Sunday-oral Presentations Oral Presentations

GMumps commonly affects children aged 5-9 years, often leading to mumps orchitisand resulting in permanent adult sterility in rare cases. However, the etiology of this long-termeffect remains unclear. Mumps orchitis results in progressive degeneration of seminiferousepithelium and sometimes leads to Sertoli cell-only syndrome. Thus the left Sertoli cells may becritical to spermatogenesis recovery after orchitis healing. We found that the protein prenylationalteration in Sertoli cells during childhood might determine adulthood sterility. Mumps infectionduring childhood decreases the expression of geranylgeranyl diphosphate synthase 1 (GGPPS)in human testes. When we altered farnesylation/geranylgeranylation balance by specificallydeleting GGPPS gene in Sertoli cell, Sertoli cell itself remained intact, while adjacentspermatogonium significantly decreased after the 5th postnatal day. The proinflammatorysignaling pathways MAPK and NF-ƒÛB were constitutively activated because the farnesylationof H-Ras was enhanced. GGPPS-/Sertoli cells synthesize an array of cytokines to stimulatespermatogonia apoptosis and chemokines to induce macrophage invasion into seminiferoustubules because the neonatal testes of mice had no BTB. Invaded macrophages further blockedspermatogonia development, thus resulting in a long-term effect on adult infertility. Our resultssuggest a novel mechanism by which mumps viral infection during human childhood results inadult infertility. Conclusions: In summary, inactivating GGPPS in testicular Sertoli cell resulted in sustainedactivation of H-RAS proteins and spontaneous hyper-inflammation response in the early stageof testis development when there was no BTB formation between Sertoli cells of mice. Ourresults indicated that the protein prenylation in Sertoli cell was critical to regulate ochidis beforepuberty. This phenotype can mimic the assault against Sertoli cell during human childhood likemumps infection that would result in adulthood infertility. 1569Relationship of testicular androgen receptor protein expression with in vitro fertilizabilityof epididymal sperm in mice.O. Suzuki, M. Koura, Y. Noguchi, K. Uchio-Yamada, J. Matsuda; Lab. Animal Models forHuman Diseases, Natl Inst Biomed Innovation, Ibaraki, Japan Strain differences in in vitro fertilizability still constitute a serious problem in mouse reproduction.We previously reported (Kawai et al., J Reprod Dev 52:561-568) that the in vitro fertilizability ofsperm in five strains (mean±SEM, n=5) was 31.3±4.9%, 29.5±3.2%, 47.8±3.3%, 68.9±3.8%,and 97.4±4.3% in 129X1, C57BL/6, C3H, BALB/c (n=10), and DBA/2, respectively. To elucidatethe molecular mechanism of strain difference in the in vitro fertilizability of mouse sperm, weexamined the androgen receptor (AR) protein expression in testes of the five mouse strains. MONDAY-POSTER PRESENTATIONS Testes of five males per strain at 12 weeks of age were collected. Proteins from the testes wereextracted using ReadyPrep protein extraction kit for Total Proteins (Bio-Rad). QuantitativeWestern blotting (QWB) using testicular protein extracts was conducted with anti-AR antibodies(Millipore, rabbit) and antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH,Millipore, mouse) as an internal control. Antibody-reacted bands were visualized withcorresponding peroxidase-conjugated secondary antibodies (anti-Rabbit and anti-mouse IgG;Jackson ImmunoResearch) and chemiluminescent reagent (Pierce). Band intensities werequantified after image captures by a CCD-camera (LAS-3000, Fuji Film). AR proteinexpressions were obtained as AR/GAPDH ratios and statistically compared among strains byKruskal-Wallis one way analysis of variance (ANOVA) on ranks (SigmaPlot 12, Systat software)because normality test (Shapiro-Wilk) was OK but equal equivalence test (Levene) failed. QWBshowed significant strain difference in the AR protein expression (n=5 per strain; p<0.05). Theexpressions were 0.498±0.081, 0.526±0.042, 0.834±0.078, 0.950±0.164, 1.356±0.160 in129X1, C57BL/6, C3H, BALB/c, and DBA/2, respectively (Arbitrary units; mean±SEM). Pairwisecomparisons by Tukey test indicated significant differences (p<0.05) between 129X1 andDBA/2, and between C57BL/6 and DBA/2. Linear regression analysis by ANOVA with averagevalues between AR expression and in vitro fertilizability (SigmaPlot) showed significantassociation (p=0.002) with R=0.974, suggesting that the differential AR expression in testesmight cause strain difference in the in vitro fertilizability. 