MOLECULAR REPRODUCTION AND DEVELOPMENT 31:200-207 (1992) Sperm Capacitation in the Domestic Cat {Felis catus) and Leopard Cat (Felis bengalensis) As Studied With a Salt-stored Zona Pellucida Penetration Assay J.C. ANDREWS,?=^ J.G. HOWARD,? B.D. BAVISTER,^ AND D.E. WILDT? Tw^?LTnt??ol';^^^^^^^ '"^''^"'^''"' ^'^^'^^?'^^^'''^' ^^'- '^^P<^rtment ofVetennary Sconce, Uru.ersUy ABSTRACT The ability of domestic cat or leop- ard cat spermatozoa to penetrate zonae pellucidae (ZP) of salt-stored, domestic cat oocytes was examined as an assay for sperm capacitation. Ovarian oocytes were recov- ered after ovariectomy and matured in vitro for 18-36 h. Following removal of cumulus cells, the oocytes were used fresh, or stored (4?C, 0.5-24 weeks) in a HEPES-buffered hypertonic salt solution. Electroejaculated, washed sperm (2-4 X 10^ sperm/ml) were preincubated for 1.0 h (38?C, 5% CO2 in air) and then co-incubated (2 x 10^ sperm/mlj with fresh or stored oocytes for 6.0 h. Gametes were incubated in a protein-free, modified Tyrode's solution (TLP-PVA) or In the same medium containing 4.0 mg/ml bovine serum albumin (BSA; TALP-PVA). Treatments were compared for percentage ZP penetration (defined as sperm heads reaching more than halfway through the ZP) as an index of sperm capacitation. In both the domestic cat and leopard cat, there was no difference (P > 0.05) in sperm penetration of fresh ZP (domestic cat, 42.5 ? 5.4%- leopard cat, 38.6 ? 2.8%) or stored ZP (domestic cat, 32.4 ? 4.2%; leopard cat, 27.6 ? 2.3%). Sperm incubated in pro- tein-free medium (TLP-PVA) were less capable (P<0.05) of ZP penetration (domestic cat, 14.6 ? 5.9%; leopard cat, 7.9 ? 3.0%) than sperm incubated in medium TALP-PVA containing BSA (domestic cat, 60.3 ? 5.9%; leopard cat, 58.4 ? 3.0%). These data indicate that (1) albumin facili- tates capacitation and ZP penetrating ability of cat sperma- tozoa; (2) domestic cat ZP appear to lack a block to het- erospecific penetration by "foreign" (leopard cat) sperm; and (3) penetration of stored domestic cat ZP can be used as an index of sperm capacitation in the domestic cat and the leopard cat. Key Words: Protein-free medium, BSA, In vitro sperm penetration INTRODUCTION Spermatozoa must undergo capacitation (Austin, 1951; Chang, 1951) and functional (physiological) ac- rosome reactions before zona pellucida (ZP) penetration can occur (Austin and Bishop, 1958; Meizel, 1978; Yan- agimachi, 1981; Koehler, 1981; Bavister, 1986). Pene- tration of intact ZP can be used as a definitive endpoint for sperm capacitation during in vitro fertilization (IVF) (Andrews and Bavister, 1989; Bavister, 1986, ? 1992 WILEY-LISS, INC. 1990). In practice, however, this approach is limited because ZP penetration is monospermic, due to the block to polyspermic fertilization, which is predomi- nantly mediated through the cortical granule reaction (Wolf, 1981). Although IVF clearly demonstrates that capacitation has occurred, it only gives information on a single sperm per egg. Since the ratio of sperm to eggs during IVF is often 10,000-20,000:1, penetration of fresh ZP via IVF is not necessarily an efficient strategy for quantifying sperm capacitation (Bavister, 1986). The salt-stored ZP penetration assay, developed in the hamster (Yanagimachi et al., 1979; Boatman et al., 1988), rabbit (Fayrer-Hosken and Brackett, 1987), and the human (Franken et al., 1988; Yoshimatsu et al., 1988) is an attractive approach for evaluating sperm capacitation in vitro. In the hamster and the human (species in which the block to polyspermy is primarily at the level of the ZP), salt storage destroys the ability of eggs to mount the cortical reaction, so that numerous sperm can reach the perivitelline space (Yanagimachi et al., 1979; Boatman et al., 1988; Yoshimatsu et al., 1988). Salt-stored ZP also retain the ability to distin- guish between capacitated and noncapacitated sperma- tozoa (Boatman et al., 1988; Uto et al., 1988). The mean number of sperm in the perivitelline space (PVS) pro- vides a quantitative index of capacitation (Boatman et al., 1988). We have demonstrated that the sensitivity of the salt-stored ZP penetration assay was improved above IVF of living eggs by up to 63-fold in the hamster (mean number of sperm in the PVS = 63.5; Boatman et al., 1988). In addition, salt-stored ZP can be conve- niently banked for later use. Although IVF has been achieved in the domestic cat {Fehs catus) (Hamner et al., 1970), Indian desert cat {Fehs silvestris) (Pope et al., 1989), leopard cat {Fehs bengalensis) (Goodrowe et al., 1990), puma {Fehs con- color) (Miller et al., 1990), and tiger {Panthera tigris) (Donoghue et al., 1990), information on the physiology of gametes in any felid species is very limited. Studying the basic biological mechanisms underlying felid sperm Received August 21, 1991; accepted September 25, 1991 Address reprint requests to Dr. Jane C. Andrews, Department of Vet- erinary Science, University of Wisconsin-Madison, 1655 Linden Drive, Madison, WI 53706. ZONA PENETRATION TO ASSESS CAT SPERM CAP AGITATION 201 1 ? 