Conference Theme: The Intersection Between Genetics, Genomics, and Reproductive BiologyThe endocrine disruptor (ED) is an exogenous substance that acts on the endocrine system and modulates normal physiological functions of the body. Although EDs such as 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) affect normal reproductive function in humans and affects the growth and reproductive functions in rodents, the underlying mechanism that modulates these changes remains unclear. Accumulating evidence suggested preimplantation embryo development is controlled by ...postprintThe 43rd Annual Meeting of the Society for the Study of Reproduction (SSR), Milwaukee, WI., 30 July-3 August 2010. In Biology of Reproduction, 2010, v. 83 Meeting abstracts, p. 62, abstract no. 27

Cheung, TY

Kodithuwakku, SP

Lee, KF

Liu, WM

Tsang, H

Wong, CKC

Yeung, WSB

Biology of Reproduction

Kai-Fai Lee

Wei-Min Liu

Hilda Tsang

Tsz-Yan Cheung

Suranga P. Kodithuwakku

William S.B. Yeung

Chris K.C. Wong

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The Endocrine Disruptor TCDD Modulates microRNA Expression in Preimplantation Mouse Embryos and Spheroids Attachment on Human Endometrial Epithelial Cells In Vitro.

The endocrine disruptor (ED) is an exogenous substance that acts on the endocrine system and modulates normal physiological functions of the body. Although EDs such as 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) affect normal reproductive function in humans and affects the growth and reproductive functions in rodents, the underlying mechanism that modulates these changes remains unclear. Accumulating evidence suggested preimplantation embryo development is controlled by ...postprintThe 43rd Annual Meeting of the Society for the Study of Reproduction (SSR), Milwaukee, WI., 30 July-3 August 2010. In Biology of Reproduction, 2010, v. 83 Meeting abstracts, p. 62, abstract no. 27