1570Oocyte Growth Depends on Phosphorylation of Specific Serine Residues in the C-terminal Cytoplasmic Tail of Connexin43.P. W. Dyce, R. P. Norris, P. D. Lampe, G. M. Kidder; Physiology and Pharmacology,Western University, London, ON, Canada, Children's Health Research Institute, London, ON,Canada, Fred Hutchinson Cancer Research Center, Seattle, WA Connexin43 (CX43) forms gap junction channels that metabolically couple the granulosa cells ofdeveloping ovarian follicles. Research with knockout mice has demonstrated a requirement forthis connexin in supporting follicle and oocyte growth: CX43 null mutant follicles fail to developbeyond the primary stage on the C57BL/6 background due to failure of the granulosa cells toproliferate. We have used the CX43 null mutant mouse line and the recombinant-reaggregatedovary technique to examine the importance of specific CX43 phosphorylation sites in supportingoocyte growth. Serines at 255, 262, 279 and 282 are MAPK family substrates that becomephosphorylated in response to EGF and other growth factors, causing a decrease in couplinglevels. Mutant forms of CX43 were constructed with these serines replaced with amino acidsthat cannot be phosphorylated. These mutants, singly and in combination, were retrovirallytransduced into CX43 null granulosa cells and combined with wildtype oocytes to makerecombinant ovaries. The ovaries were grafted under the kidney capsules ofimmunocompromised female mice permitting follicle growth in vivo. Oocyte growth was retardedwhen the empty vector was introduced to CX43 null granulosa cells compared to granulosa cellsinfected with the CX43 wildtype (WT) control (35.47±1.12 μm and 61.34±1.47 μm diameterrespectively). CX43 with a single serine to alanine mutation at residue 255, or with a singleserine to aspartic acid mutation at either residue 255 or 262, were able to rescue the nullphenotype, restoring complete oocyte growth (60.43±1.69 μm, 60.96±1.22 μm, and 62.11±1.18μm respectively). In contrast, CX43 with serine to alanine mutations at either residue 279 or 282failed to permit a complete rescue, with follicle development largely limited to the earlysecondary stage and oocytes remaining significantly smaller than those in WT CX43 controls(34.84±1.20 μm and 43.49±1.74 μm respectively). Mutating all four residues from serine toalanine caused complete arrest of folliculogenesis and retardation of oocyte growth in thecontext of our system (47.05±1.63 μm). Immunofluorescence analysis revealed that the mutant MONDAY-POSTER PRESENTATIONS molecules failing to rescue folliculogenesis were largely confined to intracellular sites, with fewgap junctions visible. Using an in vitro EdU proliferation assay, we observed a decrease inproliferation in granulosa cells containing the mutated 279 and 282 construct compared to thosereceiving a non-mutated CX43 construct. These results indicate that CX43 phosphorylation isregulated by MAPK in granulosa cells and that this regulation is essential for supporting oocytegrowth. The authors acknowledge Dan Li and Kevin J. Barr for technical assistance and theCanadian Institutes of Health Research and U.S. National Institutes of Health for funding. 1571Interactions of 14-3-3 (YWHA) protein isoforms with CDC25B phosphatase in mouseoocytes.S. De, A. Reese, D. Kline; Biological Sciences, Kent State University, Kent, OH Immature mammalian oocytes are arrested at prophase I of meiosis by an inhibitoryphosphorylation on Cyclin-Dependent Kinase I (CDK1). Release from this meiotic arrest isdependent on dephosphorylation of CDK1 by M-phase inducer phosphatase 2 (CDC25B).Evidence suggests that phosphorylated CDC25B is bound to 14-3-3 (YWHA) proteins in thecytoplasm and rendered inactive. We previously reported expression of all seven mammalianisoforms of 14-3-3 in mouse oocytes and eggs. To examine the interactions of 14-3-3 isoformswith CDC25B in oocytes and eggs, we performed an in situ Proximity Ligation Assay (PLA;Duolink in cell Co-IP, Olink Bioscience) that can detect protein-protein interactions at the singlemolecule level and allows visualization of the actual intracellular sites of the interactions. Weobserved prominent interaction of all seven 14-3-3 isoforms with CDC25B throughout cytoplasmand nuclei of mouse oocytes, along with reduced interactions for each individual isoform in eggscompared to oocytes. Co-immunoprecipitation studies with extracts of mouse oocytes alsodemonstrated interaction of CDC25B with six 14-3-3 isoforms. These results suggest that any ofthe 14-3-3 isoforms may have the potential to hold CDC25B inactive in oocytes to maintain themeiotic arrest. In preliminary experiments to explore if interactions of 14-3-3 with other proteinsare important for maintaining meiosis I arrest, we microinjected oocytes with 0.5μg/μL R18, asynthetic non-isoform specific 14-3-3-blocking peptide. Injected oocytes were incubatedovernight in media containing a threshold concentration of dibutyryl cAMP (0.05mg/mL) whichnormally holds oocytes arrested through activation of Protein Kinase A (PKA) andphosphorylation of CDC25B and CDK1. We observed a marked increase in germinal vesiclebreakdown (GVBD), compared to control oocytes. To investigate which specific isoform(s) of14-3-3 is/are responsible for maintaining the meiotic arrest, we reduced the synthesis of each14-3-3 isoform in mouse oocytes by intracytoplasmic microinjection of 0.1mM translation-blocking morpholino oligonucleotide against the corresponding isoform mRNA. Injected oocyteswere held arrested at prophase I for 24 hours, and then incubated overnight in media containingthe threshold concentration of dbcAMP. GVBD was observed in 70% of oocytes microinjectedwith morpholino against 14-3-3 eta, despite the presence of dbcAMP. Injection of morpholinostargeting other 14-3-3 isoforms caused little or no GVBD. Thus, reduction in 14-3-3 eta/CDC25Binteraction releases the mouse oocyte from meiotic arrest. These results suggest that, while all14-3-3 isoforms interact with CDC25B in mouse oocytes, 14-3-3 eta is essential for maintainingthe prophase I meiotic arrest. MONDAY-POSTER PRESENTATIONS