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In Silico Identification of a New Oocyte-Specific Gene Expression in the Mouse Ovary
central NPY tone which then inhibits the pulsatility of the GnRH neurons. Hyperleptinemia, central leptin resistance, NPY elevation, GnRH suppression, and subfertility associated with dietary-induced obesity in female DBA/2J mice appear reversible through dietary modification. Supported by: None
P-636 Steroid Sensitivity and Proliferative Potential of the Primate Ovarian Surface Epithelium. J. W. Wright, R. L. Stouffer. Oregon National Primate Research Center, Beaverton, OR. OBJECTIVE: To characterize the ovarian surface epithelium (OSE) on the primate ovary, including the effects of local disruption of OSE continuity and estrogen on proliferation and growth arrest of these cells. DESIGN: Comparative immunohistochemical analysis of the OSE of ovaries obtained after defined experimental protocols. Antibodies are used that distinguish the OSE from underlying tissue layers, and include estrogen receptor-␣ (ER), progesterone receptor (PR), keratin, -catenin, and N- and E-cadherin. Cell proliferation or growth arrest are indicated by the presence of PCNA, phosphorylated retinoblastoma (pRb), or p53 (a mediator of cell cycle arrest and apoptotic pathways). MATERIALS AND METHODS: Rhesus macaque ovaries were collected by laparoscopy and handled in a manner to preserve the surface epithelium. Control ovaries were collected and immediately fixed. In a second group, ovaries were gently brushed during laparoscopy to remove the OSE from a portion of the ovarian surface, and collected 2-4 days later. In a third group, ovaries were removed and cultured in DMEM ⫹ 1% fetal calf serum, in the presence or absence of 2 g/ml estradiol (a concentration reported in the periovulatory primate follicle), for 1-2 days after removal of a portion of the OSE. Sectioned tissue was probed with primary antibody and a phosphatase-conjugated secondary antibody, and visualized by a NBT/BCIP colorimetric reaction. RESULTS: We observed a generally uniform pattern of expression of keratin, ER-␣, PR, and -catenin throughout the entire OSE. Both N- and E-cadherin were detected within the OSE, with E-cadherin being the prevalent form; however, each cadherin was absent in some areas of the OSE. The localization of cadherins in regions of the OSE did not obviously correlate to underlying structures (such as a developing follicle or regressed corpus luteum). Markers for proliferation, PCNA and pRb, were almost entirely absent from intact OSE (⬍ 5 cells per section examined). Importantly, disrupting a portion of the OSE resulted in expression of PCNA and pRb by the OSE in regions where the ovarian surface had been brushed. A low level of p53 was detected in the OSE. However, culturing ovaries in the presence of 2 g/ml estradiol caused a several-fold increase in the number of p53-positive OSE cells. Preliminary analysis showed a nonuniform distribution of p53-positive cells, suggesting zones of enhanced estrogen sensitivity within the OSE. CONCLUSION: The OSE is a monolayer surrounding the ovary and expresses receptors for ovarian steroid hormones. The absence of markers for proliferation in the intact OSE is consistent with its role as a stable epithelium. Significantly, when a portion of the OSE is removed, the remaining cells show the potential to proliferate and rebuild this layer, possibly a normal activity following ovulatory disruption of the OSE. The ability of estradiol to increase p53 levels indicates potential regulation of OSE growth or survival by ovarian products in vivo. The heterogenous pattern of cadherin expression, as well as the variable response of OSE cells to estrogen suggests this tissue is not as simple as previously supposed. The underlying factors that result in differentiation among individual OSE cells could be significant in regards to ovarian function, health, and pathology. Supported by: HD18185 (Proj. 3), HD20869, RR00163
P-637 Inverse Relationship of Polydimethylsiloxane Thickness and Embryo Development Is Due to Elevated Media Osmolality. L. M. Cabrera, Y. Heo, S. Takayama, G. D. Smith. University of Michigan, Ann Arbor, MI. OBJECTIVE: Microfluidic technologies hold promise for future gamete and embryo culture and analysis. A microfluidic device has been devised that precisely controls fluid flow inside elastomeric capillary networks made of polydimethylsiloxane (PDMS) by using multiple computer-controlled, piezoelectric, movable pins. Each pin acts as a valve by pushing upwards
FERTILITY & STERILITY威
