 48 h of incubation. At the various stages of maturation between PI and MII, the number and the size of microvilluses on the oocyte membrane increased as MII approached and decreased after full maturation. Oocyte activation was related to oocyte membrane maturation and has an effect on the oocyte sperm penetration.     

Evaluation of the developmental potential of metaphase I oocytes from stimulated intracytoplasmic sperm injection cycles

The objective of the present study was to evaluate the developmental potential and clinical application value of metaphase I (MI) oocytes obtained from stimulated intracytoplasmic sperm injection (ICSI) cycles. ICSI was performed on MI oocytes immediately after denudation (Group A), or on in vitro-matured (IVM) oocytes following culture; oocytes in culture were further divided into two groups, being cultured for either 3-5 h (Group B) or 24-28 h (Group C). Metaphase II oocytes from the same cycle(s) isolated for ICSI served as the control group (Group D). The rates of normal fertilisation, cleavage and high-quality embryos were compared among the four groups. High-quality embryos were transferred whenever possible, and pregnancy rates were evaluated. Results showed that normal fertilisation rates for Groups B, C and D were significantly higher than that of Group A (68.6%, 57.8%, 74.5% and 30.1%, respectively; P<0.01). The rate of high-quality embryos in Group B was comparable with Group D; the rate for Group C was significantly lower than that of the other groups (P<0.05). Two clinical pregnancies were achieved after transfer of embryos from IVM oocytes. In vitro maturation of MI oocytes for a short period of time may increase the number of available embryos; however, overnight in vitro culture of MI oocytes did not improve results.     

SCHEDULE TO INJECT IN VITRO MATURED OOCYTES MAY INCREASE PREGNANCY AFTER INTRACYTOPLASMIC SPERM INJECTION

To ascertain the value of using immature oocytes in an intracytoplasmic sperm injection (ICSI) program, the authors designed a schedule, at 5 p.m. on day 1 (the day of oocyte retrieval) and at 8 a.m. and 2 p.m. on day 2, to recognize and inject the in vitro matured (IVM) oocytes. For the 1166 oocytes retrieved in 107 ICSI cycles, 128 (11.0%) were at the stage of metaphase I (MI) and 113 (9.7%) at germinal vesicle. Routine ICSI for metaphase II oocytes was performed at 2 p.m. on day 1 (initial ICSI). In culture medium of human tubal fluid with 15% maternal serum, 85.1% (205/241) immature oocytes progressed to maturation in which 16.4% (21/128) of MI oocytes matured at 5 p.m. of day 1. The rate of normal fertilization for IVM oocytes (58.5%) was not significantly different from that of initial ICSI (64.0%). One patient received a transfer of two fertilized IVM oocytes alone that were injected at 5 p.m. of day 1, maturing from the MI stage, and achieved a normal pregnancy. The fertilized IVM oocytes were replaced along with the embryos from initial ICSI for 40 cycles that led to 14 (35%) clinical pregnancies. In 43 fertilized IVM oocytes donated for research, we observed that cleavage (95.3%) to the 2- to 4-cell stage was not distinct from that of initial ICSI (94.6%); however, the percentage of embryos of grade I and II morphology was significantly smaller (24.4% vs. 62.5%). Only five (11.6%) developed to blastocysts in vitro. Twenty-one fertilized IVM oocytes were frozen for future transfer. A schedule to inject IVM oocytes in ICSI cycles may generate more accessible embryos for fresh transfer or cryopreservation to increase the chance of pregnancy, although the embryo quality was relatively poor.     

Role of the MAPK cascade in mammalian germ cells

The mitogen-activated protein kinase (MAPK) cascade is one of the most important of the intracellular signaling pathways that play a crucial role in cell proliferation, cell differentiation and cell cycle regulation. Since the first report in 1993 of MAPK's involvement in the functional regulation of mammalian oocytes, much work has been done on the role of the MAPK cascade in germ cells in different species of mammals. This review describes the possible involvement of the MOS/MEK/MAPK/RSK cascade in spermatogenesis, sperm function, oocyte meiotic re-initiation, spindle assembly, metaphase II arrest, pronuclear formation and the entry of first mitosis, as well as the cross-talk of this cascade to maturation-promoting factor (MPF) and other signal molecules in mammals.     

