In Vitro Cell. Dev. Biol.--Animal 37:581 588, October 2001 �9 2001 Society for In Vitro Biology 1071-2690/01 $10.00+0.00

ESTABLISHMENT AND CHARACTERIZATION OF IMMORTALIZED OVINE SERTOLI CELL LINES

RAGHIDA ABOU MERHI, LAURENT GUILLAUD, CLAUDE DELOUIS, AND CORINNE COTINOT l

Unitd de Biologie du ddveloppement et Biotechnologies, INRA, 78350 Jouy-en-Josas, France (R. A. M., C. C.), Laboratoire de C4ndtiq~ Moldculaire et CeUulaire, INRA-ENVA, 94704 Maisons-AlJbrt, France (L. G., C. D.)

(Received 22 March 2001; accepted 18 July 2001)

SUMMARY

The objective of this study was to generate immortalized Sertoli cell lines from prepubertal lamb testes to facilitate investigations during the course of testicular differentiation. The Sertoli cells were enzymatically isolated and immortalized by transfection, with the sequences coding for the SV40 large T-antigen fused downstream of regulatory elements from the human vimentin gene. The different cell lines were positively stained with antibodies to vimentin and transferrin, in agreement with their Sertoli origin. Reverse transcriptase polymerase chain reaction was used to analyze the specific expression of molecular markers (clusterin/sulfated glycoprotein [SGP-2], follicle-stimulating hormone [rFSH], c~-inhibin, anti-Mtillerian hormone, Wilms' tumor gene [WT-1], steroidogenic factor 1 [SF-1], SRY-related HMG box gene g [SOX9], and sex-determining region of Y chromosome) normally expressed in this cellular type. All were shown to express messenger ribonucleic acids for SGP-2, c~-inhibin, WT-1, SOX9, and SF-1 (except SF-1 for clone no. 1). Moreover, we performed alkaline phosphatase and receptor tyrosine kinase p145 (c-kit) detection to ensure the absence of contamination by peritubular, germ cells, and Leydig cells. Both tests were negative for all the seven cell lines. These ovine Sertoli cell lines are the first ones obtained from livestock that exhibit specific Sertoli cell characteristics resembling different stages of phenotypic development. They provide useful in vitro model systems for toxicological investigations, coculture, and transfection experiments, making it possible to study signal transduction pathways, cell-cell interactions, and gene expression in species other than rodents.

Key words: immortalization; Sertoli cell lines; testis; sheep.

INTRODUCTION

The Sertoli cells are considered as the first cell type to differ- entiate in the fetal testis, and it is assumed that SRY (sex-deter- mining region of Y chromosome), the switch gene on the Y chro- mosome, is expressed in their precursors (Swain et al., 1998). Through the SRY action, major aspects of testicular development are then directed by Sertoli cells. Because of the pivotal role the Sertoli cells play in organogenesis, differentiation, and function of the testis, it would be useful to develop an in vitro system where the genes are expressed, as in native cell types. As a consequence, several immortalized cell lines from rat (Roberts et al., 1995; Pog- nan et al., 1997) and mouse (Mather, 1980; Hofmann et al., 1992; Peschon et al., 1992; Rassoulzadegan et al., 1993; Capel et al., 1996; Wahher et al., 1996; Dutertre et al., 1997; Bourdon et al., 1998) Sertoli cells have previously been obtained.

In addition to SRY, several transcription factors have been found to be expressed in Sertoli ceils and to have participated in early testicular differentiation, such as the Wilms' tumor gene (WT-1) (Armstrong et al., 1992; Gessler et al., 1993), the steroidogenic factor (SF-t) gene Ftz-F1 (Luo et al., 1995), and the SRY-related

l To whom correspondence should be addressed at Unit(5 de Biologic du d6veloppement et Biotechnologies, Bat. Biotechnologies, INRA, 78350 Jouy en Josas, France. E-mail: cotinot@diamant.jouy.inra.fr

gene SOX9 (Kent et al., 1996; Morais da Silva et al., 1996). All these genes are expressed very early during gonadal differentiation, and their expression patterns have been determined in mice (Hack- er et al., 1995; Kent et al., 1996) and some of them have also been determined in sheep (Payen et al., 1996). The correct combination of these factors is needed to induce and maintain testis differenti- ation, but the exact interrelationships between these different genes are still unknown.

