Isolation of mouse embryonic stem cells using vero cells—as a feeder layer—and their differentiation into neuron-like cells

P-430 Incubator management: Effect of dual versus triple gas incubation on in vitro fertilization of mammalian embryos. Homer L. Higdon III, Jennifer E. Graves, William R. Boone. Greenville Hosp System, Greenville, SC. Objective: The purpose of this study was to determine the influence of incubator environment on in vitro fertilization of mammalian embryos. Design: Prospective, randomized study. Materials/Methods: This study was performed using 607 bovine oocytes between June 2001 to August 2001. In vitro production was conducted in two dual gas incubators (95% air and 5% CO2) and two triple gas incubators (88% N2, 7% CO2, and 5% O2). In vitro maturation and fertilization were performed by methods described by Parrish and coworkers (Theriogenology, 1986; 25: 591-600) with cleavage stage embryos being cultured to blastocysts with the use of KSOM in microdrops under oil. The study was replicated three times with each replicate representing approximately 200 oocytes divided equally among the four incubators. Fertilization, cleavage and blastocysts rates were calculated. Statistical analysis was performed using chi-square tests with P ⬍ .05 considered statistically significant. Results: No differences among replications were observed. Comparison between the two incubator environments showed statistical differences for fertilization and blastocysts rate and no difference for cleavage rate. Results are presented in the table below.

Conclusions: These data suggest that fertilization of bovine oocytes is accomplished at higher rates with 5% CO2 in air compared to triple gas incubation at 88% N2, 7% CO2 and 5% O2. In addition, culture of cleavage stage embryos to blastocysts is enhanced in a triple gas environment as compared to dual gas environment. Factors governing these observations remain to be elucidated.

P-431 The morphometric changes of endometrium and serum leptin levels during the implantation period of the embryo in the rat in response to exogenous ovarian hyperstimulation by human menopausal gonadotropin and recombinant follicle stimulating hormone. Aysin Dursun, Fatih Sendag, Cosan Terek, Huseyin Yilmaz, Kemal Oztekin, Meral Baka. Ege Univ, Izmir, Turkey. Objective: The aim of this study is to determine the changes of endometrium and serum leptin levels during the peri-implantation period of the ovum in the rat in response to exogenous ovarian hyperstimulation by human menopausal gonadotropin (hMG) and recombinant follicle stimulating hormone (rFSH). Material(s) and Method(s): A total of 100 female rats were hyperstimulated with low- and high-doses of hMG (Group II and III, respectively) and low- and high-doses of rFSH (Group IV and V, respectively) prior to mating after the cycle control had achieved. The control group of animals was not injected (Group I). The endometrium of the pregnant rats (n⫽53) were analyzed by morphometric method and serum samples obtained for leptin and estradiol levels at baseline and at the day of implantation. Mitotic index was determined separetely for endometrial surface and glandular epithelium and endometrial stroma by light microscope. Morphometric analysis of endometrium, mitotic index, and serum leptin and estradiol levels were compared between the groups. Results: The mean number of corpora lutea was significantly higher in Group III and V (p⬍0.05). The measurement of mucosal depth was found to be significantly higher in Group V compared to control group (0.36163⫾0.03 versus 0.289-63⫾0.04, p⫽0.014). The depth of endometrial surface epithelium was found to be significantly higher in all study groups (Group II, III, IV and V) compared to control group (p⬍0.001). The depth of surface epithelium was found to be significantly lower in Group II compared to other study groups (Group III, IV and V) (p⬍0.001). The depth of surface epithelium was also significantly lower in group III compared to group IV (21.58-63⫾3.56 versus 28.21-63⫾3.56, p⬍0.001). Mitotic indices

FERTILITY & STERILITY威

that were determined at the endometrial surface epithelium and stroma were all significantly lower in study groups (Group II, III, IV and V) compared to control group (p⬍0.01). There was no significant incerase for the baseline leptin levels compared to levels measured at the day of implantation (p⬎0.05). Conclusion: In an animal model, exogenous administration of gonadotropins significantly affects the morphology of the endometrium and the mitotic index at the implantation period of the embryo. These morphological effects become more pronounced as we increase the administered dose of exogenous gonadotropins. However, no differences in leptin levels were observed related to different gonadotropin type and dose regimens.

