Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer

Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.     

In vitro fertilization and development of frozen-thawed bovine oocytes.

Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%), but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in pregnancy and the birth of twin calves.     

Geminin deletion in mouse oocytes results in impaired embryo development and reduced fertility

Geminin controls proper centrosome duplication, cell division, and differentiation. We investigated the function of geminin in oogenesis, fertilization, and early embryo development by deleting the geminin gene in oocytes from the primordial follicle stage. Oocyte-specific disruption of geminin results in low fertility in mice. Even though there was no evident anomaly of oogenesis, oocyte meiotic maturation, natural ovulation, or fertilization, early embryo development and implantation were impaired. The fertilized eggs derived from mutant mice showed developmental delay, and many were blocked at the late zygote stage. Cdt1 protein was decreased, whereas Chk1 and H2AX phosphorylation was increased, in fertilized eggs after geminin depletion. Our results suggest that disruption of maternal geminin may decrease Cdt1 expression and cause DNA rereplication, which then activates the cell cycle checkpoint and DNA damage repair and thus impairs early embryo development.     

影响山羊体外受精的因素

以屠宰山羊卵母细胞为材料研究了公羊个体、附睾不同部位精子、成熟培养和受精时卵丘存在与否、卵丘扩展程度及卵龄对山羊体外受精的影响。结果表明 :1)不同公羊精液在受精、卵裂和桑椹 /囊胚率上都有显著差异 ;2 )附睾尾精子和鲜精的受精、卵裂和桑椹 /囊胚率无显著差异 ,但显著高于附睾体和附睾头精子 ;3)成熟培养 2 4和 2 7h卵母细胞的的桑椹胚 /囊胚率显著高于培养 2 1和 30h卵母细胞 ;4 )卵丘扩展 3和 4级卵母细胞受精和桑椹胚 /囊胚率显著高于扩展 0和 1级卵母细胞 ;5 )成熟培养前机械去卵丘严重影响卵母细胞体外受精和桑椹胚 /囊胚率 ;6 )受精前完全去掉卵丘显著影响桑椹胚 /囊胚率     

Bisphenol A exposure in utero disrupts early oogenesis in the mouse

Estrogen plays an essential role in the growth and maturation of the mammalian oocyte, and recent studies suggest that it also influences follicle formation in the neonatal ovary. In the course of studies designed to assess the effect of the estrogenic chemical bisphenol A (BPA) on mammalian oogenesis, we uncovered an estrogenic effect at an even earlier stage of oocyte development—at the onset of meiosis in the fetal ovary. Pregnant mice were treated with low, environmentally relevant doses of BPA during mid-gestation to assess the effect of BPA on the developing ovary. Oocytes from exposed female fetuses displayed gross aberrations in meiotic prophase, including synaptic defects and increased levels of recombination. In the mature female, these aberrations were translated into an increase in aneuploid eggs and embryos. Surprisingly, we observed the same constellation of meiotic defects in fetal ovaries of mice homozygous for a targeted disruption of ERβ, one of the two known estrogen receptors. This, coupled with the finding that BPA exposure elicited no additional effects in ERβ null females, suggests that BPA exerts its effect on the early oocyte by interfering with the actions of ERβ. Together, our results show that BPA can influence early meiotic events and, importantly, indicate that the oocyte itself may be directly responsive to estrogen during early oogenesis. This raises concern that brief exposures during fetal development to substances that mimic or antagonize the effects of estrogen may adversely influence oocyte development in the exposed female fetus.     

Term development of caprine embryos derived from immature oocytes in vitro

Ovaries were surgically removed from female goats (Toggenburg, Nubian and Saanen breeds). Oocytes were collected by follicular aspiration or after ovaries were minced, then matured in mTCM-199 with 100 mug LH + 0.5 mug FSH + 1.0 mug estradiol 17-beta/ml for 27 h prior to in vitro fertilization (17). Although more oocytes were made available by mincing than by aspiration, higher proportions of aspirated oocytes were fertilized and developed to morulae. Proportions that fertilized and reached morulae were 82 102 (80.4%) and 50 102 (49.0%) versus 77 126 (61.1%) and 27 126 (21.4%) for oocytes obtained by aspiration and after ovarian mincing, respectively (P<0.05). Proportions of inseminated ova undergoing cleavage and continuing development to the morula stage differed significantly (P<0.05) among 5 co-culture treatment groups, with higher proportions of cleavage (23 27 , 85.2%) and morulae (14 27 , 51.9%) obtained by co-culture on caprine cumulus cells (cCC). Some oocytes reached the blastocyst stage (4 54 , 7.4%) following oocyte collection by aspiration and culture on caprine oviduct epithelial cells (cOEC). After 4- and 8-cell stage embryos obtained by aspiration and culture on cCC were transferred pregnancy resulted. Twin male kids (developed from different embryos) were born on August 6, 1993, and have developed into normal bucks. Conditions reported here provided an adequate environment for support of oocyte maturation, fertilization and early embryonic development in vitro (IVMFC) with normal development after embryo transfer.     

Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. II. Chromosome aberrations induced in mature oocytes and fertilized eggs at the pronuclear stage following X-irradiation

Cytological analysis of the first-cleavage metaphase of eggs exposed to X-rays at the mature oocyte stage or the pronuclear stage 4 h after fertilization was performed using the in vitro fertilization technique. The frequency of chromosome aberrations in irradiated mature oocytes increased exponentially with dose, the dose-response relationship being best fitted to the linear-quadratic model. On the other hand, in eggs irradiated at the early pronuclear stage, the frequency increased linearly with dose and the dose-response relationship was best fitted to the linear model. The aberrations were mainly chromosome-type (mature oocytes: 86.0% and pronuclear stage: 88.5%) and the majority were fragments in both cases. Eggs in the early pronuclear stage were markedly more radiation-sensitive than mature oocytes. A comparison of the present results with the previous ones (Matsuda et al., 1985b) showed that the sensitivities to induction of chromosome aberrations were in the order: egg at early pronuclear stage (highest) greater than mature oocyte greater than mature sperm.     

Successful production of piglets derived from vitrified morulae and early blastocysts using a microdroplet method

This study was conducted to determine the efficiency of vitrification using the microdroplet (MD) method for early stage porcine embryos. Embryos at compacted morulae to early blastocyst stage were vitrified in a vitrification solution containing 40% (v/v) ethylene glycol, 0.6M sucrose and 2% (w/v) polyethylene glycol in M2 (ESP) without any pretreatment. The equilibration and dilution were carried out in third and fourth steps, respectively, at 38 degrees C. The survivability of the cryopreserved embryos was assessed for both in vitro culture (Experiment 1) and by embryo transfer (Experiment 2). In Experiment 1, the embryos were vitrified within a microdroplet or 0.25 ml straw (ST) and fresh embryos were used as a control group. The survival rates after 24h culture in the MD, ST and control groups were 21/23, 14/20 and 20/20, respectively. The hatching rates of the embryos after 48 h incubation were 14/23, 4/20 and 16/20, respectively. In Experiment 2, 171 vitrified embryos were transferred to 5 recipient gilts, and 17 healthy piglets were produced from 2 recipients (3 recipients aborted) in Group 1. In Group 2, 81 vitrified embryos and 16 fresh embryos in total were transferred to 4 recipient gilts, and 10 healthy piglets from the vitrified embryos were produced from 3 recipients. These results indicated that porcine embryos of compacted morulae to early blastocyst stage can survive cryopreservation using the microdroplet method without any special intracellular manipulation or treatment.     

精子和卵母细胞质量控制对山羊卵胞质内精子注射（ICSI）受精的影响

ABSTRACT Effects of sperm and oocyte quality control on the efficiency of ICSI of in vitro matured goat oocytes were studied in this paper. The results showed that when injected intracytoplasmically, spermatozoa from caput, corpus and cauda epididymidis resulted in similar rates of fertilization, cleavage and morulae/blastocysts, but when injected subzonally, spermatozoa from caput and corpus gave rise to significantly lower rates of fertilization and embryo development than spermatozoa from the cauda epididymidis and ejaculates. When dead spermatozoa collected from semen that had been preserved in different ways were used for ICSI, those dead from liquid storage at 20 degrees C for 24 h gave rise to the best, but those dead from liquid storage at 5 degrees C for 15 days produced the poorest fertilization and embryo development. When spermatozoa were treated with different concentrations of Triton X-100 before ICSI, significantly higher rates of fertilization, cleavage and morulae/blastocysts were obtained with 0.0005% Triton X-100 than with other concentrations and manual immobilization. Oocytes were classified as of good and poor qualities by treatment in hypertonic sucrose solution, and rates of fertilization and embryo development were significantly higher in the good than in the poor oocytes after ICSI. Post-injection activation of oocytes with either A23187 or ionomycin/6-DMAP significantly increased the rates of fertilization, cleavage and morulae/blastocysts after ICSI. It is therefore concluded that (i) epididymal maturation mainly endowed spermatozoa with the capacity to fuse with the egg plasma membrane; (ii) different methods of semen storage caused different impairment of sperm fertilizing capacity; (iii) pre-injection treatment of spermatozoa with proper concentrations of Triton X-100 might be used to replace manual immobilization for ICSI; (iv) oocyte quality was a major factor influencing the efficiency of ICSI; (v) post-injection activation treatment of oocytes improved fertilization and embryo development after ICSI.     

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.     