into microchannels, yet necessitates use of a thin flexible PDMS platform. Experiments were performed to evaluate influence of PDMS platform thickness on embryo development and media osmolality. DESIGN: Experimental animal-model embryo and media analysis. MATERIALS AND METHODS: Glass slides were drilled to form 6.5mm diameter holes and mounted on PDMS of 10, 1.0 or 0.1mm thickness to form wells. Pronuclear stage zygotes were collected from 6-8 week-old B6C3F1 female mice following gonadotropin stimulation and breeding to B6C3F1 males. All procedures performed on animals were approved by the University of Michigan Animal Care and Use Committee. Zygotes were placed into 50l of Potassium Simplex Optimized Medium (KSOM; Specialty Media, Phillipsburg, NJ) ⫹ 0.1% Serum Substitute Supplement (SSS; Irvine Scientific, Santa Ana, CA) overlaid with mineral oil in either organ culture dishes (control), or glass-wells with PDMS platforms of 10 (PDMS-10), 1.0 (PDMS-1), or 0.1 (PDMS-0.1) mm thick. Zygotes and culture devices were placed into a humidified environment of 5% CO2 in air at 37°C. Embryos were assessed at 24hr intervals. Timeappropriate development to ⱖ 2-cells at 24hrs and ⱖ 4-cells at 48hr were compared by chi-square analysis. Glass-PDMS wells with varying PDMS thickness were used as prepared or pre-soaked for 24hrs with KSOM or Oil before KSOM was added to wells, overlaid with oil, and assessed for osmolality at 24hr intervals. Changes in osmolality were tested for statistical difference by a mixed linear regression model for repeat measures. RESULTS: There was no significant difference in ability of zygotes to develop to/beyond the 2-cell stage at 24hrs of culture in control (73%), PDMS-10 (81%), PDMS-1 (88%), or PDMS-0.1 (80%). At 48hrs significantly fewer (P⬍0.01) had developed to/beyond the 4-cell stage in PDMS0.1 (22%) compared to control (67%), PDMS-10 (67%) and PDMS-1 (72%). In order to determine why embryos in PDMS-0.1 had compromised development, media were evaluated for osmolality in each culture device. KSOM osmolality drifted from 280 to 287 mmol/kg during 96hrs of incubation in control wells, and this was not significantly different from osmolality shifts in PDMS-10 or PDMS-1. However, KSOM osmolality increased from 280 to 460 mmol/kg in PDMS-0.1, which was significantly different from all other groups. Pre-soaking of PDMS with media or oil did not prevent this osmolality change over time in PDMS-0.1 devices. CONCLUSION: Embryo development is similar to controls on PDMS platforms of at least 1mm thickness. Embryo development is compromised when cultured on PDMS platforms of 0.1mm thickness and this is due to absorption/evaporation resulting in elevated osmolality to levels incompatible with embryo survival. If 0.1mm PDMS platforms are necessary for embryo culture in the future, they must be modified to protect against media osmolality shifts. Supported by: NICHD grant HD049607-01 to ST and GDS.
P-638 In Silico Identification of a New Oocyte-Specific Gene Expression in the Mouse Ovary. K. Lee, C. Park, S. Han, I. Kwak, S. Lee, K. Cha. Infertility Medical Center, CHA General Hospital, Graduate School of Life Science and Biotechnology, Pochon CHA University College of Medicine, Seoul, Republic of Korea. OBJECTIVE: Arrest at primordial follicle (PMF) stage and growth initiation into primary follicles (PRIF) are the crucial process for the female fertility. However, factors and mechanisms regulate these processes are poorly understood. Previously, using suppression subtractive hybridization (SSH) between day1 and day5 mouse ovaries, we found abundantly expressed 15 ESTs (expressed sequence tags) in day5-subtracted cDNA library, by comparison with day1-subtracted cDNA library. Among 15 ESTs, 5-25 clone (RIKEN cDNA E330009P21 gene) was a novel EST contig and selected for further study. The present study was conducted by EST clustering to obtain the full-length sequence containing that novel EST contig and further evaluated the expression of the novel gene in the mouse ovary. DESIGN: Experimental study to find novel gene by the in silico method and characterize its expression in ovary. MATERIALS AND METHODS: We performed EST clustering and cloned long sequence of 5-25 clone by 5’ Rapid Amplification of cDNA Ends (RACE). This novel sequence was similar to the registered BC085247 gene (F-box and WD40-repeat-containing protein; FBWD40). Differential expression of FBWD40 mRNA was evaluated at various postnatal stage by real-time PCR. To determine the tissue distribution of FBWD40 expression, we performed Northern blot and in situ hybridization.