The Sertoli cells also fulfill essential functions in the production of male germ cells, controlling their number and supporting their differentiation (Fritz, 1994; Griswold, 1995). Many of these regu- latory events are brought about by direct cell-cell interaction, and the exact nature of the stimuli that is effective in vivo is difficult to analyze. Nevertheless, numerous Sertoli cell products have been characterized, and some of them are specifically and actively ex- pressed in mature Sertoli cells, such as the peptide hormone inhibin (Moore et al., 1994), the secreted protein SGP-2 (Law and Griswold, 1994), transfen'in (TF) (Suire et al., 1997), and the FSH receptor (Ranniko et al., 1996). They confirm fully differentiated Sertoli cell phenotype.

We chose to develop a cellular model specific to the ruminant and worked at the 35-d post partum, before the onset of the sper- matogenesis stage, where the testis is sufficiently developed, and the Sertoli cells are easily separated from germ cells, the major contaminant cellular type. We report the use of a recombinant vector

581

582 ABOU MERHI ET AL.

composed of the SV40 large T-antigen-encoding deoxyribonucleic

acid (DNA) driving the truncated vimentin promoter for immortal-

izing Sertoli cells from prepubertal sheep testis. Seven independent

cell lines that retained characteristic properties corresponding to

native Sertoli cells were established and cultured successfully over

a period of 12 too.

MATERIALS AND METHODS

Sertoli cell isolation and culture. Testes from a 35-d-old Pr~alpes lamb were excised following castration, cut up into small fragments, and treat- ed with 0.25% (w/v) trypsin type III (Sigma) and 20 U/ml of deoxyribo- nuclease I (DNase I) (Roche Diagnostics, Meylan, France) for 30 min at 34 ~ C. The medium was replaced by Hanks' balanced salt solution (HBSS) medium containing trypsin inhibitor 0.034% (w/v) (Sigma), and the mixture was aspirated several times through a lO-ml pipette. Centri- fugation was carried out at 430 • g for 10 min to obtain a pellet of seminiferous tubules. After three washings in HBSS, seminiferous tubules were incubated in HBSS containing collagenase type II S 200 U/ml (Sig- ma), DNase I 20 U/ml, and 5% (v/v) heat-inactivated fetal calf serum (FCS) (Life Technologies, Cergy Pontoise, France) for 60 min at 34 ~ C with horizontal shaking. The cell suspension was filtered through a strain- er and then a sieve (200-~m mesh). A typical preparation of seminiferous tubule ceils contained 3% peritubular ceils, 5% germ cells, and 82% Sertoli cells. The cells were cultured in Dulbecco modified Eagle medium (DMEM) containing high glucose (4.5 g/L) and high sodium bicarbonate (4.4 g/L), and 10% FCS at 37 ~ C under a 10% atmosphere of carbon dioxide in air cultured for the first 24 h in the absence of any serum or hormones. During this time, the Sertoli cells attached to the plastic cul- ture dish and began to flatten out, whereas germinal cells continued to float in the medium. After 24 h, most germinal ceils were discarded by medium change. The enrichment in Sertoli cells was, at that time, around 90%.

Sertoli cell transformation and selection of clones. One day after iso- lation, Sertoli cells were washed with OPTI-MEM medium (Life Tech- nologies). Two micrograms of the DNA construct, composed of control elements of the promoter of the human vimentin gene fused upstream of the sequences coding for the large T and small t SV40 antigens (Schwartz et al., 1991), described in detail elsewhere (Moura-Neto et al., 1996), were diluted into 100 fxl of Opti-MEM | Twenty microliters of lipofectin (Life Technologies) were diluted into 80 ILl of OPTI-MEM and mixed with the DNA solution. After 15 min at room temperature, the mix was diluted with 1.8 ml of OPTI-MEM and pipetted into the T25 flask which there- after was maintained overnight at 37 ~ C. Sertoli cells were collected using trypsin-ethylenediaminetetraacetic acid (EDTA) (0.05:0.02%, in HBSS) and cultured in Petri dishes until the appearance of colonies (3-4 wk) which were collected using trypsin-EDTA. The population of Sertoli cells was checked for the presence of immunoreactive large T-antigen. Cloning by limiting dilution made it possible to select seven clones, SM1-SM7, from positive large T-antigen Sertoli cells.