Survival of frozen-thawed sheep embryos cryopreserved at cleavage stages

This study evaluated the effect of freezing-thawing procedures on the viability of sheep embryos cryopreserved at various developmental stages. The survival rates of frozen-thawed embryos were compared with non-frozen counterparts. Embryos were recovered from the oviduct and uterus, at different days of the early luteal phase, and were classified at six different developmental stages: 2- to 4-cell (n = 72), 5- to 8-cell (n = 73), 9- to 12-cell (n = 70), early morulae (n = 42), morulae (n = 41), and blastocyst (n = 70). For each early cleavage stage and blastocysts, approximately half of the embryos, were frozen immediately by slow freezing with an ethylene glycol-based solution. The remaining embryos were cultured to the hatched blastocyst stage. All morulae and compact morulae were frozen after recovery with the same protocol. Cryoprotectants were removed using 1M sucrose solution, and then warmed the embryos were cultured to the hatched stage in a standardized in vitro culture. Embryo developmental stage had a significant effect on the ability to hatch following freezing (P<0.0001). The cryotolerance of the embryos fitted a regression (r2 = 0.908), increasing linearly from 2- to 4-cell embryos (17.1%) to morula stage (46.3%) and in a quadratic regression from the morula to the blastocyst stage (83.7%). Frozen early cleavage stage embryos had a significantly lower viability than their fresh counterparts (23.1 vs 83.1%; P<0.0001), with a similar rate of viability between fresh or frozen blastocysts (92.5 vs 83.7%). In conclusion, early sheep embryos are very sensitive to freezing per se and the survival rates following conventional freezing improve as embryo developmental stage progresses.     

Vegetalization of Sea Urchin Larvae Induced with Cycloheximide

The embryos, kept at 20°C for 3 hr–6 hr from the time of fertilization (at the morula stage), were cultured in sea water containing cycloheximide (10–16 mM) for successive 3 hr and then transferred to normal sea water. The embryos, thus treated, became vegetalized larvae. With the same treatment performed at a developmental stage prior to 3 hr of fertilization, most of embryos developed to small blastulae filled with mesenchyme-like cells. The treatment at a stage after 6 hr of fertilization yielded normal plutei. From the embryos exposed to both ¹⁴C-leucine and ³H-thymidine during the treatment, labelled chromatin was isolated. Only in the presumptive vegetalized embryos obtained by the cycloheximide treatment of morulae, ratio of ¹⁴C-radioactivity found in proteins of chromatin to ³H-radioactivity in DNA was markedly lower than that observed in chromatin from control embryos. The rate of ³H-radioactivity-decrease by DNase I treatment was higher in chromatin isolated from the presumptive regetalized embryos than that observed in chromatin isolated from control ones. Probable failure of chromatin structure formation, due to cycloheximide-inhibition of chromatin protein synthesis, seems to disturb the determination in the embryos at the morula stage, resulting in an induction of vegetalized embryos.     

Fertilization of Chinese hamster ova in vitro and in vivo and their subsequent development in culture

Oocytes from superovulated Chinese hamsters can be fertilized in vitro using the culture medium BWW (70% of 112 ova) or a modified BWW designated as MBWW (76% of 122 ova) when either medium is supplemented with 4 mg/ml of bovine serum albumin. Ova fertilized in vitro will also cleave to the 2-cell stage in either medium (52% in BWW, 87% in MBWW), but fail to develop any further in culture. Oocytes fertilized in vivo and recovered at the late 2-cell or early 4-cell stages from females on Day 3 of pregnancy have the capacity to develop into expanded blastocysts in MBWW. When early embryos that developed into morulae and early blastocyts in culture were transferred to surrogate females, eight normal young were born.     

In vitro maturation, fertilization and culture to blastocysts of bovine oocytes in protein-free media