Timing and regulatory aspects of oocyte maturation in vitro in the tammar wallaby (Macropus eugenii)

Oocytes from a marsupial, the tammar wallaby (Macropus eugenii), resemble those of eutherian mammals in their ability to resume meiosis in vitro when cultured under suitable conditions. Culture for 42-48 h in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum, and 10 microg mL(-1) porcine luteinizing hormone (pLH) was required in order for oocytes, collected from the large antral follicles (> 2 mm diameter) of tammar wallabies (primed with 6 mg of porcine follicle stimulating hormone twice daily for four days), to proceed to metaphase II (MII) of meiosis. Under these conditions, chromatin condensation was observed within 4-8 h of culture in 61% of oocytes; metaphase I (MI) chromosomes were observed from 18-30 h of culture (66%); and most oocytes (76%) progressed to MII by 42 h in vitro. The addition of cycloheximide, a protein synthesis inhibitor, at concentrations of 1-100 microg mL(-1), prevented maturation of tammar wallaby oocytes in vitro. This effect was reversible, as oocytes washed free of cycloheximide after 4 h of incubation were able to progress to MII. The addition of cycloheximide to wallaby oocytes at MI of meiosis prevented normal progression to MII suggesting that proteins critical for nuclear maturation are synthesized throughout the maturation process. Genistein, a protein kinase inhibitor decreased maturation of wallaby oocytes in a dose dependent manner. However, the concentration required to significantly inhibit maturation of wallaby oocytes (60 microg mL(-1)) was greater than that required for eutherian species. Most wallaby oocytes were able to undergo germinal vesicle breakdown (GVBD) in the presence of high concentrations of genistein but produced abnormal chromatin configurations and were unable to progress to MII. Future studies will examine whether cytoplasmic changes occur in marsupial oocytes in vitro and their temporal relationship to nuclear maturation.     

Effect of serum on the in vitro maturation of canine oocytes

The meiotic competence of canine oocytes cultured for 72 h in medium supplemented with three different concentrations (5, 10 and 20%) of anoestrous, oestrous or metoestrous bitch serum, or with 0.3% bovine serum albumin (BSA), was examined. The oestrous serum supplement had a positive effect on the resumption of meiosis, compared with the other supplements (P<0.05). The number of oocytes that reached metaphase I (MI) and metaphase II (MII) was significantly higher (P<0.05) with the oestrous serum supplement than with the anoestrous serum supplement. There were no significant differences among the three different concentrations in each serum type with respect to the proportion of oocytes that completed meiosis (MI to MII). The number of oocytes that resumed meiosis in the 10% oestrous serum supplement was significantly higher (P<0.05) than those of each concentration of the anoestrous and metoestrous serum supplements, and of the 0.3% BSA supplement. Moreover, a higher number of oocytes reached MII in the presence of the 10% oestrous serum supplement than with the 10% anoestrous serum supplement. These results suggest that supplementation of the culture medium with 10% oestrous serum is the optimal treatment for in vitro maturation of canine oocytes.     

Cyclopiazonic acid, an inhibitor of calcium-dependent ATPases, induces exit from metaphase I arrest in growing pig oocytes

