Conference Theme: The Intersection Between Genetics, Genomics, and Reproductive BiologypostprintThe 43rd Annual Meeting of the Society for the Study of Reproduction (SSR), Milwaukee, WI., 30 July-3 August 2010. In Meeting Abstracts, 2010, p. 38, abstract no. 17

Chiu, PCN

Lee, KF

Wong, BST

Yeung, WSB

Biology of Reproduction

William S.B. Yeung

Philip C.N. Chiu

Kai-Fai Lee

Ben S.T. Wong

Crossref

Glycodelin-A Primes Zona Pellucida-Induced Acrosome Reaction of Human Spermatozoa via Downregulation of Extracellular Signal Regulated Kinases and Enhancement of Zona Pellucida-Induced Calcium Influx.

postprintThe 43rd Annual Meeting of the Society for the Study of Reproduction (SSR), Milwaukee, WI., 30 July-3 August 2010. In Meeting Abstracts, 2010, p. 38, abstract no. 17

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TitleGlycodelin-A primes zona pellucida-induced acrosome reactionof human spermatozoa via downregulation of extracellular signalregulated kinases and enhancement of zona pellucida-inducedcalcium influxAuthor(s) Yeung, WSB; Chiu, PCN; Lee, KF; Wong, BSTCitationThe 43rd Annual Meeting of the Society for the Study ofReproduction (SSR), Milwaukee, WI., 30 July-3 August 2010. InMeeting Abstracts, 2010, p. 38, abstract no. 173Issued Date 2010URL http://hdl.handle.net/10722/129900Rights Creative Commons: Attribution 3.0 Hong Kong Licensenursing after 48 h from birth did not restore uterine MMP9 or ESR1 signals at PND14 to levels observed for colostrum-fed gilts. Zymographic analyses indicated anincrease (P,0.05) in uterine MMP9 gelatinolytic activity in animals that nursed frombirth to PND 14 in comparison to gilts that were fed replacer over the same period.Overall, results indicate that normal induction of porcine uterine MMP9 and ESR1requires ingestion of colostrum during the first 48 h of life. Data support the idea thatmaternally-driven lactocrine signaling for two days from birth may be essential toestablish an optimal developmental program in neonatal uterine tissues. Deprivingneonates of such lactocrine-acting factors could alter the developmental trajectory ofuterine tissues with negative reproductive consequences in adulthood. (SupportUSDA-NRI 2003-35203-1357 and 2007-35203-18098; NSF-EPS 0814103)170. Transforming Growth Factor b Receptor Type 1 Is Essential forFemale Reproductive Tract Development and Function. Qinglei Li, Luz M.Martinez, Julio E. Agno, and Martin M. Matzuk. Baylor College of Medicine,Houston, TX, USAThe transforming growth factor b (TGFb) superfamily, the largest family ofgrowth factors in mammals, plays key roles in numerous biological processes. TGFbligands bind to their type 1 and type 2 receptors and activate intracellular SMADtransducers to initiate signal transduction. Although recent studies have achievedtremendous insights into this growth factor family in female reproduction, thefunctions of the receptors in vivo remain poorly defined, partially due to receptorredundancy or lethal phenotypes of genetically engineered ubiquitous null mousemodels. TGFb type 1 receptor (TGFBR1), also known as activin receptor-like kinase5 (ALK5), is the type 1 receptor for TGFb ligands. Tgfbr1 null mice dieembryonically, precluding functional characterization of TGFBR1 postnatally. Tostudy TGFBR1-mediated signaling in female reproduction, we generated a mousemodel with conditional knockout (cKO) of the TGFBR1 in the female reproductivetract using anti-Mullerian hormone receptor type 2 (Amhr2) promoter-driven Cre-recombinase. We found that Tgfbr1 cKO females are sterile during a 6-month fertilitytest. To uncover the causes of the sterility, we performed histological, functional, andmolecular analyses of the female reproductive tract. Strikingly, we discovered that theTgfbr1 cKO females developed oviductal diverticuli which phenocopy the cKO ofDICER1, the RNase III that processes microRNAs in the cytoplasm. These findingssuggest that the microRNA pathway is derailed in the oviduct in the absence ofTGFBR1. Further studies demonstrated that embryo development and transit to theuterus are severely compromised in the Tgfbr1 cKO mice due to the formation of theoviductal diverticuli. Thus, we identified essential roles of TGFb family signalingthrough TGFBR1 in female reproductive tract development and function. Furtherunderstanding of the regulatory significance of TGFBR1-mediated signaling in thefemale reproductive tract may help to discover novel therapeutic approaches forinfertility treatment. Supported National Institutes of Health Grant HD33438.171. Estrogen Stimulates De Novo Synthesis of Cholesterol in MouseUterus During Implantation. Yuechao Zhao, Quanxi Li, Indrani C. Bagchi, andMilan K. Bagchi. University of Illinois at Champaign-Urbana, Urbana, IL, USAIn mice, the coordinated actions of