1 fi 1 fertilizing ability is imperative for understanding the comparative similarities and differences among species and eventually for developing artificial breeding tech- niques. There are 37 felid species, all of which except the domestic cat are listed as endangered or threatened by extinction. Howard and Wildt (1990) recently demonstrated the ability of leopard cat sperm to penetrate heterologous, zona-intact domestic cat oocytes following a 1-h sperm preincubation and 3-h sperm-egg co-incubation in the presence of bovine serum albumin (BSA). This penetra- tion assay using fresh, ZP-intact oocytes was also effec- tive for determining the detrimental impact of ter- atospermia on sperm function in the domestic cat (Howard et al., 1991). The present experiments were designed to evaluate the utility of salt-stored domestic cat eggs for assessing ZP penetration by domestic cat and leopard cat sperm. The effect of BSA on capacitation of electroejaculated, washed sperm and gamete interaction was also deter- mined. In the past, this type of research has been hindered because of the need to include impure commercial prep- arations of BSA in culture media to maintain sperm motility and facilitate capacitation. As a control me- dium for the present studies, we used a chemically de- fined (protein-free) culture medium that supports sperm motility in the absence of albumin without in- ducing sperm capacitation (Bavister, 1981a; Andrews and Bavister, 1989). MATERIALS AND METHODS Animals Adult, male domestic cats and leopard cats were housed as previously described (Howard and Wildt, 1990; Howard et al., 1990). Briefly, animals were main- tained at the National Institutes of Health Animal Center (NIHAC; Poolesville, MD) and exposed to ap- proximately 12 h of natural daylight/day, 10 h of which were supplemented with artificial lighting. All animals were captive-bred, unproven breeders ranging from 4 to 13 yr of age and 2.3 to 4.2 kg in body weight. Male domestic cats were provided a dry commercial cat food (Purina Cat Chow, Ralston Purina Co., St. Louis, MO) ad libitum. Male leopard cats were fed a commercial nondomestic feline diet (Nebraska Brand Feline Diet, North Platte, NE) daily. All cats were given water ad libitum. Culture and Salt Storage Media All media and stock solutions were prepared with ultrapure water obtained by reverse osmosis followed by purification with a Milli-Q system (Millipore Corpo- ration, Bedford, MA). Water was tested for contami- nants with the hamster sperm bioassay (Bavister and Andrews, 1988). The medium for sperm preincubation and sperm-egg co-incubation consisted of a modified Tyrode's solution (TLP) containing 1 mg/ml polyvinyl alcohol (PVA, average molecular weight = 10,000; Sigma Chemical Co., St. Louis, MO) in place of BSA (TLP-PVA: Bavister, 1981b, 1989). For sperm capacita- tion, 4 mg/ml of BSA (Fraction V; Sigma Chemical Co.) was added to the medium used for sperm preincubation and sperm-egg co-incubation and was designated as TALP-PVA (Bavister, 1981b, 1989). Sodium pyruvate (P; 0.1 mM final concentration) was added to both cul- ture media immediately before use. The ovary transport and oocyte recovery medium was Dulbecco's phosphate-buffered saline (Paul, 1975), con- taining 1 mg/ml PVA (PBS-PVA), 50 |xg/ml penicillin (Sigma Chemical Co.) and 50 pig/ml streptomycin (Sigma Chemical Co.). Ovarian oocytes were matured in either Tissue Culture Medium 199 (TCM 199; Gibco Chemical Co., Grand Island, NY) containing 0.25 mM pyruvate (Sigma Chemical Co.), 10.0% heat-inacti- vated estrus cow serum (obtained from CALS experi- mental herd, UW-Madison), 10 |xg/ml follicle-stimulat- ing hormone (FSH, Vetrepharm, London, Ontario) and 10 jjig/ml gentamicin sulfate (Sigma Chemical Co.) or in modified Eagle's medium (C-MEM; Schroeder and Ep- pig, 1984) containing 0.23 mM pyruvate (Irvine Scien- tific, Santa Ana, CA), 1% heat-inactivated fetal calf serum (FCS; Gibco Chemical Co., Grand Island, NY), 3 mg/ml BSA (Fraction V; Sigma Chemical Co.), 1 |xg/ml FSH (NIADDK-oFSH-17 AFP-6446C), and 1 fig/ml luteinizing hormone (LH; NIADDK-oLH-25 AFP- 5551B). The salt storage solution consisted of 0.5 M (NH4)2S04, 0.75 M MgClg, 0.2 mM ZnClg, and 0.1 mg/ml PVA (Boatman et al, 1988), with 40 mM HEPES buffer (pH 7.4). Silicone oil (Aldrich Chemical Co, Milwaukee, WI) was extracted (Bavister, 1989) and used as an overlay of the culture media for sperm preincubation, sperm-egg co-incubation and oocyte maturation. Egg Preparation Domestic cat ovaries were obtained immediately postmortem or after ovariohysterectomy, placed in PBS-PVA, and maintained at 4?C for l-i h before pro- cessing. The ovarian follicles were punctured and aspi- rated with a pulled glass pipette tip (Unopette; VWR Scientific, Chicago, IL) attached to a 1-ml Drummond syringe (Fisher Scientific, Pittsburgh, PA) containing PBS-PVA. Oocytes with intact plasma membranes and ZP and uniform, darkly pigmented or slightly granular cytoplasm (Fig. 1) were washed 3 times in PBS-PVA and matured in equilibrated (5% CO2 in air, 38?C) mat- uration medium (as described above) for 18-36 h. After maturation, the cumulus cells were mechanically re- moved by pipetting, and the oocytes were used either immediately (fresh) or after salt storage (stored) for 0.5-24 weeks. Only high-quality oocj^es with uniform, darkly pigmented cytoplasm were used fresh. A portion of these eggs (20%) as well as eggs of suboptimal qual- ity (granular and/or mottled cytoplasm) were pooled, matured in vitro, salt-stored, and used later as stored eggs. The latter were removed from the salt solution and rinsed twice (1.0 h/rinse) in equilibrated (protein- free) TLP-PVA before insemination. 