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Generation of an equine oviductal epithelial cell line for the study of sperm-oviduct interactions
ELSEVIER
GENERATION OF AN EQUINE OVIDUCTAL EPITHELIAL CELL LINE FOR THE STUDY OF SPERM-OVIDUCT INTERACTIONS I. Dobrinski,“a
J.R. Jacob,2 B.C. Tentrant* and B.A. Ball3
‘Center for Animal Transgenesis and Germ Cell Research, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348 2 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University Ithaca, NY 14850 3Department of Population Health and Reproduction, School of Veterinary Medicine University of California, Davis, CA 95616 Received for publication: Accepted:
30 March 1999 1 July
1999
ABSTRACT Equine oviductal epithelial cells (OEC) were transformed with simian virus 40 large T antigen (SV 40 T-ag) to create a cell line for the study of the interaction of equine spermatozoa with oviductal epithelium. One cell line was established based on the expression of the SV 40 Tag and extended lifespan in culture. Immortalized equine OEC retained the characteristics of differentiated OEC such as the formation of monolayers with characteristic epithelial morphology and cell polarization as well as expression of cytokeratin and equine major histocompatibility complex I. Monolayers of immortalized equine OEC retained their functional competence to bind equine spermatozoa in a dose-dependent manner comparable to that of primary equine OEC cultures. This immortalized cell line of equine OEC provides a uniform, readily available system for sperm-OEC co-cultures, and may be a useful model for the study of sperm-oviduct interactions in the horse. 0 1999 by Elsevier
Science
Inc.
Key words: SV40, oviduct, sperm binding, horse Acknowledgments We thank Dr. J. Lopez for performing the preliminary cell transformation experiments and Dr. D.F. Antczak for the antibodies against equine MHC I and II. This study was supported by the Harry M. Zweig Memorial Fund for Equine Research. I. Dobrinski was a recipient of a Graduate Research Assistantship awarded by the College of Veterinary Medicine, Cornell University. a Correspondence and reprint requests: Dr. I. Dobrinski, Center for Animal Transgenesis and Germ Cell Research, 147 Myrin Bldg., New Bolton Center, University of Pennsylvania, 382 W. Street Rd., Kennett Square, PA 19348.
Theriopnology 52:8?&885,1999 Q 1999 by Ekevier Science
Inc.
0093-691 X/99/$+%x% front matter PII SOO93-691 X(99)00179-X
876 INTRODUCTION The mammalian oviduct is the site of sperm storage and capacitation (1, 18, 30, 3 l), fertilization and early embryonic development. Contact of spermatozoa with OEC maintains viability of spermatozoa stored in the oviduct (3 1) and prolongs their ability to fertilize (26,32). Storage of spermatozoa in the oviduct may be particularly important in the horse, in which fertilization can occur several days after breeding (11). To study the interaction of spermatozoa and OEC in the horse, in vitro systems using tissue explants of oviductal epithelium (22, 35) or monolayers of OEC (12, 36) have been described. A cytofluorescent assay was developed to more accurately quantify adhesion of equine spermatozoa to OEC monolayers (34). Equine spermatozoa preferentially bind to epithelium recovered from the oviductal isthmus of mares in the preovulatory phase of the estrous cycle (35). Recovery of fresh oviductal epithelial tissue from estrous mares for explant cultures or establishment of monolayer cultures is costly, and the use of OEC from different mares within or between experiments introduces a largely undefined source of variation. Age-dependent differences in oviductal function and fertility have been described in mares (7). Establishment of an equine oviductal epithelial cell line would provide a more uniform, readily available system for the study of sperm-oviduct interactions and, potentially, for embryo co-culture in the horse. A spontaneous cell line derived from porcine oviductal epithelium has been described (6). Human epithelial cells from different organs have been experimentally transformed (8) using human papilloma virus or simian virus 40 (SV40). Transformation of equine fibroblasts with bovine papilloma virus (38) and transformation of equine skin cells with type-C sarcoma viruses (29) has been described. Transformation of equine oviductal epithelial cells, however, has not been reported. The objective of this study was to generate a cell line by transformation of equine oviductal epithelial cells with the SV 40 large T antigen (SV 40 T-ag) and to investigate its potential use for the study of sperm-oviduct interactions in the horse. MATERIALS
AND METHODS