estrogen (E) and progesterone (P) via theirrespective nuclear receptors lead to successful establishment of embryo implantation.It is known that ovarian E stimulates the P-primed uterus to undergo receptivetransformation to initiate the attachment of blastocyst to luminal epithelium.Subsequently, uterine stromal cells undergo a unique differentiation process, knownas decidualization. However, the molecular mechanisms underlying the actions of Eduring implantation remain unclear. In this study, we sought to identify the E-inducedgene networks that operate in uterine stromal cells during implantation. Ovariecto-mized mice were treated with a well-established regimen of E and P that mimics thehormonal sequence prior to implantation and creates a receptive uterus. The globalgene expression profiling was performed using stromal cells isolated from P-primeduterus treated with or without E. We identified several hundred genes whoseexpression was significantly altered in response to E. We grouped differentiallyexpressed genes into biological pathways using Ingenuity Pathway Analysis.Strikingly, we found that the expression of 18 genes associated with cholesterolbiosynthesis was significantly up regulated in stromal cells upon administration of Eto P-primed uterus. These genes included several key enzymes that participate in thecholesterol biosynthesis as well as the sterol regulatory element-binding protein 2(Srebp2), which is the master regulator that controls the expression of these enzymes.To explore the potential role of Srebp2 in implantation, we monitored its uterineexpression during early pregnancy using immunohistochemistry. The Srebp2 proteinwas induced in uterine stromal cells on day 4 of pregnancy, and its expression wasfurther enhanced in differentiating stromal cells at days 5-7 of gestation, suggestingthat regulation of cholesterol biosynthesis is potentially critical for the decidualizationprocess. To investigate this possibility, we utilized an in vitro decidulization systemin which primary undifferentiated stromal cells, isolated from mouse uterus prior toimplantation, were allowed to undergo differentiation in the presence of E and P. Weobserved that the expression of mRNAs encoding Srebp2 and several cholesterolbiosynthesis-related enzymes was markedly elevated during the in vitro differenti-ation. Importantly, the level of endogenous cholesterol was markedly increased instromal cells undergoing decidualization. Furthermore, the treatment of these cellswith inhibitors of cholesterol biosynthesis resulted in the down-regulation of decidualbiomarkers, such as decidual prolactin-related protein and connexin 43, indicatingimpairment in uterine stromal cell differentiation. Collectively, these studies uncovera unique pathway in which E stimulates the de novo biosynthesis of cholesterol bypromoting Srebp2 expression. It is conceivable that the cholesterol synthesized withinthe pregnant uterus acts as the precursor of local production of hormonal signalscritical for stromal decidualization during early pregnancy.172. Protein Phosphatase-Type 2A (PP2A) Is Involved in the InitialEvents of Human Sperm Capacitation. Patricio Morales, Janetti R. Signorelli, andEmilce S. Diaz. University of Antofagasta, Antofagasta, ChileDuring mammalian sperm capacitation, an increase in protein phosphorylation intyrosine and serine/threonine residues has been described. The role of protein kinases,including tyrosine kinases and PKA, in this process is well documented. However, littleis known about the role of protein phosphatases in this event. PP2A is serine threoninephosphatase whose activity has been detected in human and primate sperm extracts. Itsrole during sperm capacitation is not known. The aim of this work was to study theinvolvement of PP2A in the regulation of human sperm capacitation. To accomplishthis, human sperm samples, obtained from normal donors according to the WHOguidelines, were selected by a Percoll gradient and then resuspended in a non-capacitating medium (modified Tyrode medium without BSA and bicarbonate). ThepH was 7.4 and the osmolarity was adjusted between 280 and 300 mmol/kg.Immediately thereafter, some sperm aliquots were incubated at 378C and 5% CO2 asfollow: a) in non-capacitating medium plus 90 nM endothal; b) in capacitating medium(2.6% BSA and 25 mM bicarbonate); c) in capacitating medium plus 90 nM endothal;d) capacitating medium plus inhibitor solvent. At different periods (0, 15, 30, 60 and300 min), sperm aliquots were withdrawn to evaluate the percent of capacitated spermusing the chlortetracycline fluorescence assay. The results indicate that incubation withendothal very rapidly increased the percent of capacitated sperm. Similar results wereobserved using 1 nM okadaic acid. The effect of the inhibitors took