P-432 Molecular cloning of novel temperature-induced expressed sequence tags related to apoptosis in spermatogenic cells of mouse. Mo Yaqin, Li LuYun, Fu JunJiang, Liu ShangFeng, Lu GuangXiu. Institute of Human Reproduction & Stem Cell Engineering, Central South University, Changsha, China. Objective: Spermatogenesis needs the relative cool environment of the scrotum and Cryptorchidism could cause human male infertility .It is most likely due to the cease of spermatogenesis and induced apoptosis of spermatogenic cells when the testes were exposed to the mild hyperthermia environment of abdomen. In this study, we have established a unilateral operative cryptorchid mouse model and used the method of suppression subtractive hybridization (SSH) to screen temperature-induced genes related to apoptosis in Spermatogenic Cells and to investigate the mechanism of spermatogenesis arrest in experimental cryptorchidism. Design: Scrotal testis was removed surgically into abdomen to established unilateral operative cryptorchid mouse model. On day 3 after surgery, SSH was performed between scrotal testis (‘driver’) and cryptorchid testis (‘tester’) to isolate expressed sequence tags (ESTs) which represent genes overexpressed in cryptorchid testis. Novel ESTs will be used to clone full-length cDNA in the future. Materials and Methods: Male C57BL/6 mice at 8-10 wk of age were anesthetized and a small incision was made in the abdomen. The testes on right side were displaced into the abdomen and the inguinal canals on right side were suturing to prevent testes decent. On day 3 after surgery, three mice were killed and testes were removed. mRNA from cryptorchid testes and scrotal testes were extracted and pooled each. SSH was performed between scrotal testes (‘driver’) and cryptorchid testes (‘tester’) using PCR-SelectTM cDNA Substraction Kit (Clontech) according to the manufacturer’s recommendations. The substracted library cDNA was cloned into pGEM-T vector (Promega) and total 168 individual recombinant clones were picked. After screening by reversed northern blots, total 54 recombinant clones were sequenced to analyze insert sequences. Results: 4 novel ESTs were isolated. Homology searches revealed that among them 3 ESTs were unknown cDNA and one EST was identical to a hypothetical protein whose function had not been reported. Conclusion: Experimental unilateral cryptorchidism provide us suitable model for studying temperature-induced alteration of gene expression in the process of hypospermatogenesis and spermatogenic cell apoptosis, because the environmental temperature was the only difference between cryptorchid and scrotal testes in experimental cryptorchidism mice. By using SSH technique, we have identified 4 novel ESTs overexpressed in cryptorchid testis. In further study, we can begin from these ESTs to clone full-length cDNA represent novel genes which may play important roles in temperature-induced germ cell apoptosis.

P-433 Isolation of mouse embryonic stem cells using vero cells—as a feeder layer—and their differentiation into neuron-like cells. Esmaili Fariba, T. Tiraihi, M. Movaheddin, S. J. Mowla. Tarbiat Modarres University, Tehran, Iran (Islamic Republic of). Objective: Mouse embryonic stem (ES) cells are pluripotent cells conventionally isolated from the inner cell mass of preimplantation embryos. ES cells can be maintained as stable, proliferative and undifferentiated cell lines if cultured in presence of leukemia inhibitory factor (LIF) or on feeder layers (primary embryonic fibroblasts or STO fibroblasts). Feeder layers