Calcium plays an important role in the regulation of meiotic maturation in mammalian oocytes. In the present study, mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases, was used to mobilize intracellular calcium deposits in growing pig oocytes, which had not attained full meiotic competence and in which maturation is thus spontaneously blocked at the metaphase I stage. CPA treatment significantly increased the ratio of growing oocytes that are able to overcome the spontaneously occurring metaphase I block to complete their maturation at the metaphase II stage. CPA treatment of a least 2 hours' duration is necessary to overcome the metaphase I block in growing oocytes. A similar effect upon release from the spontaneous meiotic block at the metaphase I stage was observed after treatment of growing pig oocytes with thapsigargin, another inhibitor of endogenous calcium-dependent ATPases. Numerous calcium deposits in vacuoles, the mitochondria and on the surface of yolk granules in growing pig oocytes were observed. CPA treatment is able to mobilize calcium from the mitochondria, but deposits in vacuoles and deposits on the surface of yolk granules seem to remain intact after CPA treatment. A microinjection of heparin, which is known to bind with the inositol trisphosphate receptors, significantly decreased the ratio of CPA-treated growing oocytes overcoming the block at the metaphase I stage. This indicates that CPA might mobilize calcium in growing pig oocytes through inositol trisphosphate receptors. On the other hand, a microinjection of procaine or a microinjection of ruthenium red, both inhibitors of ryanodine receptors, did not prevent the overcoming of the metaphase I block, induced by CPA treatment. The calcium channel blocker, verapamil, significantly reduces the proportion of CPA-treated growing oocytes that overcome the metaphase I block. This indicates that the influx of calcium from extracellular sources is necessary to overcome the metaphase I block. The calmodulin inhibitors ophiobolin A and W7 also reduce the proportion of CPA-treated growing oocytes overcoming the metaphase I block.     

Male sexual dysfunction associated with coronary heart disease

A pilot study of 18 males (age range 38–68) hospitalized for an acute myocardial infarction (MI) revealed that 44% were impotent and 28% had had premature ejaculation prior to the MI. A subsequent research project is the basis of this article. During a 10-month period, 131 male patients (age range 31–86), while hospitalized for an acute MI, were interviewed about their pre-MI sexual functioning. Two-thirds of the males had—by their own definition—a significant sexual problem. Among the sexually dysfunctional group, 64% were impotent, 28% had a significant (> 50%) decrease in sexual frequency, and 8% had premature ejaculation. No adverse side effects occurred from a detailed sexual history being taken while the male was recovering from an acute MI. One implication for cardiac rehabilitation is that returning to the pre-MI level of sexual functioning is not enough.     

Follicular fluid supplementation during in vitro maturation promotes sperm penetration in bovine oocytes by enhancing cumulus expansion and increasing mitochondrial activity in oocytes

The aim of this study was to examine the effects of bovine follicular fluid (bFF) on mitochondrial activity in in vitro-matured (IVM) oocytes and to assess its importance for fertilisation and embryo development. Bovine follicular oocytes were subjected to IVM in medium supplemented either with polyvinylpyrrolidone, bovine serum albumin, calf serum or bFF. Nuclear maturation, cumulus expansion, mitochondrial distribution and ATP content in oocytes were compared between groups along with subsequent in vitro fertilisation (IVF) and embryo development. Compared with other supplements, bFF generated significantly enhanced re-distribution of active mitochondria in oocytes and this effect was associated with elevated intracellular ATP content. Furthermore, bFF significantly improved cumulus expansion, which was associated with improved fertilisation rates when cumulus-enclosed oocytes were subjected to IVF; however, its promoting effect was neutralised when denuded oocytes were inseminated. Elevating ATP content in oocytes by bFF did not affect maturation or embryo development but promoted fertilisation when mitochondrial electron transport was blocked in oocytes before IVF by Rotenone. In conclusion, supplementation of IVM medium with bFF promotes sperm penetration both by the improvement of cumulus expansion and by enhancing ATP levels in oocytes, which maintains their ability to be fertilised after mitochondrial stress.     

Effect of safe concentrations of some pesticides on ovarian recrudescence in the freshwater murrel, Channa punctatus (Bl.): A quantitative study

The effects of safe concentrations of carbofuran 3% G (5 ppm) and fenitrothion 30% E.C. (1.5 ppm) on ovarian recrudescence of Channa punctatus during the maturing, prespawning, and spawning phases (mid-April through mid-August) of the annual reproduction cycle were studied. The results reveal that, compared to carbofuran treatment, fenitrothion was more effective not only in lowering ovarian weights, but also in retarding the growth of previtellogenic (stage I) oocytes into vitellogenic (stage II) and yolky (stage III) oocytes. In addition, more previtellogenic and vitellogenic oocytes underwent atresia in fenitrothion-treated fish and only atretic oocytes were observed in the ovaries of these fish after 120 days of exposure. The fenitrothion treatment also caused a significant decline in the diameter of stage III oocytes, and in the number of nucleoli in stage I oocytes.     