S383
RESULTS: Expression of FBWD40 transcript was ovary-specific and highly expressed in 5-day mouse ovary and decreased thereafter. Moreover, localization revealed that the expression of FBWD40 transcript is oocytespecific from primordial to the preovulatory follicles, and detected more in nucleus. Localization of protein expression by immunohistochemistry and Western blot is in processing. CONCLUSION: We succeeded in silico finding of the novel gene that is ovary- and oocyte-specific. This is the first report of oocyte-specific expression of the FBWD40-like sequence. We suggest that oocyte-specific expression of the FBWD40 mRNA imply its significant role(s) in growth and maturation of oocytes and/or follicles during early folliculogenesis. Supported by: This work was supported by Korea Research Foundation Grant (KRF-2004-041-E00189).
P-639 Gene Expression in ESCs Derived From Cloned Blastocysts. Q. V. Neri, T. Takeuchi, H. Kang, F. Moy, Z. Rosenwaks, G. D. Palermo. Cornell University, New York, NY; Graduate School of Basic Medical Sciences, New York Medical College, New York, NY. OBJECTIVE: Once harvested from the inner cell mass (ICM), embryonic stem cells (ESCs) undergo extended proliferation in culture as undifferentiated cells poised for multilineage differentiation. Confirmation of the pluripotent status of these cells involves both morphological criteria and specific molecular markers, and expression of typical marker genes. Nanog, a divergent homeodomain protein that directs propagation of undifferentiated cells, is down-regulated during early de-differentiation and becomes silent in completely differentiated cells. DESIGN: RNA from cloned and in vivo derived ESCs was extracted and qRT-PCR performed to assess their stemness. Molecular markers and morphological criteria were used to confirm ESC pluripotency. MATERIALS AND METHODS: MII oocytes retrieved from B6D2-F1 mice were enucleated, injected with cumulus cell nuclei, and then activated using SrCl2. Blastocysts generated in vivo were similarly cultured and served as controls. Embryos were evaluated daily for their growth, hatching, and attachment to a feeder cell layer. After reaching an adequate size, ICMs were isolated mechanically 4 days later and trypsinized to provide single cells. RNA was isolated from ESCs derived from in vivo and cloned embryos, with mouse embryonic fibroblasts (MEF) serving as a negative control. qRT-PCR was performed with custom-designed primers for the target sequences of Nanog, Oct-4 (pluripotent markers), and Ttr genes, while Act- and Gapdh were used as normalizers. Gene expression was reported as a proportion, calculated from the cycle threshold against Actconsidered at 100% expression. Quality and stemness were assessed according to morphological characteristics such as cell size, nucleus/cytoplasm ratio, and nucleolar patterns. In addition, pluripotency markers such as alkaline phosphatase activity, Oct-4 and TROMA-1 expression were also assessed. RESULTS: Of 17 oocytes that survived cloning, 12 constructs displayed two pronuclei, 4 developed further to produce fully expanded blastocysts, and 2 produced ESC lines of good quality. On the other hand, 40 (87.5%) in vivo-derived zygotes developed into blastocysts, and once plated 7 (20.0%) produced ESC lines. ESC colonies of both origin contained at least 60% of undifferentiated cells according to morphological characteristics and molecular markers. Between 10,000 and 30,000 cells processed generated 0.3 to 0.9 g of total RNA that displayed two ribosomal subunits. Reverse transcription and qRT-PCR were successful in all specimens. As expected, Nanog was not expressed in MEFs whereas the in vivo and cloned-derived ESC cells revealed a similar 74.6% and 66.6% expression, respectively (2 ⫽ 1.19). Similarly, Oct-4 