This study examined the role of protein supplementation at the various steps of the in vitro production of bovine embryos derived from two different morphological categories of COC. The basic medium was TCM 199 and was supplemented with hormones during maturation in vitro and either estrous cow serum (ECS), bovine serum albumin (BSA) at various concentrations or polyvinyl-alcohol (PVA). Fertilization in vitro was carried out using frozen-thawed semen or one bull in Fert-talp containing heparin, hypotaurin and epinephrine and either 6 mg/ml BSA or 1 mg/ml PVA. In vitro culture up to the blastocyst stage was performed in TCM 199 supplemented with either ECS, BSA or PVA. The first experiment investigated the influence of different medium-supplements (ECS, BSA or PVA) on nuclear maturation and revealed no significant differences among treatment groups nor between categories of COC (63.9% to 74.9% and 48.9% to 77.0%, respectively). The time course of in vitro fertilization was elucidated in Experiment 2 in medium supplemented with either protein or PVA during maturation and fertilization. Penetration was not affected (70.9% to 79.3% penetration 12 h after onset of oocyte-sperm-co-incubation), but formation of pronuclei was decreased (P < 0.05) 12 and 19 h after onset of oocyte-sperm-co-incubation and was retarded in medium supplemented with PVA (12 h: 63.8 vs 21.4 %; 19 h: 57.5 vs 20.8 %, respectively) while cleavage was not affected. In Experiment 3, six treatment groups were formed in which the two different morphological categories of cumulus-oocyte-complexes (COC) were incubated in basic medium supplemented with 1) ECS during maturation and embryo culture and BSA during fertilization; 2) PVA during maturation and embryo culture, fertilization medium with PVA; 3) PVA during maturation and embryo culture, fertilization medium with BSA; 4) BSA (1 mg/ml) during maturation, fertilization and embryo culture; 5) BSA (6 mg/ml) during maturation, fertilization and embryo culture; and 6) BSA (10 mg/ml) during maturation, fertilization and embryo culture. The rates of cleavage and the development to morulae or blastocysts did not differ (P > 0.05) among treatment groups and between both categories of COC and were showing a high degree of variability (cleavage 54.0% to 65.1% and 41.3% to 55.7%, respectively; morulae 25.3% to 53.0% and 26.0% to 51.2%, respectively; blastocysts 5.4% to 24.7% and 0.6% to 20.3%, respectively). Parthenogenetic activation only rarely occurred in medium containing PVA throughout all steps of in vitro production of bovine embryos (Experiment 4) and led to early cleavage stages (8%), but no development to morula- or blastocyst-stages was observed. It is concluded that 1) formation of pronuclei was retarded in medium lacking protein-supplementation, indicating that BSA is required for regular fertilization in vitro and 2) under our experimental conditions, protein-supplementation is not necessary for maturation and development up to the blastocyst stage in vitro.     

Vitrification of Mouse Embryos at Various Stages by Open-Pulled Straw (OPS) Method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure—that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method—that is, embryos were first pretreated in 10%E+10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E+10%D for 0.5 min, exposed to EDFS30 for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

Superovulation of rabbits with FSH alters in vivo development of vitrified morulae

Morulae were flushed from the oviducts and uteri of New Zealand White (NZW) rabbits superovulated with either 6 (3 d) or 8 (4 d) injections of FSH and from non-superovulated controls. The percentages of embryos recovered from 4 d (100%, n = 8) donors was significantly higher (P < 0.01) than that of 3 d (76%, n = 16) and control (87%, n = 22) donors. Overall, fertilization rates were significantly lower for the 3 d embryos (P < 0.01). Most (86 to 90%) morulae were morphologically suitable for vitrification in an ethylene glycol-based solution. Following storage in liquid nitrogen, morulae were rapidly thawed and transferred to the uteri of pseudopregnant recipients. The total number of kits born for the 3 d, 4 d, and control groups was 40, 61 and 48, respectively. The percentage of live kits from morulae transferred was significantly lower for the 3 d (20%, n = 201) than either the 4 d (36%, n = 169; P < 0.01) or the control (31%, n = 157; P < 0.05) group. The mean number of kits born/recipient for the 3 d (2.4 +/- 2.9), 4 d (4.7 +/- 3.5), and control (3.0 +/- 2.2) protocols did not differ (P > 0.05). The estimated overall efficiency of producing kits based on normal morulae collected for control and 4 d groups, however, was nearly two-fold that for females given 6 FSH treatments. We conclude that the 4 d FSH superovulation regimen enhances the efficiency of rabbit reproductive biotechnology after embryo cryopreservation. These findings have important implications for rabbit colony management using embryo cryopreservation.     

Chromosome aberrations induced by tritiated water or 60Co gamma-rays at early pronuclear stage in mouse eggs

The induction of chromosome aberrations in mouse eggs by exposure to HTO beta-particles and 60Co gamma-rays at the early pronuclear stage was examined at the first-cleavage metaphase by using an in vitro fertilization technique. Eggs at the pronuclear stage were exposed to beta-particles in a chemically defined medium containing tritiated water (HTO) for 2 h at 3-5 h after insemination. Other eggs at the same stage were exposed to gamma-rays from 60Co during the same period. The dose-response relationships for frequencies of chromosome aberrations per egg were fitted to a linear-quadratic model for HTO beta-particles, and to a linear model for 60Co gamma-rays. The chromosome aberrations were mainly chromosome-type, and the majority of all aberrations were fragments. RBE values of HTO beta-particles relative to 60Co gamma-rays and acute X-rays, which were estimated from the ratio of the linear regression coefficients over 0.05-Gy range, were 2.0 and 1.6, respectively.     

Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.     

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

Vitrification of mouse embryos at various stages by open-pulled straw (OPS) method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.