Mass spectroscopy analysis of the human follicular fluid proteome

P-141 Tuesday, October 23, 2012 MASS SPECTROSCOPY ANALYSIS OF THE HUMAN FOLLICULAR FLUID PROTEOME. A. M. Zamah,a M. Hassis,b K. Williams.b a Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco Medical Center, San Francisco, CA; bObstetrics, Gynecology and Reproductive Sciences, University of California San Francisco Medical Center, San Francisco, CA. OBJECTIVE: The maturing ovarian follicle provides a unique microenvironment where endocrine, paracrine and autocrine signaling is required for normal follicular function. A proteomic approach to comprehensively assess the soluble proteins present in human follicular fluid at various stages of follicle maturation may offer new insights into signaling pathways involved in follicular health and development, or may reveal novel biomarkers of follicular maturation. Here, we investigated in an unbiased fashion the proteome of human follicular fluid obtained during either the follicular phase or peri-ovulatory phase. DESIGN: Mass Spectroscopy analysis of human follicular fluid. MATERIALS AND METHODS: Follicular fluid was obtained from consented patients undergoing IVF treatment. Samples were depleted of abundant serum proteins by MARS-14 column, and samples trypsinized for analysis. Liquid Chromatography Mass Spectometry was performed on a Velos Orbitrap mass spectrometer. Comparison of follicular phase and peri-ovulatory follicular fluid was performed by i-TRAQ labeling. Data were analyzed by two bioinformatics database search engines with integrated peak picking. RESULTS: Analysis of pooled post-hCG follicular fluid revealed a total of approximately 1000 unique proteins present across all samples, with each peptide fragment identified with P<0.05 identification confidence. iTRAQ labeling studies comparing pre-hCG and post-hCG follicular fluid revealed 10 proteins which were significantly changed in abundance after hCG administration. Significant intra-individual differences were noted in protein levels for many of the identified peptides within either comparison group. CONCLUSION: Sensitive mass spectroscopy may be useful in identifying proteins involved in follicular signaling events during follicular development and the peri-ovulatory time period. The abundance of serum proteins present in follicular fluid is a limitation for this technology. Supported by: NIH grant K12 HD001262-12 to AMZ. P-142 Tuesday, October 23, 2012 PROTEASOME DYNAMICS DURING OOCYTE MATURATION AND THEIR RELATIONSHIP WITH CYTOPLASMIC M. Baronio,a F. Nodar,a MORPHOLOGY. C. Alvarez Sedo,a a c b a I. Mancisidor, C. Wojcik, V. Rawe. CEGYR (Centro de Estudios en Ginecologıa y Reproducci on), Capital Federal, Buenos Aires, Argentina; b Reprotec, Capital Federal, Buenos Aires, Argentina; cIndiana University School of Medicine, Evansville, IN. OBJECTIVE: Explore the distribution and gene expression of proteasomes (P) and poly-ubiquitinated proteins (Pubp) at different human oocyte maturation stages and during inhibition of proteasome functions with MG132 and Bortezomib (Velcade), and explore their relationship with the formation of cytoplasmic granules (CG). DESIGN: Experimental. MATERIALS AND METHODS: A total of 593 human oocytes were included. Oocytes were incubated with MG132 (24hrs) and Bortezomib (4hrs) under in vitro maturation conditions (IVM Controls¼culture media+vehicle). After culture, oocytes were fixed and evaluated to identify P and Pubp using monoclonal antibodies at different oocyte stages. qRT-PCR was performed to assess gene expression of genes related to proteasome activity (PSMB5, RPT2, HERP, and HSP70). Ultrastructure was evaluated by TEM. RESULTS: At GV oocytes, P were immunodetected inside the nucleus; at GVBD and MII, P presented a cytoplasmic distribution and markedly related to MII plate. Pubp had a cytoplasmic distribution in all maturation stages. After incubation with proteasomes inhibitors, 15% depicted a meiosis interruption at GV stage, 75-80% at GVBD stage, and only 5-10% progressed to MII. All of the oocytes that reached MII stage depicted the formation of CG. Gene expression analysis showed an increase of PSMB5 and RPT2 during GV GVBD transition. MII oocytes with CG presented a co-localization and a major expression of P and Pubp at granules area, moreover, visualization by electron microscopy depicted that granules were composed by a dense population of mitochondria that surround specific areas of endoplasmic reticulum. CONCLUSION: Proteasome dynamics during human oocyte maturation, suggest that they have an important role in nuclear and cytoplasmic events. Proteasome disruption seems to affect oocyte meiosis and protein degradation, inducing the formation of CG. We propose that granulated oocytes pres-