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synthesize and secrete LIF into the culture medium. Since, based on several evidences LIF is secreted by mitomycin-pretreated Vero cells (a cell line isolated from Green monkey kidney epithelial cells), our purpose of this study was to demonstrate that ES cells can be isolated using Vero cells as feeder layer. The criteria used for evaluation of undifferentiated ES cell phenotype in this study have included morphology,spontanous differentiation of embryoid bodies (EBs) and enzyme stain (alkaline phosphatase activity). Previous studies have shown that ES cells can be induced to differentiation into neurons and gelia in vitro. Induction protocols are involved culture in the presence of retinoic acid ( RA ). A key step in all of these protocols is remowal of factors which prevent ES cell differentiation. In response, ES cells form embryonic aggregates (embryoid bodies : EBs). Undifferentiated mouse ES cells derived on mitomycin C-treated Vero cells (as feeder layer) in our laboratory were aggregated and induced to differentiation using all trans RA in vitro by 4-/4⫹ protocol. After EBs adhesion and further growth, some of the resulting cells possess neuron- like cell morphology. Design: Experimental study Materials and Methods: Fully expanded blastocysts were obtained by mating superovulated NMRI female mice and cultured individually on mitomycin C-inactivated Vero cells. After 4-5 days in culture, ICMs were plucked off and partially disaggregated in a 0.25% trypsin/EDTA solution. Three to four days later compact ES cell colonies could be identified. Colonies were dissociated and reseeded on a freshly-treated Vero feeder layer. Cultures were scanned periodically and all ES cell colonies were passaged sequentially weekly intervals. Alkaline phosphatase was detected histochemically-following fixation of colonies with 100% ethanol- using ␣-naphtyl phosphate as a substrate. Induction protocol (on The cells established in our laboratory)was the 4-/4⫹ protocol as follow: EBs were produced by suspending cells in hanging drops. Culture was then continued for 4 additional days in the presence of 0.5 ␮M all-trans RA. The neuronlike cells were appeared in several days. Results: The resulting cells had a high ratio of nucleus to cytoplasm, prominent nucleuli, and a colony morphology similar to that of murine ES cells. The ES cell colonies expressed cell surface markers that characterize undifferentiated ES cells, including alkaline phosphatase. After the 8-day induction period, aggregates ( EBs ) were transferred to adhesive tissue culture. Approximately after 4 to 5 days further growth, some of the resulting cells possess neuron-like cells morphology. Conclusion: We concluded that Vero cells as feeder layers may present an alternative avenue to obtaining pluripotent ES cell lines. Although, application of Vero cells in ES cell culture still requires further investigations. Furthermore,our results demonstrated that mouse ES cells established on Vero cells (as feeder layer)can be differentiated to morphologically neuronlike cells in vitro.

P-434 Dynamic methylation changes in the centromeric regions of chromosomes of mouse preimplantation embryos. John J. Zhang, Yanwen Xu, Jamie Grifo, Lewis Krey. NYU School of Medicine, New York, NY. Objective: In mammals centromeric DNA is highly methylated in the chromosomes of somatic cells but is relatively hypo-methylated in the chromosomes of germ cells. How centromeric DNA methylation patterns change from differentiated germ cells to totipotent embryonic cells and finally to differentiated somatic cells is still unclear. This study investigates the dynamics of the methylation changes in centromeric DNA during early embryogenesis in the mouse. Design: Descriptive immunostaining study of DNA from mouse oocytes and embryos. Materials and Methods: Metaphase II (MII) oocytes were collected from superovulated female CB6F-1 mice. Some mice were mated with males overnight and zygotes were recovered from the oviducts and cultured in G2.1 and G2.2 sequential culture media until the hatched blastocyst stage. Metaphase chromosome spreads were collected at different stages of embryogenesis after incubating the embryos with colchicine (1 ␮g/ml at 37°C) for 1-9 h before an anticipated cleavage. After removing the zona pellucida and dissociating the blastomeres, chromosome spreads were fixed with an air-drying technique. At each cell stage, 20-35 well-prepared metaphases were stained with an antibody generated against 5-methylcytosine (Barbin et al, 1994, Human Molecular Genetics 94, 684-692) to detect methylation. Results: At 1-2 cell stage embryos the chromatids from half of centro-