Immortalization of Oviductal Epithelial Cells Unless otherwise mentioned, all reagents were supplied by Sigma Chemical Company (St. Louis, MO). Monolayers of OEC were derived from the oviductal isthmus of mares in the preovulatory stage of the cycle (37). Briefly, oviducts were opened longitudinally, and epithelial cells were mechanically dissociated from the underlying stroma. Epithelial cells were cultured in 50% Dulbecco’s Modified Eagle’s Medium and 50% Ham’s F- 12 (50:50 DMEM:F- 12) with 10% fetal bovine serum (FBS; Hyclone, Langhorne, PA), 10 ng/mL epidermal growth factor, 5 pg/mL insulin, 5 pg/mL transferrin, 5 ng/mL selenium, 50 IUmL penicillin, and 50 pg/mL streptomycin. Monolayers were grown to confluence, then trypsinized and plated into 6-well plates at a density of 75,000 viable cells/ml. Cell viability was determined by exclusion of trypan blue. All cultures were incubated at 38S”C in 5% CO;! in air. Cells were immortalized as described previously (19). Briefly, the pSV3neo plasmid vector (ATCC 37150, American Type
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Culture Collection, Rockville, MD), expressing the SV40 T-ag and neomycin resistance genes, was transfected into OEC 24 h after plating using liposome fusion techniques (Lipofectin,R Gibco Life Technologies Inc., Grand Island, NY). Cell cultures were maintained in selective medium containing 50 pg/mL G418 (Geneticin,R Gibco) for >l mo until proliferative cell foci were observed. Individual colonies were isolated and passaged with trypsin/EDTA solution. Two clones (EOC##2 and EOC#8) maintained to the 8th passage after selection were expanded and passaged at a density of 1 x lo6 cells/25 cm* flask, every 6 d. Primary OEC from 6 mares of unknown breeding history were utilized in separate experiments for these studies. Characterization of Transformed Cells Expression of SV40 large T-antigen. Primary OEC cultures and immortalized cell lines were analyzed by immunoblot procedure to detect the SV40 T-ag as previously described ( 19). Cell monolayers were extracted, then proteins were separated by 10% SDS-PAGE (21) and transferred electrophoretically to PVDF membrane (Immobilon-P; Millipore, Bedford, MA). Immunologic detection of the SV40 T-ag was performed with monoclonal antibody Pab4 19 (Oncogene Science Inc., Uniondale, NY), followed by a goat antibody to mouse IgG conjugated to horse radish peroxidase (Cappel, Organon Teknika, Durham, NC). Membranes were saturated in luminescent substrate (ECL, Amersham Life Sciences, Arlington Heights, IL), followed by chemiluminescent detection of signal on X-ray film (XAR-5, Kodak, Rochester, NY). Immunoblots were scanned and processed for printing (Adobe Photoshop 3.0, Adobe Systems Inc., Mountain View, CA). Extended lifespan. Cells from clones EOC#2 and EOC#8 were passaged every 6th day by trypsinization and plating at 5 x lo5 to lo6 cells/25 cm2 culture flasks. At various passages, cells were frozen in 10% dimethylsulfoxide and 50% FBS (13). If poor growth was observed, cells were plated at higher density (2 x lo6 cells/25 cm2 culture flasks). Evaluation of cell morphology. Cell morphology was examined by phase-contrast and Hoffman Modulation Contrast (Modulation Optics Inc., Greenvale, NY) microscopy, and digital images wire recorded (34). For examination of ultrastructure, cells grown to confluence in 25 mL culture flasks and on Thermanox coverslips (Electron Microscopy Sciences, Fort Washington, PA) were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 30 min at room temperature and 1.5 h at O’C. Cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS; Gibco), scraped from the culture flasks and pelleted by centrifugation. The cells grown on coverslips were processed in situ. Cells were postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated and embedded in Epon. Ultrathin sections stained with lead citrate/uranyl acetate were then examined by transmission electron microscopy. hnmunocvtochemistrv. To confirm the epithelial nature and the equine origin of the transformed OEC, the ceils were grown to confluence in slide chamber wells (Lab-Tek 4chamber slides; Nunc Inc., Naperville, IL). Cells were processed for immunocytochemical detection of cytokeratin expression (37) with the modification that a monoclonal anti-Pan cytokeratin FITC conjugate (Sigma) was used at a dilution of 1:25. The cells were then examined with epifluorescence (420-490 nm) microscopy.
Theriogenology For immunocytochemical detection of equine major histocompatibility complex I (MHC I) as a marker for equine cells, monolayers were fixed in situ in 95% ethanol for 10 min, blocked with 10% normal goat serum, and incubated with monoclonal antibodies directed against equine MHC I and II (10,20). A monoclonal antibody directed against canine parvovirus served as a negative control. Specific antibody binding was detected using a peroxidase-coupled goat-anti mouse second antibody and aminoethyl carbazol (ACE) reagent, and cells were counterstained with hematoxylin (Histostain-SP Kit, Zymed Laboratories Inc., South San Francisco, CA). Chromosome counts. Cells growing in culture for 3 d were arrested in metaphase by incubation with 200 ng/mL colcemid (Demecolcine; Sigma) for 2 h. Cells were trypsinized, incubated in 0.075 M KC12 for 30 min and fixed (3 parts methanol : 1 part galcial acetic acid) as previously described (15; modified). Cells were spread on cooled, precleaned