丝裂原活化蛋白激酶通路正性调节苯并(a)芘所致细胞周期改变

目的 研究苯并（a）芘（BaP）对人胚肺成纤维细胞（HELF）的细胞周期分布及丝裂原活化蛋白激酶（MAPK）信号分子（ERK1／2、JNK1／2和p38）磷酸化水平的影响,并探讨MAPK磷酸化水平改变与细胞周期效应之间的关系。方法 用0．1、0．5、2．5和12．5μmol／L的BaP处理HELF细胞24h,用蛋白印迹方法检测ERK1／2、JNK1／2和p38蛋白激酶的磷酸化水平和蛋白总量的改变,利用流式细胞技术检测2．5μmol／LBaP处理24h后对HELF细胞周期的影响。结果 不同剂量BaP可明显提高ERK1／2、JNK1／2和p38蛋白激酶磷酸化水平;2．5μmol／LBaP处理24h,细胞周期分布与二甲基亚砜对照组相比,G0与G1期细胞数下降了11．9％（F=41．38,P〈0．01）,S期细胞数增加了17．2％（F=68．13,P〈0．01）;三种MAPK化学抑制剂（AG126、SP600125及SB203580）可明显抑制BaP处理引起的细胞周期的改变。结论 ERK1／2、JNK1／2和p38均参与了BaP所致细胞周期改变过程．并发挥币性调节作用。     

Long-term trajectory of leisure time physical activity and survival after first myocardial infarction: a population-based cohort study

The benefits of leisure time physical activity (LTPA) in cardiovascular prevention are well established. While cardiac rehabilitation programmes have been demonstrated as improving myocardial infarction (MI) prognosis, the strength of the association between LTPA and post-MI survival has yet to be quantified. We evaluated long-term survival after MI of inactive, irregularly active, and regularly active patients and examined trajectories of LTPA and their relationship to mortality risk. Consecutive patients aged ≤65 years (n = 1,521), discharged from 8 hospitals in central Israel after first MI in 1992–1993, were followed through 2005. Extensive clinical and sociodemographic data, including self-reported LTPA habits, were obtained at baseline and at 4 subsequent interviews. Pre-MI inactive patients (54%) had lower socioeconomic status, higher prevalence of risk factors and comorbidities and more severe MI. The point prevalence rate of regular LTPA at all follow-up interviews was approximately 40% and 18% were regularly active throughout the entire follow-up. Over a median follow-up of 13.2 years, 427 deaths occurred. After multivariable adjustment, no association was observed between pre-MI LTPA and death. However, with LTPA categories modelled as time-dependent variables, providing an estimation of cumulative assessment and accounting for changes in LTPA post-MI, a strong inverse graded association was revealed (multivariable-adjusted hazard ratios, 0.56 [95% CI: 0.42–0.74] for regular and 0.71 [95% CI: 0.54–0.95] for irregular activity vs. none). Similar estimates were obtained among pre-MI sedentary patients. In summary, after MI, regularly active patients had about half the risk of dying compared with inactive patients, irrespective of pre-MI habits.     

MAPK和MPF对小鼠受精卵有丝分裂期作用

目的探讨有丝分裂促进因子（MPF）和丝裂原活化的蛋白激酶（MAPK）在小鼠受精卵第一有丝分裂期（M期）中的作用及相互关系.方法分别用MAPK激酶特异性抑制剂（U0126）和MPF特异性抑制剂（roscovitine）抑制受精卵细胞MAPK和MPF的活性,通过同位素测定MAPK和MPF的活性变化,利用显微镜进行形态观察来判定MAPK活性变化对受精卵发育的影响.结果在受精卵第一次有丝分裂期,正常组小鼠受精卵MAPK和MPF的活性均有短暂升高.在U0126处理组,MAPK的活性受抑制会导致受精卵处于M期而不发生分裂,但MPF的活性未受到影响.在roscovitine处理组,MPF的活性受到抑制导致受精卵处于M期不发生分裂,此时MAPK不发生活化.结论 MAPK和MPF的活化对于小鼠受精卵完成有丝分裂是不可缺少的,在有丝分裂期MPF参与MAPK的活化.     

