Poster 1 Characterization and purification of sperm progesterone receptor(s) JT Wu1, PS Tsai1, HP Fung1, SL Lee2 & FP Cheng1 1 Department of Veterinary Medicine, Nation Chung-Hsing University, Taichung City, Taiwan 2 Department of Animal Science, Nation Chung-Hsing University, Taichung City, Taiwan Introduction: Little is known about the molecular and pharmacological characteristics of nongenomic sperm progesterone receptor(s) (P4R). This study demonstrated the molecular characterization of these putative nongenomic P4R. Methods: Sperm-ligand binding experiments were performed using [3H]-P4 as tracer; unlabeled P4, 11βOH-P4, 11αOH-P4, estradiol, testosterone and GABA as competitors. The topography of P4R was identified by immunofluorescent staining with different mAbs (C262, NCL-PGR, h-151) in membrane intact and permeabilized sperm. A purification scheme of nongenomic P4R proteins was developed based on ligand-receptor affinity chromatography procedures. Results and Discussion: P4 exhibited the greatest specificity to P4R as compared to others competitors. Typical scatchard plot analysis for [3H]-P4 binding to canine sperm revealed the presence of two distinct binding sites. The affinity constant and concentration of the high affinity site were 1.0+/-0.7 nmol/L and 10+/-1 million sites/cell, while the low affinity site were 732.2+/-501.8 nmol/L and 2,449+/-1,017 million sites/cell. C262 (against C-terminus of genomic P4R) and h-151 (estrogen receptor) recognized antigenic determinant over acrosomal domain of pre-capacitated membrane intact and permeabilized sperm. Both mAbs failed to recognize non-capacitated sperm, regardless of membrane permeabilization. NCL-PGR (N-terminus of genomic P4R) recognized acrosomal domain of membrane permeabilized sperm, regardless of capacitation. Only mAb C262 and h151 had significant inhibition on P4-induced acrosome reaction (AR). The 74 and 63 kDa of boar membrane proteins from elution fractions of P4-immobilized affinity chromatography were recognized by C262. Sequencing of 74 kDa (undiscovered?) protein showed a significant identity in the amino acid sequence (XPXNIVLIFADDLGY) to residues 2-15 of arylsulfatase A (92 %) and to residues 2-10 of a 72 kDa (88 %) protein from boar sperm membrane. However, neither of above proteins has an identical sequence recognized by C262. Present results suggest sperm and cytosol P4R may share at least in part the structural homology but differ in topography and exposures of P4R are associated with sperm capacitation and induction of AR. (Supported by NSC 89-2313-B-005-165; 90-2313-B-005-114, Taiwan) Poster 2 The fate of cryo-preserved progesterone receptor(s) of canine sperm and its subsequent response to ligand stimulation FP Cheng1, JT Wu1, PS Tsai1, CLT Chang1 & SL Lee2 1 Department of Veterinary Medicine, Nation Chung-Hsing University, Taichung City, Taiwan 2 Department of Animal Science, Nation Chung-Hsing University, Taichung City, Taiwan Introduction: Cryo-modification of membrane progesterone receptor (P4R) has not been investigated to explore its functional change. This study was designed to demonstrate the freezing effect on sperm P4R at molecular level. Methods: To explore the functional and conformational changes of P4R, pre-incubated (2-hours in CTM medium, 38℃ fresh and frozen-thawed (Rota et al., 1997) sperm of fertile dogs (n=6) were treated with 10 ug/ml P4 to induce the acrosome reaction (AR). To detect the molecular evidence of P4R and its pattern changes after cryo-modification, western blot analysis of digitonin-treated sperm with antibodies directed against