Cytogenetic method. Cultures were synchronized by double thymidine block during the S phase to increase the metaphase yield (Hayes et al., 1991). They were then incubated at 37 ~ C until the number of mitotic cells reached a maximum. The cells were treated with colchicine at a final concentration of 0.04 ~g/ml tbr 20 min at 37 ~ C and detached with 0.05% trypsin and 0.02% EDTA (Boehringer Mannheim) for 10 min at 37 ~ C. Hypotonic treatment was performed with diluted (1:6) newborn calf serum and EDTA (Sigma) at a final concentration of 0.4 txg/ml for 13 rain at 37 ~ C. Fixation and spreading followed standard methods. Standard sheep karyotype (Ovis aries) is referenced in International Sys- tem for Chromosome Nomenclature of Domestic Bovids 2000 (Di Ber- nardino et al., in press).

Immunocytochemistry. In the case of the large T SV40 antigen, cells were cultured to confluency on glass slides, rinsed three times with phosphate- buffered saline (PBS) and then fixed with methanol-acetone (7:3, v/v) for 6 min at room temperature. Slides were dried for 30 s and then rinsed with PBS. Anti-large T monoclonal antibody (undiluted hybridoma culture me- dium) (Schwartz et al., 1991) was incubated for 1.5 h at 37 ~ C. Cells were then rinsed three times with PBS-bovine serum albumin (BSA) (0.5% BSA, w/v). The second antibody, an anti-mouse immunoglobulin G (IgG) (Fab

specific) fluorescein isothiocyanate (FITC) conjugate (Sigma, F 5262) di- luted 1:200 in PBS-BSA was incubated for 30 rain at 37 ~ C. For negative controls, incubation with a standard culture mediuni was used as a sub- stitute to the hybridoma culture medium; for positive controls, large T- expressing mouse endothelial cell lines (Schwartz et al., 1991; Vicart et al., 1994) were used instead of Sertoli cells.

With regard to vimentin, ceils were fixed with paraformaldehyde and phos- phonoformic acid (PFA) (4%, v/v; Merck Eurolab, Fontemays/Bois, France), for 10 rain at room temperature, and rinsed twice with PBS-BSA (0.5% w/ v) for 5 min. Antivimentin monoclonal antibody (mouse ascite fluid, clone V9; Sigma V6630) was diluted 1:40 and incubated for 1 h at room temper- ature. The second antibody was used as reported previously for large T im- munoassay. Negative controls were performed using culture medium instead of ascite fluid.

In the case of TF, cells were cultured to confluency in DMEM medium containing 10% FCS on glass chamber slides (Nunc, Roskilde, Denmark), rinsed three times in DMEM medium without FCS and fixed with PFA, as reported for vimentin. A rabbit polyclonal TF antibody (Monet-Kuntz et al., 1992) (kindly provided by Dr. Florian Guillou, INRA, Nouzilly, France) di- luted 1:20 in PBS-BSA was incubated for 1 h at 37 ~ C. The cells were then washed three times with PBS and incubated with the second fluorescein- labeled antibody, (L 4300, Tebu, Le Perray en Yvelines, France) diluted 1: 100, for i h at room temperature in a dark chamber and washed again several times. Negative controls for TF imnmnoassay consisted in replacing the pri- mary antibody with PBS-BSA.

With regard to the c-kit, cells cultured on glass slides were fixed as re- ported for vimentin. The first antibody was a polyclonal rabbit IgG (C-19 cat #sc-168, Santa Cruz Biotechnology, Tebu, Le Perray en Yvelines, France) diluted 1:50 in PBS-BSA, and the second antibody was a goat anti-rabbit IgG FITC conjugate (L 4300, Tebu, France) diluted 1:100 in PBS-BSA and incubated for 1.5 h at 37 ~ C.

