n~po~ twenty-s i hth rnnurl conference - the Society for Reproductive ...

CHARACTERIZATION OF IN VITRO SYNTHESIZED PROTEINS SECRETED BYPORCINE NON-ATTACHED OVIDUCTAL EPITHELIAL CELLSPHERES TREATED WITH ESTROGEN.Ping Xia, Jean Rutledge, Andrew J. Watson and David T. AnnstrongDept. of Obstetrics and GynaecoIogy, The University of Western Ontario, London, ON,Canada N6A 5A5INTRODUCTIONRecent studies by Buhi et a1. (1992) have demonstrated thatestrogen is responsible for induction ofde novo synthesis andsecretion of certain oviductal secretory proteins (OSP) andinhibition of other OSP in porcine oviductal explant cultures. Inthe baboon (Boice 1990), sheep (Gandolfi 1991), hamster (Kan1988) and mouse (Kapur 1988), E:2-dependent oviductalglycoproteins contribute to the extracellular matrix ofthe embryoby associating with the zona pellucida and to the exclusivemicroenvironment of the embryo by entering the perivitellinespace. Studies in our laboratory have shown that E:2-treatedoviductal epithelial cells facilitate early porcine embryonicdevelopment in vitro (Xia and Armstrong, 1994). The presentwork was undertaken to detect in vitro synthesis of secretoryproteins by ~-treated porcine oviductal epithelial cells used forcoculture with early embryos.MATERIALS AND METHODSEpithelial cell sheets were squeezed from ampullae of oviductsand cultured in TCM 199 with 10% FCS with or without E 2 (1~g/ml) for 48 hr, after which the oviductal cell aggregatesformed epithelial cell spheres (ECS). The ECSs (40 ECSs) werecultured in 200 ~I MEM without methionine containing 5 mg/mlglucose, 500 ~Ci/ml L)SS-Methionine and with or without E:2 (1~g/ml) under paraffin oil for 6 hr. The culture medium wasdialysed against 10 mM Tris-HCI buffer, pH 8.2, and deionizedwater. Dialysed culture medium (10,000 or 50,000 cpm) wasanalyzed by both ID- and 2D-SDS-PAGE.RESULTSThe results from ID-SDS-PAGE showed that the in vitrosynthetic pattern of secretory proteins by ECS was altered by E 2treatment. These alterations included the appearance of onepolypeptide (33,000 Mr) and the disappearance of a secondpolypeptide (82,000 Mr) in the E:2-treated group, compared to the200-200-116-97-116-97- 66-82~tV 45-ctVC ~~45-31-33~31- 21-I I I2 3 4 7.6 7.0 6.0Figure 1. ID-SDS-PAGE.patterns observed for the controls. Additional proteins ofapproximate Mr 45,000 and 97,000 were increased in abundanceby E:2 treatment (Figure 1). Upon analyses by 2D-SDS-PAGE ofECS culture medium, the Mr 97,000 protein band was resolvedinto two acidic proteins of PI 4.5 and PI 5.1. The PI 5.1 proteinwas clearly suppressed in the ~-treated group (arrow 7) whereasthe PI 4.5 protein was increased by E:2 treatment (arrow 8). TheMr 45,000 protein complex in the ~-treated group was resolvedinto three acidic proteins, Mr 45,000 and 43,000, both with PI 5.5(arrow 4,5) and a Mr 36,000-45,000 protein complex with PI 4.8(arrow 6). Two basic proteins with PI 8.0 (Mr 36,000) (arrow 1)and PI 6.8 (Mr 25,000) (arrow 3) were both inhibited by E:2treatment. Another protein (Mr 25,000, PI 7.6) was increased inthe E:2-treated group (arrow 2).CONCLUSIONIn vitro synthetic patterns of secretory proteins by porcineoviductal epithelial cells used for embryonic coculture areinfluenced by E:2 treatment.REFERENCESBuhi WC et aI., 1992 J Exp ZooI262:426-435.Boice ML et aI., 1990 Bioi Reprod 43:340-346.Gandolfi C et aI., 1991 Eur J Basic Appl Histochem 35:383-392.Kan FWK, (1988) J Histochem Cytochem 36:1441-1447.Kapur RP and Johnson LV (1988) Anat Rec 221:720-729.Xia P and Armstrong DT (1994) Therigenology 41 :340.Figure 1: 1. ECS conditioned medium, control.2. E:2-treated ECS conditioned medium.3. ECS, control.4. E:2-treated ECS.Figure 2: A: ECS conditioned medium, control.B: E:2-treated ECS conditioned medium.Figure 2. 2D-SDS-PAGE11'1 A2 3I I I L5.1 4.5 7.6 7.0pHI6.0IBI5.1 4.5-CRYOPRESERVATION OF HUMAN SPERM WITH PENTOXIFYLLINEMonash IVF, 185 RoddIe Street, Richmond, Victoria 3121INTRODUCTIONAddition of 3mM pentoxifylline (PF), a cyclicadenosine 3'-5'-monophosphate (cAl\.1P) phosphodiesteraseinhibitor, to cryoprotectant has been shownto increase recovery of motile sperm post-thaw (1).This improvement may be due to (i) increasedintracellular cAl\.1P levels and direct stimulation ofmotility or (ii) PF acting as an antioxidant therebyreducing membrane damage associated with thefr~eze-thaw process. We have previously shown thatmotility and normal acrosome morphology (AM) aresignificantly reduced by cryopreservation and thatdecreases in normal AM may be due to cell death or tosubletllal damage including membrane disruption (2).The aim of this study was to investigate the effect ofPF on sperm during cryopreservation and to assess itspotential usefulness in improving the recovery offunctional sperm post-thaw.MATERIALS AND METHODSSemen samples from donors (n=15) with normalsemen characteristics (WHO, 1992) were assessed forprogressive and total motility and AM. Motility wasassessed on a warm-stage-equipped phase contrastmicroscope. AM was assessed by labelling ethanolfixed sperm with fluorescein-conjugated concanavalinA lectin (2). Samples were diluted 1: 1 with HumanSperm Preservation Medium (3) and divided into 4aliquots to which PF was added to the following finalconcentrations; 0 (control), 1, 3 and 10mM. Thesealiquots were mixed, aspirated into O.5m1 straws,frozen in liquid N 2 vapour and stored in liquid N 2 •Samples were thawed at 25°C for 10 minutes for reassessmentofmotility and AM which were reported asa percentage oftheir initial values (mean±sem).RESULTSPost-thaw progressive motility was found to beincreased in the presence of all concentrations of PFcompared to control (Fig. I). However, this increasewas only significant (p