glass microscope slides, and chromosome spreads were examined (x 400). Images of complete spreads were digitized (NM Image Analysis software; version 1.59; 28), and chromosome numbers were counted in 80 individual spreads. Fluorescent Sperm Binding Assay To evaluate the functional capability of the transformed oviductal epithelial cells to bind equine spermatozoa, confluent monolayers of transformed cells were grown in 24-well culture plates (Sarsted Inc., Newton, NC) and used for a cytofluorescent sperm binding assay as described previously (34). Briefly, semen (2 ejaculates from each of 3 stallions on a regular semen collection schedule) was collected with an artificial vagina, diluted to 10’ sperm/ml in modified Tyrode’s solution (25) and transported to the laboratory. Spermatozoa were loaded with the fluorescent nuclear dye Hoechst 33342 (5 pg/mL), washed by centrifugation (300 x g, 6 min) and resuspended in culture medium. Confluent monolayers were inseminated with 500 pL culture medium containing 4 different concentrations of spermatozoa: 5 x 105, 106, 2.5 x lo6 and 5 x lo6 cells/well. Triplicate wells were inseminated for each concentration. Co-cultures were incubated at 38.5’C in 5% CO2 in air for 30 min, and supematant containing unbound spermatozoa was removed. The co-cultures were then rinsed twice with 500 pL culture medium/well, and the medium was replaced. After equilibration for3 min, the cultures were examined on an inverted Nikon Diaphot-TMD microscope, fluorescent videoimages of spermatozoa were digitized, and the numbers of attached spermatozoa were determined (34). Statistical Analysis Characterization of SV40 T-ag expression, cell morphology, immunocytochemistry and chromosomal numbers were observational. In the sperm binding assay, a complete randomized block design was used, with inseminate concentrations applied within ejaculates and the data blocked by stallion for analysis of variance (ANOVA), using Linear Models of Statistix (Analytical Software, Tallahassee, FL). Data were log-transformed to obtain a Gaussian distribution. The dependent variable was the number of bound spermatozoa. Data were examined for the effect of ejaculate-within-stallion, treatment, replicate and their interactions. Pairwise
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comparisons of means were made using Tukey’s honestly significant difference (HSD). The relationship between the log of number of spermatozoa inseminated and the number of spermatozoa bound was described by linear regression. RESULTS Immortalization of Oviductal Epithelial Cells Oviductal epithelial cells isolated from 6 mares were transfected in separate experiments with the SV40 T-ag. Several foci were observed after selection; however, only 2 clones expanded upon passage. Cell line EOC#K?was passaged on plastic 12 times but could not be passaged further. Cell line EOC#8 grew efficiently to Passage 12, reaching confluence within 4 to 5 d. After passage 12, the cells grew at a reduced rate and did not form a confluent monolayer after 6 d in culture. Large multinuclear cells were observed more frequently at this time. By plating at a higher density (2 x lo6 viable cells/25 cm’flask), cell survival and proliferation appeared to improve, and cultures reached confluence by day 6. Cells were maintained until Passage 29, at which time they were frozen and stored in liquid nitrogen. Only cells from the EOW8 line were found to express the SV40 T-ag by immunoblot analysis (Figure 1). Therefore, all subsequent
kD 120= 90= 705040Figure 1. One-dimensional immunoblot of cell lysates from primary and transfected equine OEC incubated with monoclonal antibody Pab419 against SV40 T-antigen followed by horseradish peroxidase conjugated goat-anti mouse IgG. Lane 1: primary OEC; Lane 2: transfected cell line EOCM; Lane 3: transfected cell line EOC#8. Arrow indicates Tantigen at 97 kD.
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characterizations were performed with cells from this clone. Cells grown to confluence showed the characteristic, rounded, polygonal appearance of epithelial cell monolayers in the majority of cells (Figure 2).
Figure 2. Hoffman modulation contrast micrograph of oviductal epithelial cell line EOC#8 (passage 14) at day 6 of culture. Bar = SOpm. Occasionally, some large multinucleated cells were observed. On ultrastructural examination, cilia were absent but microvilli were apparent at the luminal cell surface (Figure 3). Immunocytochemistry revealed that the transformed cells at passage 14 expressed cytokeratin as expected for epithelial cells, and that all cells expressed equine MHC I, but not equine MHC II antigens, confirming their origin from equine tissue (data not shown). Chromosome counts showed that the cells were highly aneuploid. Only 19% of cells examined had the normal diploid number of chromosome, 11% had a haploid number of chromosomes, and 6% a tetraploid number of chromosomes. The remaining spreads had chromosome numbers ranging from 13 to more than 140 chromosomes.