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TitleThe endocrine disruptor TCDD modulates microRNA expressionin preimplantation mouse embryos and spheroids attachment onhuman endometrial epithelial cells in vitroAuthor(s) Lee, KF; Liu, WM; Tsang, H; Cheung, TY; Kodithuwakku, SP;Yeung, WSB; Wong, CKCCitationThe 43rd Annual Meeting of the Society for the Study ofReproduction (SSR), Milwaukee, WI., 30 July-3 August 2010. InBiology of Reproduction, 2010, v. 83 Meeting abstracts, p. 62,abstract no. 278Issued Date 2010URL http://hdl.handle.net/10722/140981Rights Creative Commons: Attribution 3.0 Hong Kong Licensemeiotic spindle) if those oocytes were exposed in an wrong window of exposure.Therefore, it can be expected that, a chronic exposure of woman to environmentalestrogens may affect spindle formation and chromosome segregation in vivo as well.Our hypothesis is that the in vitro oocyte maturation system could contribute for anintegrated testing strategy (ITS) for screening chemicals that affects female fertility.Furthermore, by using ovaries from slaughterhouse waste, it could also contribute toreduce the number of experimental animals being used for those studies. We haveselected compounds with a known mechanism of action (i.e. cycloheximide,estradiol, DES, cadmium chloride) to be tested in this system. As the first step toinvestigate this proof of principle, we cultured bovine oocytes, harvested fromslaughterhouse ovaries, in a M199 media at 398C during 22 h , under 5%CO2 and inthe presence of the following concentrations of cycloheximide (CHX),a knownprotein synthesis inhibitor: 0.75; 0.60; 0.45; 0.30; 0.15 and 0 lM. The toxicologicalendpoint was the ability of the oocyte to reach the metaphase II stage (i.e. the finalstage of maturation). In addition, we investigated the general cytotoxicity of CHX byusing MTT-test. Our results show no general cytotoxicity for CHX in all of theconcentration used. The meiosis was inhibited in a dose-response manner and theEC50 for CHX was 0.55lM, much lower than the concentration used to block proteinsynthesis in somatic cells,indicating the high sensivity of the oocyte to expresstoxicity. The following step is to extend this investigation to the other selectedcompounds and to combine functional parameters with the use of proteomics andtranscriptomics to obtain mechanistic insights in oocyte maturation and the effect ofchemicals hereon. It is expected that markers derived, along with functionalparameters, can form a powerful assay for the in vitro prediction of reproductivetoxicity. In conclusion, we do believe that the use of the IVM assay can grant apowerful and physiological model in an ITS of effects of chemicals on femalefertility. Research supported by TNO Research Program on Hazardous Substancesand Occupational Safety; and The Netherlands Toxicogenomics Center.275. Calbindin-D9k Expression in GH3 Cells Is a Biomarker ofXenoestrogenic Potential of Parabens. Thuy T.B. Vo and Eui-Bae Jeung.Chungbuk National University, Cheongju, Republic of KoreaThe potential adverse effects of parabens were reported in both in vivo and invitro systems, but the molecular mechanism(s) and long-term consequences ofparabens exposure are largely unknown. In this study, we further examined theinduction of an estrogenic biomarker gene-calbindin-D9k (CaBP-9k) to investigatethe estrogenic activity of parabens (methyl-, ethyl-, propyl-, isopropyl-, butyl-, andisobutylparabens) in the rat pituitary GH3 cell line. Following 24 h exposure,significant increases in CaBP-9k transcript and protein were observed depending onthe concentration treated and the linear length of the alkyl chain from methyl- toisobutyl parabens, whereas co- treatment with fulvestrant, a pure antiestrogen, largelyreversed the paraben-induced expressions of CaBP-9k mRNA and protein in GH3cell line. To better understand the mechanism(s) of CaBP-9k induction by theseendocrine disrupting compounds, we measured the levels of estrogen receptor(ERalpha) and progesterone receptor (PR) expression following parabens exposure.In the GH3 cells, a great increase in PR mRNA and protein was observed in aconcentration-dependent manner after parabens treatment. Paraben-induced expres-sion of CaBP-9k was effectively blocked in the presence of antagonist of 17beta-estradiol (fluvestrant). To confirm whether progesterone receptor signaling isinvolved in parabens derived induction of CaBP-9k mRNA and protein, we treatedGH3 cells with antiprogesterone (mifepristone). In the study, parabens-induced up-regulation of CaBP-9k was completely reversed by mifepristone. Taken together,these results indicate that CaBP-9k may be induced by parabens via the PR-involvedpathway in addition to ERa pathway in GH3 cell line.276. Potential Estrogenic Effect(s) of Parabens at the Neonatal Stage ofan Immature Female Rat Mode. Kyung-Chul Choi and Eui-Bae Jeung. ChungbukNational University, Cheongju, Republic of KoreaThis study was performed to examine the estrogenic effects of parabens onhormonal responsiveness and on the morphology of reproductive tissues during acritical developmental stage of female rats. Two hundred immature female Sprague-Dawley rats (n = 10/group) were orally treated with methyl-, ethyl-, propyl-,isopropyl-, butyl-, and isobutylparaben from postnatal day 21 to 40 in a dose-dependent manner based on our previous study [62.5, 250, and 1000 mg/kg of bodyweight (BW) per day]. A high dose of methyl- and isopropyl paraben (1000 mg/kg ofBW per day) resulted in a significant delay in the date of vaginal opening and adecrease in length of the estrous cycle. In measurements of organ weight and bodyweight, we observed significant weight changes (i.e., decresed in ovaries, kidneys andincreased in adrenal glands, thyroid glands, liver) conversely, body weight was notaltered following paraben treatment. In all groups exposed to paraben treatment,histological analysis of the ovaries from the immature rats revealed interstitial celldisorders, decreased corpora lutea, an increase in the number of cystic follicles, andthinning of the follicular epithelium, which occurred in a dose-dependent manner. Inaddition, morphological study of the uterus revealed the myometrial dysplasia such asmyometrial hyperplasia in the highest dose of propyl- and isopropyl paraben and inall dose of butyl- and isobutyl parabens. We also observed a significant decrease inserum estradiol and T4 concentrations in methyl-, ethyl-, propyl-, isopropyl-, andisobutylparaben-treated groups (P , 0.01 and 0.05). Taken together, long-termexposure to parabens can produce suppressive effects on hormonal responsivenessand can disrupt the morphology of reproductive target tissues during this critical stageof development in immature female rats.277. Evidence That Mammary Gland Infection/Injury During Preg-nancy in Dairy Cows May Have a Negative Impact on Size of the OvarianReserve in Their Daughters. James J. Ireland, Danielle M. Scheetz, FerminJimenez-Krassel, Joseph K. Folger, George W. Smith, Francesca Mossa, andAlexander C.O. Evans. Michigan State University, East Lansing, MI, USA;University College Dublin, Dublin, IrelandThe effect of disease on maternal environment, which has a crucial role inembryo development and subsequent health of offspring, has not been examinedextensively in cattle. Therefore, the purpose of this study was to determine if apersistent mammary gland infection such as mastitis and correspondingly achronically high (. 