Positive controls were performed using frnzen slides (7 txm) of a 35-d-old lamb testis. Slides were warmed at room temperature and rinsed once with acetone for 10 min and with PBS three times for 5 min. Thereafter, slides were processed as described for cell cultures. Negative controls consisted of replacing the primary antibody with PBS-BSA.

All the slides were coverslipped with Mowiol mounting medium (Hoechst, Frankfurt, Germany) containing n-propyl gallate (Sigma) as the antitading agent. The observations were made using a polyvar Reichert or a Zeiss axi- omatic fluorescence microscope.

Detection of alkaline phosphatase activity. Cells and tissue sections were fixed with 4% PFA-PBS for 15 min, rinsed 2 • 5 rain in PBS, then stained by incubation in reaction buffer (25 mM Tris-malate, pH 9.0, 0.4 mg/ml sodium-ct-naphthyl-phosphate, 1 mg/ml Fast-Red TR, 8 mM MgCI2) for 15 rain at room temperature. This staining solution had to be prepared im- mediately prior to use, mixed vigorously, and filtered. Samples after stain- ing were rinsed 2 • 5 rain in PBS.

Reverse transcriptase-polymerase chain reaction assays. Total ribonucleic acid (RNA) from cell lines was extracted using RNA-plus solution (Bioprobe Systems, Montreuil s/Bois, France) and treated with DNase for 2 h at 37 ~ C with 2 U/Ixg DNase-RNase-fi-ee (Boehringer Mannheim). Five micrograms of the RNA were reverse transcribed at 42 ~ C for 50 rain with 200 U of Su- perscript IX (Life Technologies) in a final volume of 20 txl containing 7.5 mM of random hexamers (Pharmacia, Upsala, Sweden) and 20 U of ribonuclease inhibitor (Boehringer Mannheim). Negative controls were pertbrmed by omit- ting reverse transcriptase.

One-tenth of this reaction was amplified tbr 30 cycles using a Perkin- Elmer apparatus in a final volume of 100 p~l containing 0.2 nrM diethylni- trophyenyl thiophosphate (Ultrapure Solution, Pharmacia), 150 mM specific primer, 2 U of Taq polymerase (Perkin-Ehner-Cetus, Boston, MA), 50 mM KC1, 10 n~/Tris-HC1 (pH 9.3), 2.5 mM MgCI~, and 0.1 mg/ml gelatin (Sig- ma). One-tenth of the polymerase chain reaction (PCR) product was loaded on 2% agarose gels for electrophoresis and visualized with ethidium bronfide. Primer sequences and PCR conditions are given in Table 1.

RESULTS

Establishment and morphological characterization o f immortalized Sertoli cell lines. Cell colonies surviving 3 -4 wk after immortaliza-

tion were tested for the presence of immunoreactive large T-antigen.

As the resulting immortalized colonies were not pure, extensive

IMMORTALIZED SERTOLI CELL LINES

TABLE 1

583

SEQUENCES OF PRIMERS AND PCR CONDITIONS

Genes Primers PCR Conditions Species References

WT-1

SF-I

SRY

SOX-9

AMH

rFSH

SGP-2

c~-inhibin

Transfen'in

5'-CAGTGTAAAACTTGTCAGCG-3' 5'-CTGATGCATGTTGTGATGGC-3'

5'-GATCGCCAGGAGTTCGTCTG-3' 5'-GTGCTTGTGGTACAGGTACTC-3'

5'-GCAATCGTATGCTTCTGCTA-3' 5'-TGTCTCGGTGTATAGCTAG-3'

5'-ATGTAGTGTATCACTGAGTC-3' 5'-AGATTAAGGTCTGTCAGTGG-3'

5'-CTATGAGCAGGCCTTCCTGG-3' 5'-CCTCCAGGTGCAGGACCACC-3'

5'-ATGGCCTTGTTCCTGGTG-3' 5'-AACCTTTTGGAGAGACTG-3'

5'-GACAATGAGCTCCA(G/A)GAA(A/C)TG3' 5'-CAGGCATCCTGTGGAGTT(G/A)TG-3'

5'-CTTGTTCTGGCCAAGGTGAGG-3' 5'-GCTGGGCTGAAGTCACCTGG-3'