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Figure 3. Transmissionelectronmicrographof ultra thin sectionof equineoviductal epithelial cell line EOC#8 (Passage14)monolayer.Bar = 2pm. In the fluorescentspermbinding assay,the transformedoviductal epithelialcells bound spermatozoain a dose-dependent mannerwith a log-linearrelationshipbetweenthe numberof spermatozoainseminatedand the numberof spermatozoabound (P
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n Stallion
1
q Stallion
2
Cd Stallion
3
ef
0.5 Spermatozoa
1
I
2.5 loaded/well
5 (xl 06)
Figure4. Numbersof equinespermatozoaboundto monolayersof oviductal epithelialcell line EOC#8 (meansGEM) for different stallionsandinseminateconcentrations(a,b,c,d,e,f: P
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Based on immunocytochemistry for cytokeratin and equine MHC I, the immortalized OEC were confirmed to be of equine origin and of epithelial nature. The immortalized OEC were aneuploid, as described for human epithelial cells transformed in vitro (9), and aneuploidy has been used as a marker for successful transformation (8). The cells maintained characteristics of primary equine OEC cultures in that they remained anchorage-dependent and formed confluent monolayers with the characteristic, rounded, polygonal appearance of cultured epithelial cells (37). There was ultrastructural evidence of microvilli on the apical cell surface. Cilia were absent from the immortalized epithelial cells. Disappearance of cilia from cultured OEC was reported to occur already after the first passage (2,37). Maintenance of different degrees of differentiation and cell function is commonly observed in cells transformed with SV40 (9, 14,27, 39). Immortalized equine OEC maintained normal morphology and expressed both cytokeratin and MHC I. More importantly, the equine oviductal epithelial cell line also retained the ability to bind equine spermatozoa in a dosedependent manner and to detect differences between individual stallions in sperm attachment to OEC (34). Comparison of absolute numbers of spermatozoa bound per area of monolayer between primary and immortalized OEC, however, is not meaningful because spermatozoa from different stallions were used in our present study and in the study reported by Thomas and Ball (34). A porcine oviductal epithelial cell line has been described earlier (4,6). The cell line was derived spontaneously from the epithelium of an entire pig oviduct and underwent a morphological change to fibroblast-like appearance at higher passage numbers. This cell line was characterized for its use in virus replication experiments (3,4,5). The present study is the first report of in vitro transformation of equine oviductal epithelial cells. Use of cell lines derived from equine oviductal epithelium will provide more uniform material to study the molecular basis of sperm binding to the oviductal epithelium, and it will reduce the need for animal tissue. In conclusion, immortalization of epithelial cells from the equine oviductal isthmus with the SV40 large T antigen oncogene resulted in the selection of a single cell type with extended lifespan, displaying morphological and functional characteristics of equine OEC. These immortalized equine OEC may provide a useful model for the study of sperm-oviduct interactions in the horse. REFERENCES 1. Boatman DE. Oviductal modulators of sperm fertilizing ability. In: Bavister BD,Cummins J, Roldan ERS (eds), Fertilization in Mammals. Norwell MA: SeronoSymposia, 1990; 223-238. 2. Bongso A, Ng SC, Sathananthan H, Ng PL, Rauff M, Ratnam SS. Establishment of human ampullary cell cultures. Hum Reprod 1989; 4:486-494. 3. Boullant AM, Dulac GC, Willis N, Girard A, Greig AS, Boulanger P.Viral susceptibility of a cell line derived from the pig oviduct. Can J Comp Med 1975b; 39:450-456. 4. Boullant AM, Genest P, Greig AS. Growth characteristics of a cell line derived from the
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8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22. 23. 24.
Theriogenology pig oviduct. Can J Microbial 1975a; 21:2094-2097. Boullant AM, Greig AS. Type C virus production by a continuous line of pig oviduct cells (Pm). J Gen Virol 1975; 27:173-180. Boullant A, Greig AS, Genest P. Biological characterization of a cell line derived from the pig oviduct. In Vitro 1973; 9:92-102. Brinsko SP, Ignotz GG, Ball BA, Thomas PGA, Currie WB, Ellington JE. Characterization of polypeptides synthesized and secreted by oviductal epithelial cell explants obtained from young, fertile mares and aged, subfertile mares. Am J Vet Res 1996; 57: 1346- 1353. Chang SE. In vitro transformation of human epithelial cells. Biochim Biophys Acta 1986; 823:161-194. Chou JY. Establishment of clonal human placental cells synthesizing human choriogonadotropin. Proc Nat1 Acad Sci (USA) 1978; 75:1854-1858. Grump AL, Donaldson WL, Miller JM, Kydd JH, Allen WR, Antczak DF.) Expression of major histocompatibility complex (MHC) antigens on horse trophoblast. J Reprod Fertil 1987; 35 (Suppl):379-388. Day FT. Survival of spermatozoa in the genital tract of the mare. J Agr Sci 1942; 32:108-l 11. Ellington JE, Ball BA, Blue BJ, Wilker CE. Capacitation-like membrane changes and prolonged viability in vitro of equine spermatozoa cultured with uterine tube epithelial cells. Am J Vet Res 1993; 54:1505-1510. Ellington JE, Carney EM, Farrell PB, Simkin ME, Foote RH. Bovine 1- to 2-cell embryo development using a semi-defined medium in three oviduct epithelial cell coculture systems. Biol Reprod 1990; 43:100-107. Fitz TA, Wah RM, Schmidt WA, Winkel CA. Physiologic characterization of transformed and cloned rat granulosa cells. Biol Reprod 1989; 40:250-258. Freshney RI. Culture of animal cells. New York: AR Liss Inc. 1987; 176- 177. Girardi AJ, Jensen FC, Koprowski H. SV40-induced transformation of human diploid cells: Crisis and recovery. J Cell Comp Physiol 1965;65:69-84. Halvorsen TL, Leibowitz G, Levine F. Telomerase activity is sufficient to allow transformed cells to escape from crisis. Mol Cell Biol 1999;19: 1864-1870. Ito M, Smith TT, Yanagimachi R. Effect of ovulation on sperm transport in the hamster oviduct. J Reprod Fertil 199 1;93: 157- 163. Jacob JR, Tennant BC. Transformation of immortalized woodchuck hepatic cell lines with the c-Ha-ras proto-oncogene. Carcinogenesis 1996;17:631-636. Kydd J, Antczak DF, Allen WR, Barbis D, Butcher G, Davis W, Duffus WPH, Edington N, Grunig G, Holmes MA, Lunn DP, McCulloch J, O’Brien A, Perryman LE, Tavemor A, Williamson S, Zhang C. Report of the first international workshop on equine leukocyte antigens, Cambridge, UK, July 1991. Vet Immunol Immunopathol 1994;42:3-60. Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriphage T4. Nature 1970;277:680-685. Lefebvre R, Samper JC. Interaction between stallion spermatozoa and oviductal epithelial cells in vitro. Eq Vet J 1993 15 (Suppl):39-41. Melber K, Zhu G, Diamond L. SV40-transfected human melonocyte sensitivity to growth inhibition by phorbol ester. Cancer Res 1989;49:3650-3655. Ouhibi N, Menezo Y, Benet G, Nicollet B. Culture of epithelial cells derived from the
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oviduct of different species.Hum Reprod1989:4:229-235. Padilla AW, Foote RI-L Extender an¢rifugationeffects on the motility patternsof slow-cooledstallion spermatozoa.J Anim Sci 1991;69:3308-3313. PollardJW, PlanteC, King WA, HansenPJ, Bet&ridge KJ, SuarezSS. Fertilizing capacity of bovine spermmay be maintainedby bindingto oviductal epithelialcells. Biol Reprod 1991;44:102-107. RaaIM, GadsonPF, AndersonE, HarnsbyPJ, MahcshVB. Characterizationof Progesterone biosynthesisin a transformedgranulosacell line. Mel Cell Endocrinol 1993;94:121-128. RasbandM. Imageversion 1.54.National Institutesof Health ResearchServicesBranch, NIMH 1992,modified 1993. Rhim JS, Ro HS, Kim EB, Gilden RV, HuebnerRI. Transformationof horseskin cellsby type-C sarcomaviruses.Int J Cancer 1975;15:171-179. Smith TT, YanagimachiR. Capacitationstatusof hamsterspermatozoain the oviduct at varioustimesafter mating.J ReprodFertil 1989;86:255-261. SmithTT, YanagimachiR. The viability of hamsterspermatozoastoredin the isthmusof the oviduct: The importanceof sperm-epithelium contact for spermsurvival. Biol Reprod 1990;42:450-457. Smith ‘IT, YanagimachiR. Attachment andrekase of spematozoa from the caudal Isthmusof the hamsteroviduct. J ReprodFertil 1991;91:567-573. SteinbergML, Defendi V. Transformationandimmortalizationof humankeratinocytesby SV40. J Invest Dermatol 1983;81:131s-136s. ThomasPGA, Ball BA. Cytofhrorescentassayto quantify adhesionof equinespermatozoa COoviduct epithelialcellsin vitro. Ma1ReprodDev 1996;43:55-6I I ThomasPGA, Bali BA, Brinsko SP.interactionof equinespermatozoawith oviduct epithelialcell explantsis affectedby estrouscycle andanatomicorigin of explant. Biol Reprod1994a;51:222-228. ThomasPGA, Ball BA, Miller PG, Brinsko SP,SouthwoodL. A subpopulationof morphologicallynormal,motile spermatozoaattachto equineoviduct epithelial cells in vitro. Bial Reprod 1994b;51:303-309. ThomasPGA, lgnotz GG, Ball BA, Miller PG, Brinsko SP,Currie WB. Isolation, culture, andcharacterizationof equineoviduct epithelialcells in vitro. Mel ReprodDev 1995;41:468-478. Wood AL, SpradbrowPB. Transformationof culturedequinefibroblastswith a bovine papillomavirus.ResVet Sci 1985;38:241-242. Zepter K, Haffner AC, Trefzer U, E!metsCA. Reducedgrowth Factorrequirementand acceleratedcell-cycle kineticsin adult humanmelanocytestransformedwith SV40 largeT antigen.J Invest Dermatol 1995;104:755-762.
GENERATION OF AN EQUINE OVIDUCTAL EPITHELIAL CELL LINE FOR THE STUDY OF SPERM-OVIDUCT INTERACTIONS I. Dobrinski,“a
J.R. Jacob,2 B.C. Tentrant* and B.A. Ball3
‘Center for Animal Transgenesis and Germ Cell Research, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348 2 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University Ithaca, NY 14850 3Department of Population Health and Reproduction, School of Veterinary Medicine University of California, Davis, CA 95616 Received for publication: Accepted:
30 March 1999 1 July
1999
ABSTRACT Equine oviductal epithelial cells (OEC) were transformed with simian virus 40 large T antigen (SV 40 T-ag) to create a cell line for the study of the interaction of equine spermatozoa with oviductal epithelium. One cell line was established based on the expression of the SV 40 Tag and extended lifespan in culture. Immortalized equine OEC retained the characteristics of differentiated OEC such as the formation of monolayers with characteristic epithelial morphology and cell polarization as well as expression of cytokeratin and equine major histocompatibility complex I. Monolayers of immortalized equine OEC retained their functional competence to bind equine spermatozoa in a dose-dependent manner comparable to that of primary equine OEC cultures. This immortalized cell line of equine OEC provides a uniform, readily available system for sperm-OEC co-cultures, and may be a useful model for the study of sperm-oviduct interactions in the horse. 0 1999 by Elsevier
Science
Inc.
Key words: SV40, oviduct, sperm binding, horse Acknowledgments We thank Dr. J. Lopez for performing the preliminary cell transformation experiments and Dr. D.F. Antczak for the antibodies against equine MHC I and II. This study was supported by the Harry M. Zweig Memorial Fund for Equine Research. I. Dobrinski was a recipient of a Graduate Research Assistantship awarded by the College of Veterinary Medicine, Cornell University. a Correspondence and reprint requests: Dr. I. Dobrinski, Center for Animal Transgenesis and Germ Cell Research, 147 Myrin Bldg., New Bolton Center, University of Pennsylvania, 382 W. Street Rd., Kennett Square, PA 19348.