202 J.C. ANDREWS ET AL. 'Ik m ^^Ksi^*- . ^ =.'%;?,. ??^ Fig. 1. A salt-stored domestic cat egg with zona pellucida (ZP) penetrated by leopard cat spermatozoa (magnification = x740; 7.4 mm = 10 (xm). Three sperm are indicated with arrows, one in the outer (O) half and two are in the inner (I) half of the ZP. Semen Collection Spermatozoa were obtained by electroejaculation us- ing a standardized procedure (Wildt et al., 1983; Howard and Wildt, 1990; Howard et al., 1990). Briefly, males were anesthetized with tiletamine hydrochlo- ride-zolazepam (Telazol; 4.5 mg/kg im, A.H. Robbins, Richmond, VA), and a probe (1 cm in diameter x 13 cm in length; P.T. Electronics, Boring, OR) with three lon- gitudinal electrodes was inserted into the rectum. A sterile polyethylene, 5.0-ml collection vial (Nalge Co., Rochester, NY; cat. #6250-0005) was placed over the penis, and 80 electrical stimuli of 2-5 V and 20-100 mA were administered with an AC, 60-Hz sine wave stimu- lator (P.T. Electronics, Boring, OR). Sperm Preparation Semen was evaluated immediately for volume, sperm concentration, percentage motility, and forward pro- gressive motility (X250, phase-contrast microscopy; Wildt et al., 1983; Howard et al., 1990). An aliquot of each ejaculate was fixed in 1% glutaraldehyde for as- sessment of sperm morphology at x 1,000 magnifica- tion (Howard et al., 1990). Ejaculates that contained at least 4 X 10? total motile sperm with an average pro- gression rating of 3.0 (0-5, low to high) and greater than 60% structurally normal sperm/ejaculate, were transferred to a 1.5-ml conical microcentrifuge tube (Sarstedt Inc., Princeton, NJ). Each ejaculate was di- luted 1:1 with equilibrated (5% COg in air, 38?C) TLP- PVA, held at room temperature (23?C) and protected from light. The diluted semen sample was centrifuged at 300g for 8 min, and the resulting supernatant was carefully removed and discarded. The sperm concentra- tion of the pellet was determined using a hemocytome- ter. The washed spermatozoa were preincubated at 4 X lO*' motile sperm/ml for 1 h in 100-|xl drops of equil- ibrated (5% CO2 in air, 38?C) TLP-PVA or TALP-PVA overlaid with 4 ml of silicone oil in 35-mm Petri dishes (Falcon #1008). Because of a limited ejaculation vol- ume on 1 day, sperm from one domestic cat male was preincubated at a concentration of 2 x 10*^ sperm/ml. Sperm motility was evaluated at 0.0 and 7.0 h of incu- bation (i.e., after 1 h of preincubation and 6 h of sperm- egg co-incubation). In Vitro ZP Penetration Prior to sperm collection, IVF dishes (Falcon #1007) were prepared. Two drops (95 jjtl/drop) of TALP-PVA ( + BSA) and two drops of TLP-PVA (-BSA) were placed in each dish, overlaid with 10 ml of silicone oil and equilibrated at 38?C in 5% COg in air for 1.0 h before egg addition. The stored or fresh eggs from each female were kept separate (in different drops of holding medium) and distributed equally between the drops of TALP-PVA ( + BSA) and TLP-PVA (-BSA) in a 2 x 2 factorial design. Each 95-JJL1 drop of medium contained 2-14 eggs, the absolute number depending on the num- ber of acceptable eggs obtained from each female/day. Five ^.1 of the corresponding preincubated sperm sus- pension (see Sperm Preparation) from the domestic cat ZONA PENETRATION TO ASSESS CAT SPERM CAP AGITATION 203 TABLE 1. Experiment I: Experimental Design for Evaluation of the Effect of Albumin on Fresh and Salt-Stored Domestic Cat Zonae Pellucidae Penetration by Domestic Cat or Leopard Cat Spermatozoaf No. of males No. of days No. of females No. of eggs Medium BSA Type of eggs DC LC DC LC DC LC DC LC TAl.P-PVA + + FRESH STORED 2 2 3 3 3 3 3 3 5 8 5 7 48 49 57 38 Total pooled for ANOVAt: FRESH STORED 13 12 97 95 TLP-PVA 2 2 3 3 3 3 3 3 5 8 5 7 59 47 58 44 Total pooled for ANOVA*,t: 13 12 106 102 tElectroejaculated, washed domestic cat (DC) or leopard cat (LC) spermatozoa were preincubated for 1.0 h (2-4 X 10^ sperm/ml) and subsequently co-incubated with eggs for 6.0 h (2 X 10^ sperm/ml). *The ANO VA demonstrated no difference (P> 0.05) between STORED and FRESH groups, so these eggs were pooledand the effect of albumin on ZP penetration evaluated (ANOVA). (Experiment I) or leopard cat (Experiment II) was added to each IVF drop (final drop volume was 100 |jil). In one case, domestic cat sperm were preincubated at 2 X 10^ sperm/ml; therefore, 10 |j.l of