5'-GAACATCCCCATGGGCCTGC-3' 5'-AAGGCCACGTCTCCCTTCTC-3'

94 ~ C% 1 min Human Gessler et al. 1992 55 ~ C, 1 min 72 ~ C, 1 min 94 ~ C, 1 rain Cow Honda et al. 1993 53 ~ C, 1 min 72 ~ C, 1 min 94 ~ C, 1 min Sheep Payen et al. 1996 55 ~ C, 1 rain 72 ~ C, 1 min 94 ~ C, I min Human Foster et al. 1994 56 ~ C, i rain 72 ~ C, 1 rain 94 ~ C ~ 1 rain Cow Cate et al. 1986 58 ~ C, I min 72 ~ C, i min 94 ~ C, 1 min Sheep Yarney et al. 1993 55 ~ C, 1 rain 72 ~ C, 1 min 94 ~ C, 1 min Human Kirszbaum eta]. 1989 55 ~ C, 1 min 72 ~ C, 1 min 94 ~ C% 1 min Mouse Albano et al. 1993 58 ~ C, i rain 72 ~ C, 1 min 94 ~ C, 1 min Human Yang et al. 1984 55 ~ C, 1 min 72 ~ C, 1 rain

5% formamide in PCR buffer.

cloning by limiting dilution was carried out, yielding a total of seven stable cell lines, named SM1-SM7. These cell lines were cultured over a period of approximately 12 mo. They were characterized according to their morphology, expression of immunocytochemical markers, production of specific messenger RNA (mRNAs). Karyo- types performed on SM1, SM4, SM6, and SM7 lines revealed that all were aneuploid and contained a Y chromosome (data not shown). SM6 and SM7 presented a more stable number of chromosomes (near normal sheep karyotype 2n = 54) than SM1 and SM4 which presented some tetraploid cells.

Under phase contrast microscopy, immortalized cell lines did not present polarity when grown separately from each other. They pre- sented two types of morphology according to the density of cultures: either flat and exhibiting long, thin cytoplasmic projections at low density, or euboidal or hexagonal when cultures were close to con- fluency (Figs. 1 and 2A). Two cell lines, SM1 and SM3, presented a different morphological aspect: SM1 had a regular epithelial as- pect and SM3 a mix of long cytoplasmic and an epithelial-like shape (Fig. 1).

Immunohistochemical analysis. All cells adhered to plastic, and the presence of the large T molecule in the nucleus was revealed by immunohistochemical staining. This analysis was carried out for all clonal lines but is only illustrated for SM4 in Fig. 2. The SM4 cells presented characteristic extensive cytoplasmic elongations when grown at low cell density. All cells stained positively in the nucleus for the SV40 large T-antigen, consistent with stable trans- formation by the simian virus (Fig. 2B). To further establish that

the cell lines have retained Sertoli cell characteristics, we tested for the presence of TF and vimentin using corresponding antibodies. All cell lines were decorated after staining for these two markers of Sertoli cells as illustrated for cell line SM4 (Fig. 2C and D). More- over, to detect possible contamination by Leydig cells, we performed immunodetection of the receptor-tyrosine kinase c-kk on each line. All were negative, whereas the testis sections at the same age were positive (data not shown).

Detection of Sertoli-specific gene expression. The seven clonal cell lines (SM1-SM7) were analyzed by reverse transcriptase PCR (RT- PCR) for the expression of known Sertoli-specific markers over the course of approximately 12 mo. RNA was collected at 3 4 and 9 - 12 mo after immortalization and tested for SRY, SOX9, WT-1, SF- 1, anti-Mtillerian hormone (AMH), SGP-2, TF, c~-inhibin, and rFSH expression. Only the genes showing a positive expression are shown in Fig. 3.