Theriopnology 52:8?&885,1999 Q 1999 by Ekevier Science
Inc.
0093-691 X/99/$+%x% front matter PII SOO93-691 X(99)00179-X
876 INTRODUCTION The mammalian oviduct is the site of sperm storage and capacitation (1, 18, 30, 3 l), fertilization and early embryonic development. Contact of spermatozoa with OEC maintains viability of spermatozoa stored in the oviduct (3 1) and prolongs their ability to fertilize (26,32). Storage of spermatozoa in the oviduct may be particularly important in the horse, in which fertilization can occur several days after breeding (11). To study the interaction of spermatozoa and OEC in the horse, in vitro systems using tissue explants of oviductal epithelium (22, 35) or monolayers of OEC (12, 36) have been described. A cytofluorescent assay was developed to more accurately quantify adhesion of equine spermatozoa to OEC monolayers (34). Equine spermatozoa preferentially bind to epithelium recovered from the oviductal isthmus of mares in the preovulatory phase of the estrous cycle (35). Recovery of fresh oviductal epithelial tissue from estrous mares for explant cultures or establishment of monolayer cultures is costly, and the use of OEC from different mares within or between experiments introduces a largely undefined source of variation. Age-dependent differences in oviductal function and fertility have been described in mares (7). Establishment of an equine oviductal epithelial cell line would provide a more uniform, readily available system for the study of sperm-oviduct interactions and, potentially, for embryo co-culture in the horse. A spontaneous cell line derived from porcine oviductal epithelium has been described (6). Human epithelial cells from different organs have been experimentally transformed (8) using human papilloma virus or simian virus 40 (SV40). Transformation of equine fibroblasts with bovine papilloma virus (38) and transformation of equine skin cells with type-C sarcoma viruses (29) has been described. Transformation of equine oviductal epithelial cells, however, has not been reported. The objective of this study was to generate a cell line by transformation of equine oviductal epithelial cells with the SV 40 large T antigen (SV 40 T-ag) and to investigate its potential use for the study of sperm-oviduct interactions in the horse. MATERIALS
AND METHODS
Immortalization of Oviductal Epithelial Cells Unless otherwise mentioned, all reagents were supplied by Sigma Chemical Company (St. Louis, MO). Monolayers of OEC were derived from the oviductal isthmus of mares in the preovulatory stage of the cycle (37). Briefly, oviducts were opened longitudinally, and epithelial cells were mechanically dissociated from the underlying stroma. Epithelial cells were cultured in 50% Dulbecco’s Modified Eagle’s Medium and 50% Ham’s F- 12 (50:50 DMEM:F- 12) with 10% fetal bovine serum (FBS; Hyclone, Langhorne, PA), 10 ng/mL epidermal growth factor, 5 pg/mL insulin, 5 pg/mL transferrin, 5 ng/mL selenium, 50 IUmL penicillin, and 50 pg/mL streptomycin. Monolayers were grown to confluence, then trypsinized and plated into 6-well plates at a density of 75,000 viable cells/ml. Cell viability was determined by exclusion of trypan blue. All cultures were incubated at 38S”C in 5% CO;! in air. Cells were immortalized as described previously (19). Briefly, the pSV3neo plasmid vector (ATCC 37150, American Type
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Culture Collection, Rockville, MD), expressing the SV40 T-ag and neomycin resistance genes, was transfected into OEC 24 h after plating using liposome fusion techniques (Lipofectin,R Gibco Life Technologies Inc., Grand Island, NY). Cell cultures were maintained in selective medium containing 50 pg/mL G418 (Geneticin,R Gibco) for >l mo until proliferative cell foci were observed. Individual colonies were isolated and passaged with trypsin/EDTA solution. Two clones (EOC##2 and EOC#8) maintained to the 8th passage after selection were expanded and passaged at a density of 1 x lo6 cells/25 cm* flask, every 6 d. Primary OEC from 6 mares of unknown breeding history were utilized in separate experiments for these studies. Characterization of Transformed Cells Expression of SV40 large T-antigen. Primary OEC cultures and immortalized cell lines were analyzed by immunoblot procedure to detect the SV40 T-ag as previously described ( 19). Cell monolayers were extracted, then proteins were separated by 10% SDS-PAGE (21) and transferred electrophoretically to PVDF membrane (Immobilon-P; Millipore, Bedford, MA). Immunologic detection of the SV40 T-ag was performed with monoclonal antibody Pab4 19 (Oncogene Science Inc., Uniondale, NY), followed by a goat antibody to mouse IgG conjugated to horse radish peroxidase (Cappel, Organon Teknika, Durham, NC). Membranes were saturated in luminescent substrate (ECL, Amersham Life Sciences, Arlington Heights, IL), followed by chemiluminescent detection of signal on X-ray film (XAR-5, Kodak, Rochester, NY). Immunoblots were scanned and processed for printing (Adobe Photoshop 3.0, Adobe Systems Inc., Mountain View, CA). Extended lifespan. Cells from clones EOC#2 and EOC#8 were passaged every 6th day by trypsinization and plating at 5 x lo5 to lo6 cells/25 cm2 culture flasks. At various passages, cells were frozen in 10% dimethylsulfoxide and 50% FBS (13). If poor growth was observed, cells were plated at higher density (2 x lo6 cells/25 cm2 culture flasks). Evaluation of cell morphology. Cell morphology was examined by phase-contrast and Hoffman Modulation Contrast (Modulation Optics Inc., Greenvale, NY) microscopy, and digital images wire recorded (34). For examination of ultrastructure, cells grown to confluence in 25 mL culture flasks and on Thermanox coverslips (Electron Microscopy Sciences, Fort Washington, PA) were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 30 min at room temperature and 1.5 h at O’C. Cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS; Gibco), scraped from the culture flasks and pelleted by centrifugation. The cells grown on coverslips were processed in situ. Cells were postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated and embedded in Epon. Ultrathin sections stained with lead citrate/uranyl acetate were then examined by transmission electron microscopy. hnmunocvtochemistrv. To confirm the epithelial nature and the equine origin of the transformed OEC, the ceils were grown to confluence in slide chamber wells (Lab-Tek 4chamber slides; Nunc Inc., Naperville, IL). Cells were processed for immunocytochemical detection of cytokeratin expression (37) with the modification that a monoclonal anti-Pan cytokeratin FITC conjugate (Sigma) was used at a dilution of 1:25. The cells were then examined with epifluorescence (420-490 nm) microscopy.
