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Effects of androgen-binding protein (ABP) on spermatid Tnp1 gene expression in vitro
Molecular and Cellular Endocrinology 198 (2002) 131 /141 www.elsevier.com/locate/mce
Effects of androgen-binding protein (ABP) on spermatid Tnp1 gene expression in vitro Julie Della-Maria a, Anne Gerard a,*, Patricia Franck b, Hubert Gerard a a
EA 3442 ‘Ge´ne´tique, Signalisation, Diffe´renciation’, De´partement de Cytologie, Histologie et Biologie du De´veloppement, Faculte´ de Me´decine, Universite´ Henri Poincare´ de Nancy, 9, avenue de la Foreˆt de Haye, BP 184, 54505 Vandoeuvre-le`s-Nancy Cedex, France b Laboratoire de Biochimie, Hoˆpital Central, CHU, 54000 Nancy, France
Abstract In vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur via a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat spermatids. Using semi-quantitative RT-PCR, the expression of the Tnp 1 gene encoding the Transition Protein 1, involved in the histone to protamine replacement during spermatid nuclear transformation, was analyzed. Our results provide clear evidence that Sertoli cell-derived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones. To our knowledge this is the first evidence of an effect of ABP itself on male germ cells. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rat androgen-binding protein (Sertoli cell ABP); Spermiogenesis; Tnp1 gene expression
1. Introduction Rat Androgen-binding Protein (rABP) is a major 90 kDa testicular glycoprotein encoded by the abp /shbg gene and produced by the Sertoli cells. ABP is known to bind, transport and concentrate sex steroids as well as to protect them from catabolism in the testicular fluids (Hagena¨s et al., 1975; Bardin et al., 1981; Joseph, 1994). In human testis, however, beside the testicular transcript encoding the entire steroid-binding protein, there are several differentially spliced transcripts encoding truncated proteins that are unable to bind steroids (Hammond et al., 1989; Joseph et al., 1996; Sullivan et al., 1993). This raises the question of potential functions for testicular ABP other than regulating the bioaviability of steroids. ABP/SHBG membrane binding sites have been demonstrated on spermatogenic cells in several animal species (Steinberger et al., 1984; Felden et al., 1992;
* Corresponding author. Tel.: /33-3-8359-2893; fax: /33-3-83592883 E-mail address: [email protected] (A. Gerard).
Porto et al., 1992; Bedjou et al., 1996). Internalized radio-labeled ABP and gold-labeled SHBG have been detected by electron microscopy within spermatogenic cells (Ge´rard et al., 1994). It was thus proposed that ABP could act as a paracrine factor produced by Sertoli cells and be involved in germ cell maturation. But, until now, demonstration of the physiological effect of ABP binding and/or internalization on spermatogenesis is thoroughly lacking. In this paper, in vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur through a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat germ cells. Paracrine/juxtacrine conditions were obtained by using bicameral chambers allowing Sertoli cells and germ cells to be cocultivated in direct contact or not. In an additional set of experiments, we investigated whether steroid adjunction (testosterone or 17b-estradiol) could modify the effects of ABP in these cocultures.
0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 2 ) 0 0 3 7 6 - 3
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Spermatids were chosen as target germ cells to test ABP action since we previously demonstrated specific binding and internalization of ABP within spermatids, as well as accumulation of cytoplasmic Sertoli ABP close to elongating spermatids (Ge´rard et al., 1994). The expression of the Tnp 1 gene encoding the Transition Protein 1 (TP1, Kistler et al., 1975, 1987), concerned in the histone-protamine exchange during the spermatid nuclear transformation, was selected as a marker to follow this critical event during spermiogenesis. Our results provided clear evidence that Sertoli cellderived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones.
2. Materials and methods
medium. The plasmid pRc/CMV-rat ABP cDNA was inserted into CHO cells using the polyethylenimine (PEI, Exgen 500, Euromedex, Strasbourg, France) transfection methodology previously used for mouse Sertoli cells (Ducray et al., 1998). Neomycin-resistant clones were selected using G418 containing culture medium during 20 days and checked for the presence of recombinant rat ABP in the culture medium by probing with a rabbit polyclonal antibody raised against rat testicular ABP or human SHBG. Transfected CHO cells producing recombinant rat ABP in the culture medium were pooled for use in the subsequent experiments and designated CHO-rABP (/). Untransfected CHO cells were also tested and designated CHO-rABP (/). Vector-only transfected Mouse Sertoli cells and CHO cells were checked for the presence of recombinant rABP and were always found negative.