One of the clonal lines, SM4, had expressed SRY gene at the first screening by 3 mo (data not shown), but over time, expres- sion declined, and we no longer detected SRY expression after 6 mo of culture. In the other six clonal lines, SRY expression was never observed. To make sure that these cell l ines had not lost their Y chromosome or their SRY gene after chromosomal rearrangement, we performed karyotypes and a PCR analysis from genomic DNA with SRY-specific primers. The Y chromo- some was present within each cell line tested, as was the SRY gene (data not shown). A recently identified gene, SOX9, member of the family of genes related to SRY, has been shown to be

584 ABOU MERHI ET AL.

FIG. 1. Phase contrast micrographs of monolayer of six immortalized ovine Sertoli cell clones. Cultures were between 8 and 11 passages after cloning by limiting dilution. At low density, cells exhibit long and cytoplasmic projections, except clone SMI that presents a more cuboidal aspect and clone SM3 that presents a mixing aspect, still maintained after subcloning. Magnification: •

expressed in mouse Sertoli cel ls . In our seven l ines of ovine Sertoli cel ls , express ion of SOX9 was clearly de tec tab le (Fig. 3). Posi t ive resul ts were also obta ined for WT-1 and the orphan ste- roid receptor SF-1 genes in six l ines; both be ing expressed nor- really in Sertoli cel ls (Fig. 3). In contrast , two genes known to

be specif ical ly expressed in Sertoli cel ls were absen t in the seven l ines: AMH and rFSH. Finally, we tes ted the p resence of tran- scr ipts cor responding to TF, a - inh ib in , and SGP-2 expression; these three genes were exp res sed in all the cell l ines.

Furthermore, 6 mo after the establishment of the lines, alkaline

IMMORTALIZED SERTOLI CELL LINES 585

FIG. 2. (A) Morphological and (B) immunocytochemical analysis of clone SM4 for SV40 large T-antigen, (C) transferrin, and (D) vimentin. Magnification: A, • B, C, D, •

phosphatase activity was examined to discriminate the possible presence of peritubular ceils in our Sertoli cell lines. Sections of testis at 35 d post partum were used as positive controls. All the seven lines were devoid of alkaline phosphatase activity (data not shown).

DISCUSSION

Prepubertal Sertoli cells were chosen for the study because many of the differentiated functions of Sertoli cells, are established by this age, whereas the number of germ cells, the major contaminant of SeI~toli cell cultures, is significantly reduced compared with sex- ually mature testes (Hochereau de Reviers et al., 1987). Further- more, most regulating factors, such as SRY, SOX9, and SF-1 are again present at this stage.

The results of the present study show that transfection of Sertoli cells with plasmid Hu ProVimT allows for the efficient inunortali- zation and establishment of Sertoli cell lines that retain morphology, differentiated properties, and morphogenetic potential, as previously

described for other cell types (Vicart et al., 1994; Schweitzer et al., 1997).

The loss of SRY expression by cells of clone SM4 and its absence from other cell lines was not caused by the absence of this gene in the genomic DNA of the immortalized cell lines, but this could be the result of a dedifferentiation process occurring with time. Anoth- er explanation could be that SRY expression requires interaction between Sertoli cells and one or several other cell types of the testis that were apparently absent in the clonal cell line culture. Fur- thermore, we could not exclude the fact that SRY expression was because of the contamination caused by a small population of germ cells, as has been described by Paquis-Flucklinger et al. (1993), who found close association of immortalized SerIoli cells and germ cells in their cultures even after repeated cloning of the cells. SRY is also expressed in germ cells in adult testis (Rossi et al., 1993; Hacker et al., 1995; Salas-Cortes et al., 1999).

In contrast to SRY, expression of WT-1, SF-1, and SOX9 was observed in all cell lines (except SF-1 in clone SM1). In the testes

586 ABOU MERHI ET AL.

FIG. 3. RT-PCR amplification of mRNA isolated from the seven immortalized Sertoli cell lines and a normal testis of 35 d post partum (T35). The expression patterns of the seven cell lines are similar, except for the absence of SF-1 expression in cells of the clone SM1. All the other seven cell lines (SM1-SM7) were positive for the expression of SGP-2, c~-inhibin, WT-1, TF, and SOX9. For intronless genes, like SRY and SOX9, samples were tested with (+) and without ( - ) reverse transcriptase in order to control genomic DNA contamination. For others genes, primers were chosen to flank an intron and produce amplification products of different size according to whether they originated from complementary DNA (cDNA) or genomic DNA. In order to compare the amount of cDNA present in each sample after

IMMORTALIZED SERTOLI CELL LINES 5 8 7

of developing mice and adults, these three transcription factors were detected in testis cords (Kreidberg et al., 1993; Hatano et ah, 1994; Morais da Silva et al., 1996). Judging by their morphological situ- ation and appearance, it has been concluded that these cells were Sertoli ceils. Given that seven of our cell lines express WT-1 and SOX9 and six expressed SF-1 gene, we can assume that these orig- inate as Sertoli cells, and that the expression of these three factors can be maintained in culture without interaction with other cell types.