was not expressed in MEFs while the in vivo and cloned-derived ESC had an upregulation of 76.9% and 67.8%. MEFs manifested a higher expression (69.6%; P ⫽ 0.001) of Ttr (a marker of differentiation) compared to the in vivo and cloned-derived ESC colonies (40.7 and 49.2%, respectively). CONCLUSION: Results coming from a genomic indicator accord with those from other molecular markers in establishing stemness of the ESC colonies. Microarray analysis showed that clone-derived ESCs have similar expression patterns to those generated in vivo, and therefore that they may be used for stem cell therapy. Supported by: Institutional
S384
Abstracts
P-640 In Utero and Lactational Exposure to Nicotine: Ovarian Effects. M. S. Neal, J. J. Petrik, A. C. Holloway. Centre for Reproductive Care, Hamilton Health Sciences, and Reproductive Biology Division, Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada; Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada; Reproductive Biology Division, Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada. OBJECTIVE: Women born to mothers who smoked during pregnancy have been shown to have an earlier onset of menopause and a shorter reproductive lifespan (Sharpe RM and Franks S. 2002. Nat Med. Suppl: S33-40) although the mechanisms underlying this association are unknown. Nicotine administration in adult animals has adverse effects on the ovary and uterus (Patil SR et al. 1998. Indian J. Physiol. Pharmacol. 42: 503-8), however effects of fetal exposure to nicotine on the offspring’s ovary have not yet been determined. The goal of this study was to assess the effect of in utero exposure to nicotine, at concentrations that are representative of human exposure, on follicle dynamics and ovarian growth factor expression in the offspring. DESIGN: Prospective in vivo animal study in a university laboratory setting. All animal procedures were approved by the McMaster University Animal Research Ethics Board in accordance with the guidelines of the Canadian Council for Animal Care. MATERIALS AND METHODS: Nulliparous female Wistar rats (200250g) were given 1mg/kg/day nicotine bitartrate for 14 days prior to mating, during pregnancy and throughout lactation until weaning. Ovaries were removed from offspring in estrous at 120 days of age, fixed in formalin, embedded in paraffin and sectioned. Sections were stained with hemotoxylin and eosin to determine the number of follicles at different stages of development and the proportion of atretic follicles. Sections were examined using immunohistochemical staining to assess the expression of insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in granulosa cells. Data were analyzed by Student’s t-test (␣⫽0.05). RESULTS: Offspring of nicotine-exposed dams had significantly more growing follicles (p⬍0.05) and more antral follicles (p⫽0.09) than the salineexposed offspring. However, in the nicotine-exposed offspring the percentage of growing follicles that were atretic was 50% higher than in the saline-exposed offspring. There was no difference in the percentage of atretic antral follicles between the two treatment groups. Fetal and neonatal exposure to nicotine significantly (p⬍0.01) decreased the number of IGF-I immunopositive cells in the granulosa cells of the ovary. Similarly there was a significant decrease in VEGF expression in the granulosa cells in the nicotine-exposed offspring (p⬍0.001) compared to saline controls. CONCLUSION: Results from this study have demonstrated that nicotine exposure during fetal and neonatal life can alter follicle dynamics and growth factor expression in the ovary of the offspring, which may result in impaired ovarian function postnatally. Supported by: Funding for this project was provided by the Canadian Institutes of Health Research.