sperm was added to 90-(jil IVF drops. Sperm (2 x 10^ sperm/ml) and fresh or stored domestic cat eggs were co-incubated for 6.0 h (38?C, 5.0% CO2 in air). Following co-incubation, the eggs were fixed in a 2% glutaraldehyde/2% formaldehyde solution and evalu- ated for penetration using differential interference con- trast microscopy (x400). Assessment categories in- cluded the proportion of eggs with (1) sperm heads in the outer one-half of the ZP (ZP??t); (2) sperm heads more than one halfway through the ZP (ZPj^); (3) more than one sperm head greater than halfway through the ZP (poly-ZPjn); (4) one or more sperm heads in the peri- vitelline space (PVS); and (5) more than one sperm in the PVS (poIy-PVS). In addition, the mean number of sperm in ZP^^t, or in ZPi? plus the PVS, was evaluated. Experimental Design and Statistical Analysis Each treatment set (fresh or stored ZP ? BSA) was duplicated 0^ times within a day using eggs from dif- ferent females as available (Table 1); each experiment was replicated over 3 days using sperm from one male for each day. A total of 2 domestic cat males and 3 leopard cat males was used. The stored or fresh eggs from each female were distributed equally and ran- domly among treatments (Table 1). The percent ZP penetration data were analyzed by female (n = 13, Experiment I; n = 12, Experiment II) using an analysis of variance (ANOVA) with an arcsin transformation for the 5 categories of ZP penetration. The data concerning the mean number of sperm per ZP were analyzed by female with an ANOVA and a logio transformation. The standard errors and probabilities were calculated using the type III MS as an error term. Fresh vs. stored eggs were used as the whole plot and ?BSA as the split plot. RESULTS Experiment I (Domestic Cat Sperm and Eggs) No interaction or differences between fresh and stored percentage ZP penetration were observed within the five penetration categories (P > 0.05; Table 2; ?BSA treatments remained pooled). Therefore, data for fresh and stored eggs were combined and analyzed for the effect of BSA on sperm capacitation as deter- mined by ZP penetration. Domestic cat spermatozoa incubated in the presence of BSA penetrated more (P < 0.05) ZP than sperm incubated in the absence of BSA (Table 3). A large number of spermatozoa re- mained in the outer half of the ZP in both the presence and absence of BSA, although the values were different (Table 3). When BSA was present throughout incuba- tion, 60.3% of the eggs were ZP?n penetrated, 43.0% were poly ZPj^ penetrated, 44.3% contained PVS sperm, and 28.9% were poly-PVS penetrated (Table 3). By con- trast, few spermatozoa reached the inner half of the ZP and/or the PVS in the absence of BSA (14.6% ZPi?, 9.7% poly ZPi?, 9.6% PVS, 3.5% poly-PVS; Table 3;P < 0.05) compared with the BSA-containing counterparts. When the data were analyzed for the mean number of spermatozoa with heads embedded in the ZP, no inter- action or difference (P > 0.05) between stored and fresh ZP was observed; therefore, stored and fresh ZP data were pooled and evaluated for the effect of BSA on the mean number of sperm within the ZP and PVS. The outer half of the ZP contained more (P < 0.05) sperma- tozoa when BSA was present than when it was absent, although the increase was less than twofold (Table 4). There was an approximately sixfold increase in the number of sperm penetrating more than halfway through the ZP in the presence of BSA than in its ab- sence (P < 0.05). There was no difference (P > 0.05) in the mean number of sperm in the PVS of stored (0.58 ? 0.14) vs. fresh (0.58 ? 0.11) eggs (?BSA re- mained pooled); the mean number of sperm in the PVS 204 J.C. ANDREWS ET AL. TABLE 2. Percentage Penetration of Fresh and Salt-Stored Domestic Cat Zonae Pellucidae by Domestic Cat or Leopard Cat Spermatozoaf Experiment I'''* (domestic cat) Experiment !!''?* (leopard cat) Penetration category" Fresh (X% +SE) Stored Fresh (X% ?SE) Stored ZPout ZPin Poly-ZPin PVS Poly-PVS 79.7 ? 7.3 42.5 ? 5.4 29.1 ? 5.0 30.6 ? 4.7 14.7 ? 3.4 87.8 ? 5.7 32.4 ? 4.2 23.7 ? 3.9 23.2 ? 3.8 17.7 ? 2.7 88.0 ? 3.3 38.6 ? 2.8 27.6 ? 5.0 15.6 ? 6.5 8.7 ? 5.5 95.5 ? 2.7 27.6 ? 2.3 26.2 ? 4.1 23.1 ? 5.3 21.2 ? 4.5 tElectroejaculated, washed spermatozoa were preincubated for 1.0 h (2-4 X10^ sperm/ml) and subsequently co-incubated with eggs for 6.0 h (2 X 10^ sperm/ml). * Categories include the percentage of eggs with (1) sperm heads less than halfway through ZP (ZPout); (2) sperm heads greater than halfway through ZP (ZPin); (3) more than one sperm greater than halfway into the ZP (Poly ZPin); (4) one or more sperm in the PVS (PVS); (5) more than one sperm in the PVS (Poly-PVS). ''Experiments I and II were not directly comparable because they are independent data sets collected on separate days. ?There was no difference {P > 0.05) in stored and fresh ZP penetration by domestic cat or leopard cat spermatozoa (+BSA treatments were grouped together). TABLE 3. Percentage Penetration of Domestic Cat Zonae Pellucidae by Domestic Cat or Leopard Cat Spermatozoa in the Presence and Absence of Albuminf Experiment P (domestic cat) Experiment IP (leopard cat) Penetration +BSA -BSA -fBSA -BSA category'' (X% +SE) (X% ?SE) ZPout 94.8 ? 