Theriogenology For immunocytochemical detection of equine major histocompatibility complex I (MHC I) as a marker for equine cells, monolayers were fixed in situ in 95% ethanol for 10 min, blocked with 10% normal goat serum, and incubated with monoclonal antibodies directed against equine MHC I and II (10,20). A monoclonal antibody directed against canine parvovirus served as a negative control. Specific antibody binding was detected using a peroxidase-coupled goat-anti mouse second antibody and aminoethyl carbazol (ACE) reagent, and cells were counterstained with hematoxylin (Histostain-SP Kit, Zymed Laboratories Inc., South San Francisco, CA). Chromosome counts. Cells growing in culture for 3 d were arrested in metaphase by incubation with 200 ng/mL colcemid (Demecolcine; Sigma) for 2 h. Cells were trypsinized, incubated in 0.075 M KC12 for 30 min and fixed (3 parts methanol : 1 part galcial acetic acid) as previously described (15; modified). Cells were spread on cooled, precleaned glass microscope slides, and chromosome spreads were examined (x 400). Images of complete spreads were digitized (NM Image Analysis software; version 1.59; 28), and chromosome numbers were counted in 80 individual spreads. Fluorescent Sperm Binding Assay To evaluate the functional capability of the transformed oviductal epithelial cells to bind equine spermatozoa, confluent monolayers of transformed cells were grown in 24-well culture plates (Sarsted Inc., Newton, NC) and used for a cytofluorescent sperm binding assay as described previously (34). Briefly, semen (2 ejaculates from each of 3 stallions on a regular semen collection schedule) was collected with an artificial vagina, diluted to 10’ sperm/ml in modified Tyrode’s solution (25) and transported to the laboratory. Spermatozoa were loaded with the fluorescent nuclear dye Hoechst 33342 (5 pg/mL), washed by centrifugation (300 x g, 6 min) and resuspended in culture medium. Confluent monolayers were inseminated with 500 pL culture medium containing 4 different concentrations of spermatozoa: 5 x 105, 106, 2.5 x lo6 and 5 x lo6 cells/well. Triplicate wells were inseminated for each concentration. Co-cultures were incubated at 38.5’C in 5% CO2 in air for 30 min, and supematant containing unbound spermatozoa was removed. The co-cultures were then rinsed twice with 500 pL culture medium/well, and the medium was replaced. After equilibration for3 min, the cultures were examined on an inverted Nikon Diaphot-TMD microscope, fluorescent videoimages of spermatozoa were digitized, and the numbers of attached spermatozoa were determined (34). Statistical Analysis Characterization of SV40 T-ag expression, cell morphology, immunocytochemistry and chromosomal numbers were observational. In the sperm binding assay, a complete randomized block design was used, with inseminate concentrations applied within ejaculates and the data blocked by stallion for analysis of variance (ANOVA), using Linear Models of Statistix (Analytical Software, Tallahassee, FL). Data were log-transformed to obtain a Gaussian distribution. The dependent variable was the number of bound spermatozoa. Data were examined for the effect of ejaculate-within-stallion, treatment, replicate and their interactions. Pairwise
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comparisons of means were made using Tukey’s honestly significant difference (HSD). The relationship between the log of number of spermatozoa inseminated and the number of spermatozoa bound was described by linear regression. RESULTS Immortalization of Oviductal Epithelial Cells Oviductal epithelial cells isolated from 6 mares were transfected in separate experiments with the SV40 T-ag. Several foci were observed after selection; however, only 2 clones expanded upon passage. Cell line EOC#K?was passaged on plastic 12 times but could not be passaged further. Cell line EOC#8 grew efficiently to Passage 12, reaching confluence within 4 to 5 d. After passage 12, the cells grew at a reduced rate and did not form a confluent monolayer after 6 d in culture. Large multinuclear cells were observed more frequently at this time. By plating at a higher density (2 x lo6 viable cells/25 cm’flask), cell survival and proliferation appeared to improve, and cultures reached confluence by day 6. Cells were maintained until Passage 29, at which time they were frozen and stored in liquid nitrogen. Only cells from the EOW8 line were found to express the SV40 T-ag by immunoblot analysis (Figure 1). Therefore, all subsequent
kD 120= 90= 705040Figure 1. One-dimensional immunoblot of cell lysates from primary and transfected equine OEC incubated with monoclonal antibody Pab419 against SV40 T-antigen followed by horseradish peroxidase conjugated goat-anti mouse IgG. Lane 1: primary OEC; Lane 2: transfected cell line EOCM; Lane 3: transfected cell line EOC#8. Arrow indicates Tantigen at 97 kD.