P-641 Role of Extracellular Matrix, Activin-A and Mitogen Activated Protein Kinase (MAPK) Signaling Pathways in Preantral Follicle Growth and Survival in the Mouse. O. Oktem, K. Oktay. Weill Medical College of Cornell University, New York, NY. OBJECTIVE: We have previously shown that ECM-Activin interactions play role in preantral follicle growth in organ cultures (1). Our current objective was to investigate the role of extracellular matrix (ECM)-activin-A interactions in in-vitro growth and survival of mouse preantral follicles under different culture conditions, and to determine mitogen activated protein kinase (MAPK) signaling pathways in preantral follicle development. MAPK family consists of c-jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK) and p38 which regulate cell growth, differentiation, and survival. DESIGN: In vitro study of isolated preantral follicles MATERIALS AND METHODS: 452 preantral follicles isolated from the ovaries of prepubertal SV129/B6 mice were cultured for 7 days with various combinations of ECM and activin-A. Follicles were cultured with or without recombinant activin-A either in wells coated with fibronectin (n⫽29),
Vol. 84, Suppl 1, September 2005
P-636 Steroid Sensitivity and Proliferative Potential of the Primate Ovarian Surface Epithelium. J. W. Wright, R. L. Stouffer. Oregon National Primate Research Center, Beaverton, OR. OBJECTIVE: To characterize the ovarian surface epithelium (OSE) on the primate ovary, including the effects of local disruption of OSE continuity and estrogen on proliferation and growth arrest of these cells. DESIGN: Comparative immunohistochemical analysis of the OSE of ovaries obtained after defined experimental protocols. Antibodies are used that distinguish the OSE from underlying tissue layers, and include estrogen receptor-␣ (ER), progesterone receptor (PR), keratin, -catenin, and N- and E-cadherin. Cell proliferation or growth arrest are indicated by the presence of PCNA, phosphorylated retinoblastoma (pRb), or p53 (a mediator of cell cycle arrest and apoptotic pathways). MATERIALS AND METHODS: Rhesus macaque ovaries were collected by laparoscopy and handled in a manner to preserve the surface epithelium. Control ovaries were collected and immediately fixed. In a second group, ovaries were gently brushed during laparoscopy to remove the OSE from a portion of the ovarian surface, and collected 2-4 days later. In a third group, ovaries were removed and cultured in DMEM ⫹ 1% fetal calf serum, in the presence or absence of 2 g/ml estradiol (a concentration reported in the periovulatory primate follicle), for 1-2 days after removal of a portion of the OSE. Sectioned tissue was probed with primary antibody and a phosphatase-conjugated secondary antibody, and visualized by a NBT/BCIP colorimetric reaction. RESULTS: We observed a generally uniform pattern of expression of keratin, ER-␣, PR, and -catenin throughout the entire OSE. Both N- and E-cadherin were detected within the OSE, with E-cadherin being the prevalent form; however, each cadherin was absent in some areas of the OSE. The localization of cadherins in regions of the OSE did not obviously correlate to underlying structures (such as a developing follicle or regressed corpus luteum). Markers for proliferation, PCNA and pRb, were almost entirely absent from intact OSE (⬍ 5 cells per section examined). Importantly, disrupting a portion of the OSE resulted in expression of PCNA and pRb by the OSE in regions where the ovarian surface had been brushed. A low level of p53 was detected in the OSE. However, culturing ovaries in the presence of 2 g/ml estradiol caused a several-fold increase in the number of p53-positive OSE cells. Preliminary analysis showed a nonuniform distribution of p53-positive cells, suggesting zones of enhanced estrogen sensitivity within the OSE. CONCLUSION: The OSE is a monolayer surrounding the ovary and expresses receptors for ovarian steroid hormones. The absence of markers for proliferation in the intact OSE is consistent with its role as a stable epithelium. Significantly, when a portion of the OSE is removed, the remaining cells show the potential to proliferate and rebuild this layer, possibly a normal activity following ovulatory disruption of the OSE. The ability of estradiol to increase p53 levels indicates potential regulation of OSE growth or survival by ovarian products in vivo. The heterogenous pattern of cadherin expression, as well as the variable response of OSE cells to estrogen suggests this tissue is not as simple as previously supposed. The underlying factors that result in differentiation among individual OSE cells could be significant in regards to ovarian function, health, and pathology. Supported by: HD18185 (Proj. 3), HD20869, RR00163