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Abstracts

meric regions were hypomethylated until early blastocyst stage. In another half of the centromeric regions chromatids showed slightly stronger staining; however, such variations were not observed in more developed embryos. By the hatching blastocyst stage even strong fluorescence was noted in all centrometric regions. Conclusion: De novo methylation of centromeric DNA started in the early blastocyst stage and accelerated throughout blastogenesis to the hatched blastocyst stage. These dynamic methylation changes in centromeric DNA may signify an important epigenetic modification(s) that takes place during early embryo development. However, the molecular mechanisms underlying the transition from meiosis-specific to mitosis-specific patterns remain to be elucidated. Further research on the centromeres of germ cells and early preimplantation embryos will provide more insights about this epigenetic process.

P-435 Ooplasm biopsy in ART: Influence of biopsy size on survival, fertlization and development in the mouse model. Anuradha P. Goud, Pravin T. Goud, Michael P. Diamond, Mark R. Hughes. Wayne State Univ, Detroit, MI. Objective: Embryo selection is the epicenter of Human ART. However the current methods to assess oocyte/embryos are limited to morphology and cleavage evaluation. We recently devised a technique to biopsy an M II stage oocyte. In our initial experiments the biopsy fragments were representative of the parent oocytes in terms of microtubule dynamics. Moreover, increased microtubule dynamics in the ooplasm biopsy reflected aging related phenomena in the parent oocyte. The current study investigates the feasibility of ooplasm biopsy in ART and compares the influence of biopsy size on fertilization and development after ICSI. Design: M II stage mouse oocytes were subjected to ICSI either following (groups A and B) or without ooplasm biopsy (group C). Influence of biopsy size on survival, fertilization and development was compared. Materials and Methods: Sibling oocytes from superovulated B6D2F1 mice (n⫽192) were micromanipulated to obtain biopsy fragments measuring ⬃10-15 and 20-25 ␮m (groups A and B respectively, measured with ocular grid), which were treated with Taxol, fixed and subjected to confocal laser scanning microscope to assess ooplasmic microtubule dynamics (Fertil Steril 2002; 76Suppl 3S: S57). The parent oocytes in groups A and B along with control oocytes (group C) were subjected to ICSI with epididymal spermatozoa using conventional technique with cooling of microscope stage to 15°C. The injected oocytes were assessed for survival and cultured in M16 for subsequent development to the blastocyst stage. Development to the 2-cell, morula and blastocyst stages were compared using the Fisher’s exact test. Results: Biopsy fragments from groups A and B measured 12.2⫾1.9 and 22.1⫾2.2 (Mean⫾SD) respectively. The rates of oocyte survival following biopsy were similar in groups A (92.6%) and B (83.3%). Also, the oocyte survival after ICSI (72.0, 71.1 and 70.2%), fertilization (91.7, 90.6 and 88.1%) and development to the 2-cell stage (100% in all three) were similar in groups A, B and C respectively. Nonetheless, the incidence of development to the morula stage was significantly lower in group B (58.6%) compared to groups A (84.8%, P⫽0.026) and C (84.6%, P⫽0.015). Finally, the incidence of blastocyst per oocyte surviving ICSI was significantly lower in group B compared to groups A (72.2%, P⫽0.026) and C (67.8%, P⫽0.043) Conclusion: Ooplasm biopsy is feasible in ART as seen from successful fertilization and development post-biopsy. However, successful development is dependent on the size of the biopsy fragment and increase in biopsy dimensions affect the incidence of blastocyst development. Further studies are underway to investigate the influence of microtubule dynamics in the biopsied ooplasm on developmental potential of parent oocyte.

P-436 Bee venom promotes in vivo follicular development of immature rats. Ali F. M. Ali, M. Mostafa, A. Gaafar, S. El-Shayeb, Z. El-Bashir. Ain Shams Univ, Cairo, Egypt. Introduction: Bee venom is rich with cytokines, the most predominant is granulocyte macrophage colony stimulating factor. It is now clear that

Vol. 80, Suppl. 3, September 2003