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characterizations were performed with cells from this clone. Cells grown to confluence showed the characteristic, rounded, polygonal appearance of epithelial cell monolayers in the majority of cells (Figure 2).
Figure 2. Hoffman modulation contrast micrograph of oviductal epithelial cell line EOC#8 (passage 14) at day 6 of culture. Bar = SOpm. Occasionally, some large multinucleated cells were observed. On ultrastructural examination, cilia were absent but microvilli were apparent at the luminal cell surface (Figure 3). Immunocytochemistry revealed that the transformed cells at passage 14 expressed cytokeratin as expected for epithelial cells, and that all cells expressed equine MHC I, but not equine MHC II antigens, confirming their origin from equine tissue (data not shown). Chromosome counts showed that the cells were highly aneuploid. Only 19% of cells examined had the normal diploid number of chromosome, 11% had a haploid number of chromosomes, and 6% a tetraploid number of chromosomes. The remaining spreads had chromosome numbers ranging from 13 to more than 140 chromosomes.
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Figure 3. Transmissionelectronmicrographof ultra thin sectionof equineoviductal epithelial cell line EOC#8 (Passage14)monolayer.Bar = 2pm. In the fluorescentspermbinding assay,the transformedoviductal epithelialcells bound spermatozoain a dose-dependent mannerwith a log-linearrelationshipbetweenthe numberof spermatozoainseminatedand the numberof spermatozoabound (P
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n Stallion
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ef
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Figure4. Numbersof equinespermatozoaboundto monolayersof oviductal epithelialcell line EOC#8 (meansGEM) for different stallionsandinseminateconcentrations(a,b,c,d,e,f: P
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Based on immunocytochemistry for cytokeratin and equine MHC I, the immortalized OEC were confirmed to be of equine origin and of epithelial nature. The immortalized OEC were aneuploid, as described for human epithelial cells transformed in vitro (9), and aneuploidy has been used as a marker for successful transformation (8). The cells maintained characteristics of primary equine OEC cultures in that they remained anchorage-dependent and formed confluent monolayers with the characteristic, rounded, polygonal appearance of cultured epithelial cells (37). There was ultrastructural evidence of microvilli on the apical cell surface. Cilia were absent from the immortalized epithelial cells. Disappearance of cilia from cultured OEC was reported to occur already after the first passage (2,37). Maintenance of different degrees of differentiation and cell function is commonly observed in cells transformed with SV40 (9, 14,27, 39). Immortalized equine OEC maintained normal morphology and expressed both cytokeratin and MHC I. More importantly, the equine oviductal epithelial cell line also retained the ability to bind equine spermatozoa in a dosedependent manner and to detect differences between individual stallions in sperm attachment to OEC (34). Comparison of absolute numbers of spermatozoa bound per area of monolayer between primary and immortalized OEC, however, is not meaningful because spermatozoa from different stallions were used in our present study and in the study reported by Thomas and Ball (34). A porcine oviductal epithelial cell line has been described earlier (4,6). The cell line was derived spontaneously from the epithelium of an entire pig oviduct and underwent a morphological change to fibroblast-like appearance at higher passage numbers. This cell line was characterized for its use in virus replication experiments (3,4,5). The present study is the first report of in vitro transformation of equine oviductal epithelial cells. Use of cell lines derived from equine oviductal epithelium will provide more uniform material to study the molecular basis of sperm binding to the oviductal epithelium, and it will reduce the need for animal tissue. In conclusion, immortalization of epithelial cells from the equine oviductal isthmus with the SV40 large T antigen oncogene resulted in the selection of a single cell type with extended lifespan, displaying morphological and functional characteristics of equine OEC. These immortalized equine OEC may provide a useful model for the study of sperm-oviduct interactions in the horse. REFERENCES 1. Boatman DE. Oviductal modulators of sperm fertilizing ability. In: Bavister BD,Cummins J, Roldan ERS (eds), Fertilization in Mammals. Norwell MA: SeronoSymposia, 1990; 223-238. 2. Bongso A, Ng SC, Sathananthan H, Ng PL, Rauff M, Ratnam SS. Establishment of human ampullary cell cultures. Hum Reprod 1989; 4:486-494. 3. Boullant AM, Dulac GC, Willis N, Girard A, Greig AS, Boulanger P.Viral susceptibility of a cell line derived from the pig oviduct. Can J Comp Med 1975b; 39:450-456. 4. Boullant AM, Genest P, Greig AS. Growth characteristics of a cell line derived from the
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