P-637 Inverse Relationship of Polydimethylsiloxane Thickness and Embryo Development Is Due to Elevated Media Osmolality. L. M. Cabrera, Y. Heo, S. Takayama, G. D. Smith. University of Michigan, Ann Arbor, MI. OBJECTIVE: Microfluidic technologies hold promise for future gamete and embryo culture and analysis. A microfluidic device has been devised that precisely controls fluid flow inside elastomeric capillary networks made of polydimethylsiloxane (PDMS) by using multiple computer-controlled, piezoelectric, movable pins. Each pin acts as a valve by pushing upwards
FERTILITY & STERILITY威
into microchannels, yet necessitates use of a thin flexible PDMS platform. Experiments were performed to evaluate influence of PDMS platform thickness on embryo development and media osmolality. DESIGN: Experimental animal-model embryo and media analysis. MATERIALS AND METHODS: Glass slides were drilled to form 6.5mm diameter holes and mounted on PDMS of 10, 1.0 or 0.1mm thickness to form wells. Pronuclear stage zygotes were collected from 6-8 week-old B6C3F1 female mice following gonadotropin stimulation and breeding to B6C3F1 males. All procedures performed on animals were approved by the University of Michigan Animal Care and Use Committee. Zygotes were placed into 50l of Potassium Simplex Optimized Medium (KSOM; Specialty Media, Phillipsburg, NJ) ⫹ 0.1% Serum Substitute Supplement (SSS; Irvine Scientific, Santa Ana, CA) overlaid with mineral oil in either organ culture dishes (control), or glass-wells with PDMS platforms of 10 (PDMS-10), 1.0 (PDMS-1), or 0.1 (PDMS-0.1) mm thick. Zygotes and culture devices were placed into a humidified environment of 5% CO2 in air at 37°C. Embryos were assessed at 24hr intervals. Timeappropriate development to ⱖ 2-cells at 24hrs and ⱖ 4-cells at 48hr were compared by chi-square analysis. Glass-PDMS wells with varying PDMS thickness were used as prepared or pre-soaked for 24hrs with KSOM or Oil before KSOM was added to wells, overlaid with oil, and assessed for osmolality at 24hr intervals. Changes in osmolality were tested for statistical difference by a mixed linear regression model for repeat measures. RESULTS: There was no significant difference in ability of zygotes to develop to/beyond the 2-cell stage at 24hrs of culture in control (73%), PDMS-10 (81%), PDMS-1 (88%), or PDMS-0.1 (80%). At 48hrs significantly fewer (P⬍0.01) had developed to/beyond the 4-cell stage in PDMS0.1 (22%) compared to control (67%), PDMS-10 (67%) and PDMS-1 (72%). In order to determine why embryos in PDMS-0.1 had compromised development, media were evaluated for osmolality in each culture device. KSOM osmolality drifted from 280 to 287 mmol/kg during 96hrs of incubation in control wells, and this was not significantly different from osmolality shifts in PDMS-10 or PDMS-1. However, KSOM osmolality increased from 280 to 460 mmol/kg in PDMS-0.1, which was significantly different from all other groups. Pre-soaking of PDMS with media or oil did not prevent this osmolality change over time in PDMS-0.1 devices. CONCLUSION: Embryo development is similar to controls on PDMS platforms of at least 1mm thickness. Embryo development is compromised when cultured on PDMS platforms of 0.1mm thickness and this is due to absorption/evaporation resulting in elevated osmolality to levels incompatible with embryo survival. If 0.1mm PDMS platforms are necessary for embryo culture in the future, they must be modified to protect against media osmolality shifts. Supported by: NICHD grant HD049607-01 to ST and GDS.
P-638 In Silico Identification of a New Oocyte-Specific Gene Expression in the Mouse Ovary. K. Lee, C. Park, S. Han, I. Kwak, S. Lee, K. Cha. Infertility Medical Center, CHA General Hospital, Graduate School of Life Science and Biotechnology, Pochon CHA University College of Medicine, Seoul, Republic of Korea. OBJECTIVE: Arrest at primordial follicle (PMF) stage and growth initiation into primary follicles (PRIF) are the crucial process for the female fertility. However, factors and mechanisms regulate these processes are poorly understood. Previously, using suppression subtractive hybridization (SSH) between day1 and day5 mouse ovaries, we found abundantly expressed 15 ESTs (expressed sequence tags) in day5-subtracted cDNA library, by comparison with day1-subtracted cDNA library. Among 15 ESTs, 5-25 clone (RIKEN cDNA E330009P21 gene) was a novel EST contig and selected for further study. The present study was conducted by EST clustering to obtain the full-length sequence containing that novel EST contig and further evaluated the expression of the novel gene in the mouse ovary. DESIGN: Experimental study to find novel gene by the in silico method and characterize its expression in ovary. MATERIALS AND METHODS: We performed EST clustering and cloned long sequence of 5-25 clone by 5’ Rapid Amplification of cDNA Ends (RACE). This novel sequence was similar to the registered BC085247 gene (F-box and WD40-repeat-containing protein; FBWD40). Differential expression of FBWD40 mRNA was evaluated at various postnatal stage by real-time PCR. To determine the tissue distribution of FBWD40 expression, we performed Northern blot and in situ hybridization.