It was also noted that only the SF-1 negative cell line (SM1) presented a morphological aspect different fi'om other cell lines, which was also partly observed in clone SM3. This particular aspect could indicate a different stage of phenotype development rather than a contamination by Leydig cells or peritubular, as demonstrat- ed by the absence of c-kit and alkaline phosphatase activity in these two cell lines.

In addition to the other Sertoli-specific genes expressed by our lines, we also found SGP-2, c~-inhibin, and TE All the three cor- respond to differentiated functions of Sertoli cells.

Two typical Sertoli-specific factors were absent from our lines: the FSH receptor and the AMH. Although it has recently been shown that SOX9 and SF-1 interact to promote AMH expression (De Santa Barbara et al., 1998; Arango et ah, 1999), it is noteworthy that the presence of these two factors in our lines was not sufficient to enable AMH transcription. A possible explanation is that SOX9 protein was not correctly translocated in the nucleus of these cells; further investigations with an antibody directed against SOX9 will be needed to test this possibility. The absence of AMH expression may also be related to the immortalization of the ceils through the T-antigen pathway as this pathway could be antagonistic to trans- forming growth factor-13-1ike factors. Similar results were also re- ported by Capel et al. (1996) on the loss of SRY and AMH ex- pression by urogenital ridge explants from transgenic mice carrying a temperature-sensitive SV40 large T-antigen. Finally, the absence of AMH transcription may be a result of DAX-1 expression in these cell lines. Indeed, it had been shown that the proper spatio-tem- poral expression of the AMH gene required the concerted action of several transcription factors that include SOX9, SF-1, WT-1, GATA- 4, and DAX-1. In mice, DAX-1 is coexpressed with SF-1 in many tissues, including the gonads, where it acts as a negative modulator of SF- l -dependent transcription. It has been reported that DAX-1 could repress AMH transcription in Sertoli cells by disrupting tran- scriptional synergism between GATA-4 and SF-1 (Tremblay and Viger, 2001). The second Sertoli-specific factor absent from our lines was rFSH. Similar absence of rFSH in a mouse Sertoli cell line (MSC-1) obtained by immortalization by SV40 T-antigen gene has previously been described by McGuiness et al. (1994). Never- theless, these cells were able to respond to high levels of cAMP, thereby attesting that the signaling pathway for rFSH was intact.

Availability of clonal Sertoli cell lines characterized at a molec- ular level will make it possible to address questions about cell interactions in the molecular and morphological differentiation of Sertoli cells. Using coculture experiments with different cell types

that are normally present in the testis, we will investigate the effects of this cell-cell interaction on the expression of different genes. Furthermore, the immortalized Sertoli cell lines described above present great advantages over primary cultures and will be useful in transfection studies to investigate further the relationship be- tween SRY and the other downstream genes required for Sertoli cell differentiation.

ACKNOWLEDGMENTS

We thank Dr. H. Hayes (INRA, Lab. de G6n6tique Biochimique et Cyto- g6n6tique) for karyotype analysis, Pr. D. Paulin (Lab. de Biologic mol6colaire de la Differentiation, Institut Pasteur et Universitd Paris 7) for the gift of the vector HuProVimT and Dr. E Guillou (INRA, Nouzilly) for the transferrin antibody. This study was supported by grant 734/93 fl'om the DGER, Min- istSre de l'Agriculture et de la P8che.

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reveise transcription, we used WT-1 RT-PCR reaction. PCR products were visible in equal amounts in the seven lines, attesting the presence of an equivalent amount of cDNA in each sample. The 35 d post-partum testis (T35) was used as positive control for the PCR reactions.

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