5.2* 72.6 ? 5.2** 99.0 ? 3.3* 84.7 ? 3.3** ZPin 60.3 + 5.9* 14.6 ? 5.9** 58.4 + 3.0* 7.9 ? 3.0** Poly-ZPin 43.0 ? 5.5* 9.7 ? 5.5** 51.1 ? 4.6* 2.7 ? 4.6** PVS 44.3 ? 5.3* 9.6 ? 5.3** 35.4 + 5.4* 3.3 ? 5.4** Poly-PVS 28.9 ? 3.4* 3.5 + 3.4** 29.3 ? 4.7* 0.6 ? 4.7** t Studies I and II were not directly comparable because the data were collected on different days. * Values are means +SEM of combined fresh and stored eggs. ''See Table 2 for definitions of sperm penetration categories. Values within species and row with different superscripts *?** differ {P < 0.05). was greater (P < 0.05) in the presence of BSA (1.02 ? 0.16) than in its absence (0.14 ? 0.16) (data not shown). The variance attributable to (1) the length of time the eggs were salt-stored, or (2) the type of oocyte matura- tion protocol used (see Materials and Methods) is nested within the variance attributable to day, fresh*stored, day*fresh*stored, and female (day*fresh*stored). Since the F values indicated that there was no effect of, or interaction between, these variables (data not shown), the effect of salt-storage time, or the type of oocyte maturation, was not investigated further. Experiment II (Leopard Cat Sperm and Domestic Cat Eggs) No interaction or difference was observed in the var- ious penetration categories for fresh vs. stored ZP (Ta- ble 2); therefore, fresh and stored ZP were pooled to examine the impact of BSA on heterologous IVF. Sper- matozoa incubated in the presence of BSA penetrated more (P < 0.05) ZP in all penetration categories com- pared with sperm incubated in the absence of BSA. As observed in Experiment I, the presence or absence of BSA had little influence on the ability of leopard cat sperm to enter the outer half of the domestic cat ZP (Table 3; -^BSA = 99.0%; -BSA 84.7%). However, pen- etration of the ZPj? was greatly influenced by BSA. In the presence of albumin, 58.4% were ZPjn penetrated, 51.1% were poly 1/2 ZPjn penetrated, 35.4% contained PVS sperm, and 29.3% were poly-PVS penetrated. By contrast, less than 8.0% ZPj^ penetration occurred in the absence of albumin (Table 3). There was no difference (P > 0.05) in the mean num- ber of leopard cat sperm between stored and fresh eggs when the ZP^^ and ZP?n plus PVS penetration were evaluated. Although there was no difference {P > 0.05) in the average number of sperm in the outer half of the ZP in the ?BSA treatments, the mean number of sperm reaching more advanced stages of ZP pene- tration (>^ ZP) was enhanced by approximately 20-fold over the control (-BSA; P < 0.05) when BSA was present (Table 4). The only category in which there was ZONA PENETRATION TO ASSESS CAT SPERM CAPACITATION 205 TABLE 4. Number of Domestic Cat or Leopard Cat Sperm in Domestic Cat Zonae Pellucidae in the Presence or Absence of Albumin Penetration Experiment P'' Experiment IP'' category BSA (domestic cat) (leopard cat) ZPo ZPi, + 5.3 ? 0.3** 3.6 + 0.3* 2.6 ? 0.5* 0.4 ? 0.5*** 13.9 ? 3.1** 10.8 ? 2.9** 3.9 ? 0.4* 0.2 ? 0.4** "Studies I and II were not directly comparable because they were independent data sets collected on separate days. "Values are means ?SEM of combined fresh and stored eggs. Within a study and penetration category, values with different superscripts *?** differ (P < 0.05). a significant difference (P < 0.05) between fresh ver- sus stored eggs was in the mean number of leopard cat sperm in the PVS, in the presence of albumin (fresh, +BSA = 0.63 ? 0.44; stored, +BSA = 3.5 ? 0.39); in the absence of BSA, the mean number of sperm in the PVS of fresh and stored eggs was identical (0.04 ? 0.39 and 0.04 ? 0.39 respectively; data not shown). Since there was no effect of, or interaction between, day, fresh*stored, day*fresh*stored, and female (day*fresh*stored), the effect of salt-storage time, or the type of oocyte maturation (nested within these vari- ances), was not further investigated (data not shown). DISCUSSION Our results demonstrate that stored domestic cat ZP were as penetrable by felid spermatozoa as fresh ZP. Therefore, as found in other species (hamster: Yanagimachi et al., 1979; Boatman et al., 1988; rabbit: Fayrer-Hosken, 1987; human: Franken et al., 1988; Yoshimatsu et al., 1988), we have confirmed that the salt-stored ZP penetration assay can be used as an al- ternative to conventional IVF as a valuable indicator for domestic cat and leopard cat sperm capacitation and the acrosome reaction. When our data were evaluated on the basis of (1) five categories of sperm penetration into the ZP (Table 3), and (2) the mean number of sperm/ZP (Table 4), th? results clearly indicated that BSA facilitated domestic cat and leopard cat sperm capacitation. Approximately 6- and 20-fold, respectively, more sperm progressed to the inner half of the ZP in the presence of BSA than in its absence. By contrast, the differential between ?BSA for eggs with sperm