200,000 units) somatic cell count (SCC) in milk of pregnantdairy cows has a potential negative impact on size of the ovarian reserve (totalnumber of healthy follicles and oocytes in ovaries) and potentially fertility of theiroffspring. This study was conducted at Green Meadow Farms Inc. in Elsie, MI andused 192, 12-mo-old Holstein heifers. Each animal was injected with 2 injections ofprostaglandin F2 alpha (PG) spaced 11 d apart. To assess relative size of the ovarianreserve in each heifer, a single blood sample was removed 4 d after the 2nd PGinjection and assayed for concentration of anti-Mu¨llerian hormone (AMH), andovarian ultrasonography was used to determine number of follicles  3 mm indiameter and ovary size. Results confirm previous observations that AMHconcentrations were highly variable among animals but positively correlated (P ,0.001) with both ovary size and number of antral follicles. To determine if a chronicmammary gland infection during pregnancy of dairy cows was associated withalterations in AMH concentrations and correspondingly size of the ovarian reserve intheir offspring, we next tested whether number of SCC measurements . 200,000 foreach heifer’s dam was associated with alterations in the serum AMH concentration ineach heifer. Number of SCC measurements . 200,000 per individual cow is anaccurate index of recurrent udder infections, injury and/or inflammation. A total of 5to 7 measurements of SCC were made for each dairy cow beginning 2 mo beforepregnancy and continuing throughout pregnancy. Number of SCC measurements .200,000 ranged from 0 to 5 per individual cows, thus cows were separated intogroups based on their number of SCC measurements . 200,000. Cows with 4 or 5SCC measurements . 200,000 were ~2 years older (P, 0.01, 6.1 vs 4.4 yrs old) andtended (P , 0.09) to produce less milk compared with cows that had 0 to 3 SCCmeasurements . 200,000. Results showed that the daughters of cows with 4 and 5SCC measurements . 200,000 also had markedly lower (P , 0.02) AMHconcentrations as young adults compared with daughters of the cows with 0 to 3 SCCmeasurements . 200,000. Although mechanisms are unclear, our results imply that achronic mammary infection or injury during pregnancy of older cows (as predicted bya high number of SCC measurements . 200,000) is associated not only with lowmilk production, but also with diminished size of the ovarian reserve and perhapsreduced reproductive performance (fertility and reproductive lifespan) of theirdaughters. Research supported by USDA-NRI 2004-01697, 2007-01289 to JJI.278. The Endocrine Disruptor TCDD Modulates microRNA Expres-sion in Preimplantation Mouse Embryos and Spheroids Attachment on HumanEndometrial Epithelial Cells In Vitro. Kai-Fai Lee, Wei-Min Liu, Hilda Tsang,Tsz-Yan Cheung, Suranga P. Kodithuwakku, William S.B. Yeung, and Chris K.C.Wong. The Unviversity of Hong Kong, Hong Kong, China; Hong Kong BaptistUniversity, Hong Kong, ChinaThe endocrine disruptor (ED) is an exogenous substance that acts on theendocrine system and modulates normal physiological functions of the body.Although EDs such as 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) affect normalreproductive function in humans and affects the growth and reproductive functions inrodents, the underlying mechanism that modulates these changes remains unclear.Accumulating evidence suggested preimplantation embryo development is controlledby concerted expression of microRNAs (miRNA) that regulate mRNA stability andprotein translation. We hypothesized that EDs affect pre-implantation embryodevelopment by modulating miRNA expression, as well as attachment (an initial stepon implantation process) of embryo onto endometrial epithelium. Treatment of mousezygote with 10nM TCDD did not affect the blastulation rate of mouse embryodeveloped in vitro. Yet, analysis of the expression of 238-plex miRNA by RT-PCR atthe blastocyst stage revealed 9 and 11, up- and down-regulated miRNA (.2-foldchanges; p,0.05) between the TCDD-treated and control embryos. RT-PCRconfirmed the differential expression on some of the selected miRNA includingmiR-133a and 199a expression in single blastocyst. Computation analysis (picTar)identified putative implantation-related targets for these miRNAs including cadherin,integrin and GSK3beta. An in vitro choriocarcinoma (Jeg-3 and BeWo) andendometrial epithelial (Ishikawa and RL95-2) cells co-culture system was establishedand used to study the effect of TCDD on spheroids attachment. The receptor ofTCDD, aryl hydrocarbon receptor (AhR), as well as estrogen receptor (ERalpha)alpha and (ERbeta) beta were detected in all the cell lines. TCDD dose-dependently(1pM - 10nM, for 24 hours) induced CYP1A1 (a biomarker gene for TCDDexposure) transcript and protein expression in these cell lines. In addition, TCDDdose-dependently suppressed attachment of BeWo spheroid onto the RL95-2monolayer (from 82% to 53%, n=6). Yet, TCDD has no effect on cell proliferation.Our results are the first demonstrating that ED modulates the miRNA expression ofpreimplantation embryos, and may in turn affect the normal expression of genes thatare important for embryo attachment and implantation in vitro. This research wassupported in part by a RGC Collaborative Research fund (CRF) Group ResearchProject (HKBU 1/CRF/08).ABSTRACTS62

The endocrine disruptor TCDD modulates microRNA expression in preimplantation mouse embryos and spheroids attachment on human endometrial epithelial cells in vitro

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The endocrine disruptor TCDD modulates microRNA expression in preimplantation mouse embryos and spheroids attachment on human endometrial epithelial cells in vitro

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