Effect of centrifugation and electrical activation on male pronucleus formation and embryonic development of porcine oocytes reconstructed with intracytoplasmic sperm injection

Oocyte centrifugation and electrical activation are commonly used in intracytoplasmic sperm injection (ICSI) of bovine and porcine oocytes, to facilitate visual identification of sperm release into the ooplasm and to support oocyte activation following injection with tail membrane-damaged sperm. The present study evaluated the necessity of these steps in porcine modified ICSI. In the first series of experiments, in vitro-matured gilt oocytes with or without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail first. Oocytes without centrifugation exhibited a significantly higher normal fertilisation rate, defined as male pronucleus (MPN) and female pronucleus (FPN) formation and the presence of two polar bodies, than centrifuged oocytes (40% v. 9%, respectively; P < 0.05). The rate of MPN formation was significantly higher in uncentrifuged oocytes compared with centrifuged oocytes (48% v. 17%, respectively; P < 0.05). The rates of survival, cleavage, blastocyst formation and total cell number in blastocysts did not differ between the two groups of oocytes. Next, the effect of electrical activation after ICSI on uncentrifuged oocytes injected with head membrane-damaged spermatozoa was determined. No significant differences were observed in the rate of MPN formation in sperm-injected oocytes regardless of electrical activation. However, the survival rates of sperm-injected or control oocytes without electrical activation were significantly higher than those of sperm-injected or control oocytes with electrical activation (88% and 84% v. 77% and 64%, respectively; P < 0.05). The cleavage rates of sperm-injected oocytes were significantly higher than those of control oocytes, regardless of electrical activation (77% and 81% v. 47% and 61% in sperm-injected and control oocytes with or without electrical activation, respectively; P < 0.05). Although development to blastocysts was similar in all experimental groups, the total cell numbers in blastocysts from control oocytes were significantly higher than those in sperm-injected oocytes, regardless of electrical activation (40 and 44 v. 22 and 26 in control and sperm-injected oocytes with or without electrical activation, respectively; P < 0.05). In conclusion, the present study clearly demonstrated that oocyte centrifugation before sperm injection is not beneficial to normal fertilisation and that electrical activation is not necessary in the modified porcine ICSI.     

Chromatin, microtubule and microfilament configurations in the canine oocyte

In the present study, we observed chromatin, microtubule and microfilament distribution in canine oocytes. The germinal vesicle (GV) chromatin of canine oocytes was classified into four configurations (GV-I, -II, -III and -IV) based on the degree of chromatin separation and condensation. Oocytes recovered from follicular phase ovaries had a greater amount (68%, P < 0.05) of GV-III or GV-IV chromatin than did those from non-follicular phase ovaries (35%). The majority (86.7%) of in vivo ovulated oocytes were at GV-IV. The rates of development to GV breakdown/metaphase I/metaphase II were higher in oocytes recovered from follicular ovaries than from non-follicular ovaries. Immunostaining results revealed cytoplasmic microtubules present in all GV-stage oocytes. Following GV breakdown, microtubular asters were produced from condensed chromatin. The asters appeared to be elongated, and encompassed condensed chromatin particles to form meiotic metaphase chromatin. Microfilaments were located in the cortex and around the GV. During meiotic maturation, a microfilament-rich area, in which the chromatin is allocated, was observed in the oocyte. Our results indicate that oocytes recovered from follicular ovaries were in an advanced stage of GV, and were more competent to complete maturation compared to those from non-follicular phase ovaries. Both microtubules and microfilaments are closely associated with reconstruction of chromatin during meiotic maturation in canine oocytes.     