S383
RESULTS: Expression of FBWD40 transcript was ovary-specific and highly expressed in 5-day mouse ovary and decreased thereafter. Moreover, localization revealed that the expression of FBWD40 transcript is oocytespecific from primordial to the preovulatory follicles, and detected more in nucleus. Localization of protein expression by immunohistochemistry and Western blot is in processing. CONCLUSION: We succeeded in silico finding of the novel gene that is ovary- and oocyte-specific. This is the first report of oocyte-specific expression of the FBWD40-like sequence. We suggest that oocyte-specific expression of the FBWD40 mRNA imply its significant role(s) in growth and maturation of oocytes and/or follicles during early folliculogenesis. Supported by: This work was supported by Korea Research Foundation Grant (KRF-2004-041-E00189).
P-639 Gene Expression in ESCs Derived From Cloned Blastocysts. Q. V. Neri, T. Takeuchi, H. Kang, F. Moy, Z. Rosenwaks, G. D. Palermo. Cornell University, New York, NY; Graduate School of Basic Medical Sciences, New York Medical College, New York, NY. OBJECTIVE: Once harvested from the inner cell mass (ICM), embryonic stem cells (ESCs) undergo extended proliferation in culture as undifferentiated cells poised for multilineage differentiation. Confirmation of the pluripotent status of these cells involves both morphological criteria and specific molecular markers, and expression of typical marker genes. Nanog, a divergent homeodomain protein that directs propagation of undifferentiated cells, is down-regulated during early de-differentiation and becomes silent in completely differentiated cells. DESIGN: RNA from cloned and in vivo derived ESCs was extracted and qRT-PCR performed to assess their stemness. Molecular markers and morphological criteria were used to confirm ESC pluripotency. MATERIALS AND METHODS: MII oocytes retrieved from B6D2-F1 mice were enucleated, injected with cumulus cell nuclei, and then activated using SrCl2. Blastocysts generated in vivo were similarly cultured and served as controls. Embryos were evaluated daily for their growth, hatching, and attachment to a feeder cell layer. After reaching an adequate size, ICMs were isolated mechanically 4 days later and trypsinized to provide single cells. RNA was isolated from ESCs derived from in vivo and cloned embryos, with mouse embryonic fibroblasts (MEF) serving as a negative control. qRT-PCR was performed with custom-designed primers for the target sequences of Nanog, Oct-4 (pluripotent markers), and Ttr genes, while Act- and Gapdh were used as normalizers. Gene expression was reported as a proportion, calculated from the cycle threshold against Actconsidered at 100% expression. Quality and stemness were assessed according to morphological characteristics such as cell size, nucleus/cytoplasm ratio, and nucleolar patterns. In addition, pluripotency markers such as alkaline phosphatase activity, Oct-4 and TROMA-1 expression were also assessed. RESULTS: Of 17 oocytes that survived cloning, 12 constructs displayed two pronuclei, 4 developed further to produce fully expanded blastocysts, and 2 produced ESC lines of good quality. On the other hand, 40 (87.5%) in vivo-derived zygotes developed into blastocysts, and once plated 7 (20.0%) produced ESC lines. ESC colonies of both origin contained at least 60% of undifferentiated cells according to morphological characteristics and molecular markers. Between 10,000 and 30,000 cells processed generated 0.3 to 0.9 g of total RNA that displayed two ribosomal subunits. Reverse transcription and qRT-PCR were successful in all specimens. As expected, Nanog was not expressed in MEFs whereas the in vivo and cloned-derived ESC cells revealed a similar 74.6% and 66.6% expression, respectively (2 ⫽ 1.19). Similarly, Oct-4 was not expressed in MEFs while the in vivo and cloned-derived ESC had an upregulation of 76.9% and 67.8%. MEFs manifested a higher expression (69.6%; P ⫽ 0.001) of Ttr (a marker of differentiation) compared to the in vivo and cloned-derived ESC colonies (40.7 and 49.2%, respectively). CONCLUSION: Results coming from a genomic indicator accord with those from other molecular markers in establishing stemness of the ESC colonies. Microarray analysis showed that clone-derived ESCs have similar expression patterns to those generated in vivo, and therefore that they may be used for stem cell therapy. Supported by: Institutional