different domains of the genomic P4R (mAb C262, NCL-PGR) and estrogen receptor (h-151) was conducted. To determine the binding sites of [3H]-progesterone, fresh and frozen-thaw sperm were identically processed as described by Ambhaikar and Puri (1998). Results and Discussion: The percentage of live AR (EthD-1 & FITC-PNA staining) in fresh sperm was 9.6+/-5.1% (pre-incubated) and 31.0+/-6.7% (P4 induced) while in frozen-thawed sperm, 10.4+/-2.7% (pre-incubated) and 21.6+/-4.1% (P4 induced) (P<0.05) was detected. The percentage of P4R staining (P4-BSA-FITC) over acrosomal region was 18.3+/-10.3% (fresh) and 36.0+/-11.9% (pre-incubated) (P<0.05) while it was barely visualized in frozen-thaw sperm regardless of pre-incubation. The protein mass recognized by all mAbs was approximately 54 kDa as the major and 65 kDa as the minor band. Cryo-process resulted in alternation of protein pattern and undetectable P4R protein mass of 65 kDa in sperm preparation of four dogs (n=6). Digitonin treated fresh and frozen-thaw sperm, labeled with [3H]-progesterone, revealed the P4 binding capacity decreased from 6.0+/-4.4 to 3.0+/-2.1 nM (n=6, P<0.05).These results indicate the reduced AR incidence in response to P4 is evident in frozen spermatozoa and suggestive to be due to the conformational/degraded changes of P4R and/or reduced P4R density derived from freezing injury. (Supported by the NSC 89-2313-B-005-054; 89-2313-B-005-165; 90-2313-B-005-114, Taiwan) Poster 3 Effect of different extenders on post-thaw sperm survival, acrosomal integrity and longevity in cryopreserved semen of Formosan Sika and Formosan Sambar deer FP Cheng1, JT Wu1, JPW Chan 1, JS Wang1, B Colenbrander2 & KC Tung1 1 Department of Veterinary medicine, National Chung-Hsing University, Taichung, Taiwan 2 Department of Farm Animal Health, Utrecht University, Utrecht, The Netherlands Introduction: Formosan Sika (FSK) and Sambar (FSM) deer are classified to be endangered Taiwan specific sub-species. This study investigated the efficiency of five potential extenders used in FSK and FSM to establish semen cryobank. Methods: Pooled semen (n = 4) of 6 ejaculates from each species was tested for efficiency of extender A, B (Dog: Rota et al., 1997), C (Eld’s deer: Monfort et al., 1993), D (Fallow deer: Mulley et al., 1988) and E (Fallow deer: Jabbour et al., 1993), frozen at a designed program (Rota et al., 1997). Results and Discussion: FSK: Post-thaw motility was A: 66 +/- 16 %; B: 71 +/- 2 %; C: 73 +/- 6 %; D: 9 +/- 4 % and E: 26 +/- 12 % while C (74 +/- 14 %) preserved more viable sperm (Sybr-14/PI staining) than others (A: 64 +/- 10 %; B: 48 +/- 11 %; D: 41 +/- 16 %; E: 47 +/6 %). Five extenders showed the equivalence capacity of acrosome protection (Fitc-PNA staining). Decline of longevity (vigorous motility in 4 h incubation) wasn’t observed in all extenders. FSM: B (74 +/- 6 %) and C (73 +/- 2 %) were superior of all extenders (A: 69 +/2 %; D: 13 +/- 6 %; E: 31 +/- 20 %) with respect to post-thaw motility. Similarly, A (70 +/- 7 %), B (76 +/- 7 %) and C (79 +/- 2 %) preserved more viable sperm than D (25 +/- 19 %) and E (29 +/- 17 %). Sperm preserved in B (86 +/- 4 %) and C (83 +/- 4 %) showed more intact acrosome than those in A (54 +/- 13 %), D (39 +/- 22 %) and E (46 +/- 22 %). Longevity in A dropped by nearly 54 % from the onset whereas only slight decrease in others (P < 0.05). Conclusively, post-thaw sperm species-specific characters revealed egg-yolk-tris-tes-glycerol based C extender containing Equex is the optimal for freezing FSK and FSM semen. (Supported by the ROC Deer Farmer Association)