terminating in the ZP^^t was only 1.3-1.5 and was only significant for the homologous domestic cat system. Without this protein, sperm from these two species remained relatively effi- cient at penetrating the outer layer of the ZP. However, BSA appeared to be vital for deeper ZP penetration! Taken together, our results indicate that sperm pene- tration into the outer half of the ZP may not accurately reflect sperm capacitation in the domestic cat or the leopard cat. Because the outer half of the cat ZP ap- peared more diffuse and porous than the inner half (Fig. 1), it is possible that many sperm, including those of inferior quality, become embedded in this region. Until it has been determined that all spermatozoa in the outer half of the ZP are robust and have undergone the physiological processes of sperm capacitation and the acrosome reaction (rather than sperm death), pene- tration of the inner half of the ZP should be considered as a more rigorous index of felid sperm capacitation. An obligate role for serum albumin in supporting sperm capacitation in vitro has been demonstrated in the mouse, rat, and golden hamster (Miyamoto and Chang, 1973; Hoppe and Whitten, 1974; Bavister, 1981a). In addition to its role in supporting capacita- tion, serum albumin is a potent stimulator of the ac- rosome reaction (Meizel, 1978; Bavister, 1986; Andrews and Bavister, 1989). Although numerous hypotheses have been formulated to explain the capacitation and acrosome reaction inducing properties of albumin (Davis, 1980; Davis et al., 1980; Langlais et al., 1981; Aonuma et al., 1982; Andrews and Bavister, 1989), the biochemical mechanisms underlying the facilitation of these processes remain unclear. This is due, in part, to the dual requirements for albumin in culture media for supporting sperm capacitation/acrosome reactions and for maintenance of sperm viability. In the present ex- periments, it was possible to maintain domestic cat and leopard cat sperm viability in the absence of albumin in a chemically defined (protein-free) culture medium (TLP-PVA). Since we now know that this medium fails to support sperm capacitation and/or the acrosome re- action efficiently (Tables 3 and 4), it will be possible in future studies to probe the mechanisms of cat sperm capacitation and the acrosome reaction in the absence of a ubiquitous protein such as BSA. Because salt storage destroys the block to polyspermy (Yanagimachi et al., 1979; Boatman et al., 1988), we expected the stored ZP to exhibit a greater incidence of polyspermic penetration than the fresh eggs. However, our results showed that there was no difference in the percentage polypenetration (Table 2) or in the mean number of sperm per egg between stored and fresh eggs, except in the case of leopard cat sperm PVS penetration: significantly more leopard cat sperm were in the PVS of stored eggs than in fresh eggs (see Results). This increase in PVS penetrability of stored eggs by leopard cat sperm may have been associated with the elimination of the block to polyspermy by salt storage. In contrast, Gelwicks et al. (1990) observed an increased incidence of polyspermy (as determined by penetration of the plasma membrane) when domestic cat oocytes contained intact germinal vesicles com- pared with mature ova that had undergone germinal vesicle breakdown (66% vs. 23% polyspermy, respec- tively). It also has been suggested that the optimal in vitro oocyte maturation interval is 30^8 h in the do- mestic cat (Johnston et al., 1989). Therefore, our rela- tively high frequency of polyspermy with fresh ZP may be attributable to our relatively short oocyte matura- tion interval of 18-36 h. Alternatively, the high inci- 206 J.C. ANDREWS ET AL. dence of poly-ZP penetration may be normal in the cat, since the level at which the block to polyspermy occurs is unknown. The cat may be like the rabbit (another induced ovulator), in which the block is at the level of the plasma membrane, where supernumerary sperm are often found in the perivitelline space (Braden et al., 1954; Kuzan et al., 1984). In the pig, the block to polyspermy is established in the inner region of the ZP shortly after egg activation by spermatozoa, but acces- sory sperm do penetrate the outer, more diffuse regions of the ZP of both fertilized and unfertilized eggs (Thi- bault, 1959; Hunter, 1977). This is interesting, since the bilayered pig ZP is morphologically similar to the cat ZP observed in the present study with the light microscope (Fig. 1). By contrast, polypenetration of the outer half of the cat ZP may have resulted from an excessive concentration of spermatozoa in the fertil- ization medium (2 x 10^ sperm/ml). Additional studies are warranted to determine the physiological sig- nificance of our polyspermic fresh ZP penetration data. Hamner et al. (1970) demonstrated that ejaculated domestic cat sperm require 0.5-24 h of incubation in utero before they acquire the capacity to fertilize cat ova in vitro. Bowen (1977) demonstrated that ductus deferens cat sperm could