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)