Apoptotic effect of IP6 was not enhanced by co-treatment with myo-inositol in prostate carcinoma PC3 cells

Inositol hexaphosphate (IP₆) is a major constituent of most cereals, legumes, nuts, oil seeds and soybean. Previous studies reported the anticancer effect of IP₆ and suggested that co-treatment of IP₆ with inositol may enhance anticancer effect of IP₆. Although the anticancer effect of IP₆ has been intensively studied, the combinational effect of IP₆ and inositol and involved mechanisms are not well understood so far. In the present study, we investigated the effect of IP₆ and myo-inositol (MI) on cell cycle regulation and apoptosis using PC3 prostate cancer cell lines. When cells were co-treated with IP₆ and MI, the extent of cell growth inhibition was significantly increased than that by IP₆ alone. To identify the effect of IP₆ and MI on apoptosis, the activity of caspase-3 was measured. The caspase-3 activity was significantly increased when cells were treated with either IP₆ alone or both IP₆ and MI, with no significant enhancement by co-treatment. To investigate the effect of IP₆ and MI of cell cycle arrest, we measured p21 mRNA expression in PC3 cells and observed significant increase in p21 mRNA by IP₆. But synergistic regulation by co-treatment with IP₆ and MI was not observed. In addition, there was no significant effect by co-treatment compared to IP₆ treatment on the regulation of cell cycle progression although IP₆ significantly changed cell cycle distribution in the presence of MI or not. Therefore, these findings support that IP₆ has anticancer function by induction of apoptosis and regulation of cell cycle. However, synergistic effect by MI on cell cycle regulation and apoptosis was not observed in PC3 prostate cancer cells.     

Oocyte quality determines bovine embryo development after fertilisation with hydrogen peroxide-stressed spermatozoa

Exposure of gametes to specific stressors at sublethal levels can enhance the gametes' subsequent performance in processes such as cryopreservation. In the present study, bull spermatozoa were subjected to H?O? for 4 h at 100-, 200- and 500-μM levels; computer-assisted sperm analysis (CASA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay were used for evaluation of subsequent sperm motility and DNA integrity, respectively. Exposure of spermatozoa to H?O? did not affect sperm motility but DNA integrity was negatively affected by 500 μM H?O? compared with mock-exposed spermatozoa, whereas both motility and DNA integrity were affected compared with untreated spermatozoa. Nevertheless, insemination of oocytes with spermatozoa exposed to 200 μM H?O? increased fertilisation, cleavage and blastocyst rates (P < 0.05). Furthermore, the higher blastocyst yield after fertilisation of oocytes with spermatozoa exposed to 200 μM H?O? was related to oocyte diameter, with large-medium oocytes yielding higher blastocyst rates, while small-diameter oocytes consistently failed to develop into blastocysts. In conclusion, the results indicate that exposure of spermatozoa to 200 μM H?O? before sperm-oocyte interaction may enhance in vitro embryo production in cattle. However, this increased embryo production is largely dependent on the intrinsic quality of the oocytes.     

Sucrose-exposed chemically enucleated mouse oocytes support blastocyst development of reconstituted embryos

This study was carried out to test the ability of sucrose-exposed chemically enucleated mouse oocytes to support the development of reconstituted embryos in vitro. Cumulus-enclosed germinal-vesicle-stage mouse oocytes were matured in vitro to metaphase I stage and were chemically enucleated with 50 microg mL(-1) etoposide in tissue culture medium 199. The chemically enucleated oocytes were grouped into two groups. Group I was exposed to 0.75 M sucrose and group II was not exposed to sucrose. The zonae pellucidae of the chemically enucleated oocytes were removed with acid Tyrode's solution (pH 2.7). They were then aggregated into couplets with karyoplasts from pronuclear-stage embryos using phytohemagglutinin-P. The couplets were electrically fused to form reconstituted embryos. The reconstituted embryos were activated with 7% ethanol and cultured in vitro in simplex optimisation medium to test their developmental ability to the blastocyst stage. Some of the reconstituted embryos that developed to the blastocyst stage were used for chromosome counts to test their ploidy. The results of the present study showed that chemically enucleated oocytes exposed to sucrose supported the development of reconstituted embryos to the blastocyst stage (21.5%), whereas those not exposed to sucrose did not. The chromosome counts showed that the reconstituted embryos had normal ploidy (40 chromosomes). It is concluded that sucrose exposure improves the quality of chemically enucleated mouse oocytes. Thus they can be used as recipients for mouse embryo cloning and nucleocytoplasmic interaction studies.