S384
Abstracts
P-640 In Utero and Lactational Exposure to Nicotine: Ovarian Effects. M. S. Neal, J. J. Petrik, A. C. Holloway. Centre for Reproductive Care, Hamilton Health Sciences, and Reproductive Biology Division, Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada; Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada; Reproductive Biology Division, Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada. OBJECTIVE: Women born to mothers who smoked during pregnancy have been shown to have an earlier onset of menopause and a shorter reproductive lifespan (Sharpe RM and Franks S. 2002. Nat Med. Suppl: S33-40) although the mechanisms underlying this association are unknown. Nicotine administration in adult animals has adverse effects on the ovary and uterus (Patil SR et al. 1998. Indian J. Physiol. Pharmacol. 42: 503-8), however effects of fetal exposure to nicotine on the offspring’s ovary have not yet been determined. The goal of this study was to assess the effect of in utero exposure to nicotine, at concentrations that are representative of human exposure, on follicle dynamics and ovarian growth factor expression in the offspring. DESIGN: Prospective in vivo animal study in a university laboratory setting. All animal procedures were approved by the McMaster University Animal Research Ethics Board in accordance with the guidelines of the Canadian Council for Animal Care. MATERIALS AND METHODS: Nulliparous female Wistar rats (200250g) were given 1mg/kg/day nicotine bitartrate for 14 days prior to mating, during pregnancy and throughout lactation until weaning. Ovaries were removed from offspring in estrous at 120 days of age, fixed in formalin, embedded in paraffin and sectioned. Sections were stained with hemotoxylin and eosin to determine the number of follicles at different stages of development and the proportion of atretic follicles. Sections were examined using immunohistochemical staining to assess the expression of insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in granulosa cells. Data were analyzed by Student’s t-test (␣⫽0.05). RESULTS: Offspring of nicotine-exposed dams had significantly more growing follicles (p⬍0.05) and more antral follicles (p⫽0.09) than the salineexposed offspring. However, in the nicotine-exposed offspring the percentage of growing follicles that were atretic was 50% higher than in the saline-exposed offspring. There was no difference in the percentage of atretic antral follicles between the two treatment groups. Fetal and neonatal exposure to nicotine significantly (p⬍0.01) decreased the number of IGF-I immunopositive cells in the granulosa cells of the ovary. Similarly there was a significant decrease in VEGF expression in the granulosa cells in the nicotine-exposed offspring (p⬍0.001) compared to saline controls. CONCLUSION: Results from this study have demonstrated that nicotine exposure during fetal and neonatal life can alter follicle dynamics and growth factor expression in the ovary of the offspring, which may result in impaired ovarian function postnatally. Supported by: Funding for this project was provided by the Canadian Institutes of Health Research.
P-641 Role of Extracellular Matrix, Activin-A and Mitogen Activated Protein Kinase (MAPK) Signaling Pathways in Preantral Follicle Growth and Survival in the Mouse. O. Oktem, K. Oktay. Weill Medical College of Cornell University, New York, NY. OBJECTIVE: We have previously shown that ECM-Activin interactions play role in preantral follicle growth in organ cultures (1). Our current objective was to investigate the role of extracellular matrix (ECM)-activin-A interactions in in-vitro growth and survival of mouse preantral follicles under different culture conditions, and to determine mitogen activated protein kinase (MAPK) signaling pathways in preantral follicle development. MAPK family consists of c-jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK) and p38 which regulate cell growth, differentiation, and survival. DESIGN: In vitro study of isolated preantral follicles MATERIALS AND METHODS: 452 preantral follicles isolated from the ovaries of prepubertal SV129/B6 mice were cultured for 7 days with various combinations of ECM and activin-A. Follicles were cultured with or without recombinant activin-A either in wells coated with fibronectin (n⫽29),
Vol. 84, Suppl 1, September 2005