place only whenthe sperm were incubated in capacitating medium. The increase in sperm capacitationwas especially impressive during the first half hour of incubation; thereafter, there wasno difference between sperm incubated in capacitating medium plus the inhibitorsversus sperm incubated in capacitating medium without the inhibitors. In addition, theeffect of endothal was evidenced by an increase in protein phosphorylation in threonineresidues by mean of western blot. Using a polyclonal rabbit antibody the presence ofPP2A was confirmed in human sperm. These results suggest that PP2A may have animportant role in regulating the initial events of the human sperm capacitation process.This research was supported by Fondecyt Project 1080028.173. Glycodelin-A Primes Zona Pellucida-Induced Acrosome Reactionof Human Spermatozoa via Downregulation of Extracellular Signal RegulatedKinases and Enhancement of Zona Pellucida-Induced Calcium Influx.William S.B. Yeung, Philip C.N. Chiu, Kai-Fai Lee, and Ben S.T. Wong. TheUniversity of Hong Kong, Hong Kong, ChinaSpermatozoa interact with different isoforms of glycodelin during their passagetowards the oocytes in the female reproductive tract. The only known action ofglycodelin-A, one of the glycodelin isoforms, is inhibition of spermatozoa-zonapellucida binding, an action paradoxical to general belief that the maternal bodyshould promote fertilization. Therefore, we studied other actions of glycodelin-A onspermatozoa. The objective of this report was to investigate the mechanism of actionof glycodelin-A on zona pellucida-induced acrosome reaction, a crucial step in thefertilization process. Glycodelin-A did not affect spontaneous acrosome reaction. Pre-treatment of human spermatozoa with glycodelin-A enhanced zona pellucida-inducedacrosome reaction, i.e. the increase in the percentage of acrosome-reacted spermatozoaafter sequential glycodelin-A and zona pellucida treatment was significantly higherthan the sum of treatments with glycodelin-A and zona pellucida alone. Native humanzona pellucida protein-3 (ZP3) and ZP4 were purified. Glycodelin-A primed ZP3-, butnot ZP4-induced acrosome reaction. Other glycodelin isoforms and deglycosylatedglycodelin-A did not have such priming activity. Glycodelin-A treatment increasedcAMP level and protein kinase-A (PKA) activity, but decreased that of extracellularsignal-regulated kinase (ERK). Treatment with inhibitors of ERK simulated thepriming activity of glycodelin-A. Calcium influx is an important event in zonapellucida-induced acrosome reaction. Therefore, the action of glycodelin-A oncalcium influx was studied by fluorescence imaging of individual spermatozoon usingFluo-4AM dye. Compared with the untreated control, glycodelin-A induced a strongerand more rapid calcium influx in spermatozoa upon exposure to solubilized zonapellucida. In conclusion, glycodelin-A sensitizes human spermatozoa for zonapellucida-induced acrosome reaction through activation of the cAMP/PKA-ERKsignaling leading to enhanced zona pellucida-induced calcium influx. (This work issupported in part by a research grant from the University of Hong Kong.)174. STIM1 Is Required for Normal Fertilization in the Pig. Kiho Lee,Chunmin Wang, John M. Chaille, and Zoltan Machaty. Purdue University, WestLafayette, IN, USAThe oscillation in the intracellular free Ca2+ and the depletion of the intracellularstores stimulates an influx of extracellular Ca2+. This Ca2+ influx is responsible forsustaining the long-lasting Ca2+ oscillation. A previous report from our lab indicatesthat STIM1, a known Ca2+ sensor in somatic cells is expressed in porcine oocytesand is essential for store-operated Ca2+ entry. Because a Ca2+ entry through theplasma membrane is critical for the maintenance of Ca2+ oscillation duringfertilization, we hypothesized that STIM1 function has implications for subsequentembryo development. In this study, the inactivation of STIM1 in oocytes and itseffect on early embryo development after fertilization was investigated using the pigas a model. Gilt ovaries were obtained at a local abattoir and immature oocytes werecollected by aspirating mid-size follicles. The oocytes were matured in vitro in aTCM199-based medium. First, matured oocytes (34 hours after the beginning ofABSTRACTS38

Glycodelin-A primes zona pellucida-induced acrosome reaction of human spermatozoa via downregulation of extracellular signal regulated kinases and enhancement of zona pellucida-induced calcium influx

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Glycodelin-A primes zona pellucida-induced acrosome reaction of human spermatozoa via downregulation of extracellular signal regulated kinases and enhancement of zona pellucida-induced calcium influx

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