fertilize domestic cat oviduc- tal ova in vitro without incubation in the female repro- ductive fluids in vivo. More recently, Niwa et al. (1985) demonstrated that epididymal domestic cat sperm could penetrate domestic cat ZP within 20 min postin- semination, and swollen sperm heads are observed in as little as 30 min after insemination. Goodrowe et al. (1988) assessed cat ZP penetration by electroejaculated sperm and suggested that optimal domestic cat sperm capacitation is achieved within 3 h of incubation in vitro. However, unlike in the present study, the depth of sperm penetration into the ZP was not evaluated. In our pilot studies, we observed that most domestic cat sperm co-cultured with cat eggs for 3 h remained in ZPout; spermatozoa reached the PVS only after 6 h of sperm/egg co-incubation. The present studies demon- strated that domestic cat and leopard cat sperm capaci- tation (as determined by sperm penetration of ZPj^) can occur within 7 h of incubation (1 h preincubation plus 6 h sperm/egg co-incubation). However, additional studies must be conducted to determine the actual tim- ing of felid sperm capacitation both within and among species. Utilization of the salt-stored ZP penetration assay, and a protein-free culture medium that main- tains sperm viability but that does not support cat sperm capacitation efficiently (TLP-PVA), will facili- tate examination of the biological mechanisms and ki- netics of cat sperm capacitation. Our results also confirmed the data of Howard and Wildt (1990) by demonstrating that leopard cat sper- matozoa can penetrate fresh domestic cat ZP. Leopard cat spermatozoa penetrated fresh and salt-stored do- mestic cat ZP with equal efficiency. This was interest- ing because it implied that the ZP receptor on felid sperm and the ligand on the ZP may be conserved across felid species; therefore, a functional block to het- erospecific ZP penetration may not exist. Because of this nonspecificity, it may be possible to use the pene- tration of salt-stored domestic cat ZP by nondomestic cat sperm in place of conventional IVF as a tool for studying sperm function in other endangered felid taxa. Understanding the basic biological mechanisms un- derlying felid sperm fertilizing ability will aid in devel- oping assisted breeding techniques including artificial insemination, IVF, in vitro oocyte maturation, and cry- opreservation. Each of these strategies may contribute to maintaining genetic diversity and promoting species survival. ACKNOWLEDGMENTS This research was supported by a fellowship from the Friends of the National Zoo (FONZ) to J.C.A., and NIH grants RR00045 (to J.G.H.) and HD23853 (to D.E.W.), and the Department of Veterinary Science, University of Wisconsin, Madison. We are grateful to Richard W. Gneiser for photographic assistance. REFERENCES Andrews JC, Bavister BD (1989); Capacitation of hamster spermato- zoa with the divalent cation chelators D-penicillamine, L-histidine, and L-cysteine in a protein-free culture medium. Gamete Res 23:159-170. Aonuma S, Okabe M, Kishi Y, Kawaguchi M, Yamada H (1982): Capacitation inducing activity of serum albumin in fertilization of mouse ova in vitro. J Pharm Dyn 5:980-987. Austin CR (1951): Observations on the penetration of the sperm into the mammalian egg. Aust J Sei Res 4:581-596. Austin CR, Bishop MWH (1958): Role of the rodent acrosome and perforatorium in fertilization. Proc R Soc Lond (Biol) 49:241-248. Bavister BD (1981a): Bovine serum albumin is required for efficient capacitation of hamster spermatozoa in vitro. Biol Reprod 24(Suppl l):37a. Bavister BD ( 1981b): Substitution of a synthetic polymer for protein in a mammalian gamete culture system. J Exp Zool 217:45-51. Bavister BD (1986): Animal in vitro fertilization and embryo develop- ment. In Gwatkin RBL (ed): "Developmental Biology: A Compre- hensive Synthesis." Vol 4. New York: Plenum Press, pp 81-148. Bavister BD (1989): A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Res 23:139-158. Bavister BD (1990): Tests of sperm fertilizing ability. In RH Asch, JP Balmaceda, I Johnston (eds): "Gamete Physiology." Norwell, MA: Serono Symposia, pp 77?105. Bavister BD, Andrews JC (1988): A rapid sperm motility bioassay procedure to quality-control testing of water and culture media. J In Vitro F?rtil Embryo Transfer 5:67-75. Boatman DE, Andrews JC, Bavister BD (1988): A quantitative assay for capacitation: Evaluation of multiple sperm penetration through the zona pellucida of salt-stored hamster eggs. Gamete Res 19:19- 29. Bowen RA (1977): Fertilization in vitro of feline ova by spermatozoa from the ductus deferens. Biol Reprod 17:144-147. Braden AWH, Austin CR, David HA (1954): The reaction of the zona pellucida to sperm penetration. Aust J Biol Sei 7:391-409. Chang MC (1951): Fertilizing capacity of spermatozoa deposited into the Fallopian tubes. Nature 168:697-698. Davis BK (1980): Interaction of lipids with the plasma membrane of sperm cells. I. The antifertilization action of cholesterol. Arch An- drol 5:249-254. Davis BK, Byrne R, Bedigian K (1980): Studies on the mechanism of capacitation: Albumin-mediated changes in plasma membrane lip- usiv^vTsfJisso"'"''^"''" ?