FERTILITY & STERILITYÒ

ent an altered competence that might be reflect of proteasome dynamics abnormalities. Supported by: CEGyR Foundation. P-143 Tuesday, October 23, 2012 INHIBITION OF FATTY ACID OXIDATION (FAO) DURING IN VITRO MATURATION ALTERS EXPRESSION OF GLYCOLYTIC, FAO AND REDOX GENES IN MURINE AND BOVINE OOCYTES. M. Paczkowski, R. Krisher, W. Schoolcraft. National Foundation for Fertility Research, Lone Tree, CO. OBJECTIVE: To evaluate the effect of inhibition of fatty acid oxidation (FAO) during in vitro murine (few intracellular lipids) and bovine (moderate intracellular lipids) oocyte maturation on expression of glycolytic, FAO and redox genes. DESIGN: Research study. MATERIALS AND METHODS: Cumulus-oocyte-complexes (COC) were collected from B6D2F1 females 48h post PMSG. Bovine COC were obtained from abattoir derived ovaries. A defined oocyte maturation medium was supplemented with 0 mM, 25 or 100 mM (bovine) or 100 or 250 mM etomoxir (murine), a FAO inhibitor. Oocytes were collected for analysis following maturation (murine 18h, bovine 24h), and frozen in 4 groups of 10 oocytes in lysis buffer. Transcript abundance was analyzed by quantitative PCR, relative to 18s rRNA. RESULTS: There were no significant differences in murine oocyte gene expression after maturation with 0, 100 or 250 mM etomoxir. Treatment with 100 mM etomoxir tended (P<0.1) to down regulate CPT2 and up regulate HK2, while 250 mM tended to down regulate CPT1B. Bovine oocytes significantly decreased expression of CPT1B, GLRX2, and HK2 when supplemented with 25 and 100 mM etomoxir, while TXNRD1 tended to be up regulated. At 100 mM etomoxir, ACADL also tended to be down regulated. We hypothesize that when FAO is inhibited during bovine oocyte maturation, glucose metabolism is up regulated and glucose is depleted from the maturation medium, resulting in down regulation of glycolytic mechanisms. CONCLUSION: The amount of intracellular lipids present in the oocyte appears to correspond to reliance on FAO for oocyte maturation. In the mouse, gene expression was not significantly altered when FAO was blocked, although there is suggestion of an increased reliance on glucose metabolism. In the bovine, significant changes in expression of genes controlling FA and glucose metabolism, and redox balance, were observed.

P-144 Tuesday, October 23, 2012 EFFECT OF MII ARREST ON IN VITRO-MATURED HUMAN GV OOCYTES. M. J. Escriba,a N. Grau,a L. Escrich,a A. Belen,a R. Jose,a M. Messeguer.a aIVI Valencia, Valencia, Spain; bIVI Valencia, Valencia, Spain. OBJECTIVE: To study different periods of MII arrest in order to determine the optimal period for maximizing the normal oocyte activation (NA) response. DESIGN: We designed a study in which IVM oocytes were parthenogenetically activated in order to study differences in activation response between E-IVM (extruded the 1st polar body (PB) at or before23.3 hrs) and L-IVM oocytes(after 23.3 hrs). MATERIALS AND METHODS: 95 GVoocytes recovered from stimulated cycles underwent IVM in a time-lapse incubator, with 88 of them reaching MII (92.5%). Retrospectively, we defined 62 E-IVM and 26 L-IVM. All MII oocytes were parthenogenetically activated with A23184 (8mM, 5 min) and puromicin (10g/Ml,5hrs). Following activation, eggs were cultured in a time-lapse incubator. Signs of activation (presence of both PB and/or at least one pronucleus (PN)) and normal activation (parthenotes with two PB and a single PN) were then assessed. All statistical analyses were performed using SPSS 17.0. RESULTS: E- and L-IVM oocytes had comparable activation rates (91.9%); however, more E-IVM oocytes displayed normal activation than L-IVM oocytes (61.3% vs 34.6% P¼0.015). E-IVM oocytes (n¼62) had a higher activation rate when they were arrested at MII for 4.3 hours than those arrested for a longer period (100% vs 83.9%; P¼0.02). However, all E-IVM oocytes displayed a NA response at a comparable rate, regardless of the period of arrest (average 61.3%). On the other hand, L-IVM oocytes (n¼26) became activated and exhibited a NA response at comparable rates, regardless of their MII aging (80.8% and 34.6%, respectively). CONCLUSION: IVM human oocytes do not require an additional period of MII arrest for the assembly and normal functioning of the meiotic spindle

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