^^^^ '''^"" '^"'- ^?' ^^^^ ^^""^ ^" I ^?"?l*l"' ^^' i"'?"^"'" LA, Seal US, Armstrong DL, Tilson RL, Wolf ?, f, Petrmi K, Simmons LG, Gross T, Wildt DE ( 1990): In vitro fertil- I ization and embryo development in vitro and in vivo in the tiger ir (Fanthera tigns). Biol Reprod 43:733-744 I Fayrer-Hosken RA, Brackett EG (1987): Use of salt-stored zonae pel- ucidae for assessing rabbit sperm capacitation for in vitro fertiliza- :. tion. Gamete Res 17:191-201. ? Franken D, Oehninger S, Veeck L, Hodgen G (1988): The evaluation of the sperm potential of salt stored human zonae pellucidae during the hemizona assay. JAndrol9:24-P(abst 17) 3? Gelwicks EJ, Pope CE, Turner JL, Keller GL, Dresser BL (1990)- An i evaluation of meiotic stages and polyspermy in uncleaved oocytes ? 33(1^221 '" ^'^'? fertilization in domestic cats. Theriogenology I Goodrowe KL, Miller AM, Wildt DE (1990): In vitro fertilization of I. gonadotropm-stimulated leopard cat (Felis bengalensis) follicular S oocytes. J Exp Zool 252:89-95. . Goodrowe KL, Wall RJ, O'Brien SJ, Schmidt PM, Wildt DE (1988)- j. Developmental competence of domestic cat follicular oocytes after S tertilization m vitro. Biol Reprod 39:355-372 i. Hamner CE, Jennings LL, Sqjka NJ (1970): Cat (Felis catus L ) sper- ; ?atozoa require capacitation. J Reprod F?rtil 23-477^80 t Hoppe PC, Whitten WK (1974): An albumin requirement for fertiliza- tion of mouse eggs in vitro. J Reprod F?rtil 39433^36 I Howard JG, Brown JL, Bush M, Wildt DE (1990): Teratospermic and ? normospermic domestic cats: Ejaculate traits, pituitary-gonadal hormones and improvement of spermatozoa! motility and morphol- ; ogy after swim-up processing. J Androl 11:204-215 Zr^r' T^^'?^ *\^'"'^ Ejaculate-hormonal traits in the leopard cat ?ehs bengalensis) and sperm function as measured by in vitro penetration of zona-free hamster ova and zona-intact domestic cat oocytes. Mol Reprod Dev 26:163-174 """ei-uc cat "Z?romi,^"'' '^'T'f' ?! *''^''?? T--'-Pe?.a in domestic cats compromises penetration of zona-free hamster ova and cat zonae pellucidae. J Androl 12:36-45 Hunter RHF (1977): Physiological factors influencing ovulation fer- tilization, early embryonic development and establishment of preg- nancy in pigs. Br Vet J 133:461.^470 Johnston LA, O'Brien SJ, Wildt DE (1989): In vitro maturation and fertilization of domestic cat follicular oocytes. Gamete Res 24:343- Kuzan FB. Fleming AD, Seidel GE (1984): Successful fertilization in ZONA PENETRATION TO ASSESS CAT SPERM CAPACITATION 207 vitro of fresh intact oocytes by perivitelline (acrosome-reacted) sper- matozoa of the rabbit. F?rtil Steril 41-766-770 ^ Km?l?; w""?' ^' ^'^"'" ^' Chapdelaine A, Bleau G. Roberts K? (1981): Localization of cholesterol sulfate in human spermato- zoa in support of a hypothesis for the mechanism of capacitation Proc Nati Acad Sei USA 78:7266-7270. Meizel S (1978): The mammalian sperm acrosome reaction, a biochem- ^a approach^In MH Johnson (ed): "Development in Mammals." Vol 3. Amsterdam: North-Holland, pp 1-64 Miller AM, Roelke ME, Goodrowe KL, Howard JG, Wildt DE (1990)- Oocyte recovery maturation and fertilization in vitro in the puma (/? elis concolor). J Reprod F?rtil 88:249-258 Miyamoto H, Chang MC (1973): The importance of serum albumin and metabolic intermediates for capacitation of spermatozoa and fertili- zation of mouse eggs in vitro. J Reprod F?rtil 32-193-205 Niwa K, Ohara K, Hosoi Y, Iritani A (1985): Early events of in vitro 74 eSeO ^^^' ^^ epididymal spermatozoa. J Reprod F?rtil %"i '' ^n?-^'-- "^'" ^"'^ '^'^^"^ C"'*"^" 5th Ed. Edinburgh: Churchill Livingstone, p 95 ^ BI n^q'??r'^''''' ^/', ^'*^ ^^' ^^"^^ ^L, Maruska EJ, Dresser Hitn H 't r,l i" '"?-.'^??^Pe^es transfer of embryos from the In- dian desert cat (Fehs silvestris ornata) to the domestic cat (Felis catus) following in vitro fertilization. Biol Reprod 40(1)-61 (abst 40) 'SlmSlir"^ ""'''-' '- '^ RecVchjA.o2qZ- Uto N, Yoshimatsu N, Lopata A, Yanagimachi R (1988): Zona-induced WiTd^oT R'-TM"? ^^'"I*??^'P'?^'?'?''- "^ ^^P Z?ol 248:113-120. FhL H p j ^7^'^ '^^' ? ^"^" SJ, Meltzer D, Van Dyk A, Africrn^h rn' ?/ ''"''' """'"^ ^^'"'"^' ''-?'ty '" the south ^wLf ^''TJP' '"'"""""^" ^^^'^ '""?k to polyspermy. In L Mastroianni JD Biggers (eds): "Fertilization and Embiyonic Devel- opment In Vitro." New York: Plenum Press, pp 183-197 Yanagimachi R (1981): Mechanisms of fertilization in mammals. In L Mastroianni JD Biggers (eds): "Fertilization and Embryonic Devel- opment In Vitro." New York: Plenum Press, pp 81-182 GL (1979). Retention of biologic characteristics of zona pellucida in hghly concentrated salt solution: The use of salt-stored eggs for 31 562-5^74 '^"'^'''"'"^ ?^^P^^ty "f spermatozoa. F?rtil Steril Yoshimatsu N, Yanagimachi R, Lopata A (1988): Zonae pellucidae of salt-stored hamster and human eggs: Their penetrabili^ by homol- ogous and heterologous spermatozoa. Gamete Res 21:115-126.