2.1. Somatic cell lines 2.1.1. Sertoli cell lines The cell line derived from 15-day-old prepubertal Balb/c murine Sertoli cells, TM4 (Mather, 1980) was obtained from ECACC (European Collection of Animal Cell Cultures, Valbonne, France) and cultured at 37 8C, in Ham’s F12-Dulbecco’s Modified Eagle’s medium (DMEM, 1:1, vol/vol) supplemented with 2 mM Lglutamine, 10 IU/ml penicillin, 10 mg/ml streptomycin, 10 mg/ml insulin, 5 mg/ml transferrin and 5 ng/ml sodium selenite and 5% fetal calf serum (FCS) (all Sigma products, L’Isle d’Abeau, France), without FSH and testosterone supplementation. This Mouse Sertoli cell line was hereafter designated MSC-rABP (/) owing to the absence of rat ABP production. This cell line was stably transfected with a rat ABP construct, the plasmid pRc/CMV-containing the cloned 1.6 Kpb rat Androgen-Binding Protein (ABP) cDNA inserted into a Hind III/Xba I-digested pRc/CMV for eukaryotic expression, the neonmycin and ampicillin resistance genes for selection in mammalian cells and Escherichia Coli, respectively (kindly provided by G.L. Hammond). Neomycin-resistant clones selected by adding to the culture medium 200 mg Geneticin/ml for 3 weeks (G418, Life Technologies, Cergy Pontoise, France) were shown to produce recombinant rat ABP as already reported (Ducray et al., 1998). Neomycinresistant clones were pooled for use in the subsequent experiments and designated MSC-rABP (/). 2.1.2. CHO cell lines (Chinese Hamster ovary) Chinese Hamster ovary (CHO) cells were used to determine the effects of somatic, non testicular, cell lines. CHO cells were obtained from ECACC and maintained in culture at 37 8C, in the same culture
2.2. Adult rat spermatid preparation Germ cells were isolated from 60-day-old Lewis rat testes according to the enzymo-mechanic method previously described (Ge´rard et al., 1996) combining 1 mg/ ml collagenase (Roche, Meylan, France) and 25 U/ml hyaluronidase (Choay, Gentilly, France) in Ham’s F12DMEM (1:1, vol/vol) supplemented with 2 mM LGlutamine, 10 IU/ml pencillin, 10 mg/ml streptomycin, (Sigma products) at 32 8C for 30 min under mechanical agitation. Germ cell suspension was washed in Ham’s F12-DMEM (1:1, vol/vol) supplemented with 2 mM Lglutamine, 10 IU/ml penicillin, 10 mg/ml steptomycin, 0.2% bovine serum albumin (BSA, Roche), 20 mg/ml soybean trypsin inhibitor and 10 mg/ml of DNase I (Roche). Sertoli cell and spermatozoa were removed by a 2-step-filtration through a nylon 0.45 mm filter (Millipore, Molsheim, France) and sterile absorbent cotton wool. Germ cells were then fractionated by centrifugation through a 30/60% Percoll density gradient (Sigma) in Ham’s F12-DMEM (1:1, vol/vol) at 325/g for 2 h. The highly enriched fractions containing spermatids were pooled and used for coculture experiments after washing in the supplemented culture medium. The purity of the spermatid fractions was analyzed by phase-contrast microscopy. The proportion of round and elongated spermatids was evaluated using a hematimeter and was in a range of 54.869/1.12 and 34.789/ 2.92%, respectively (contaminating spermatocytes and residual bodies were 5.89/2.92 and 4.559/2.25%, respectively; n/4). Cell viability was checked using the trypan blue exclusion test and was found to be 93.839/1.73% (n /4).
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2.3. Cell cultures 2.3.1. Establishment of the feeder cell monolayers MSC-rABP (/), MSC-rABP (/), and CHO-rABP (/) cells were plated in 10-cm2 bicameral culture chambers (Nunc, Noisy-le Grand, France) at the density of 6 /104 cells per cm2 for Sertoli cells and 8 /104 cells per cm2 for CHO cells in 2 ml of the somatic culture medium cited above, for 24 h, at 37 8C in a humidified atmosphere of 95% air and 5% CO2. After 24 h, the feeder layer reached 80% confluency. 2.3.2. Adult rat spermatid*/feeder cell cocultures After 24 h, adherent feeder monolayers were renewed with 2 ml serum-free coculture medium containing Ham’s F12-DMEM (1:1, vol/vol) supplemented with 2 mM L-glutamine, 10 IU/ml penicillin, 10 mg/ml streptomycin, 10 mg/ml insulin, 5 mg/ml transferrin and 5 ng/ml sodium selenite and 0.65% DL-lactic acid and 1% sodium pyruvate (all Sigma products) and grown at a reduced temperature of 34 8C. Freshly fractionated spermatids were then added to the three somatic layers according to two different modes: either 1 ml containing 3 /106 spermatids was loaded on the somatic monolayer leading to direct germ cell-somatic cell contacts, or one ml containing 3 /106 spermatids was loaded onto the microporous membrane of the apical chamber of the bicameral culture dish (0.22 mm Millicell, Millipore) so that no cell /cell contact could be established between spermatids and the somatic cell monolayers growing in the basal chamber. 2.3.3. Adult rat spermatid monocultures For comparison purpose, 3/106 freshly isolated spermatids were cultured without any feeder layer in 3 ml of the same medium as used for cocultures and in the same conditions. 2.3.4. Protocols A total of four independent cultures were grown in each of the nine following different conditions: spermatids alone, spermatids cocultivated in direct contact with MSC-rABP (/) or MSC-rABP (/) or CHO-rABP (/) feeder layer, spermatids cocultivated without contact with MSC-rABP (/) or MSC-rABP (/) or CHO-rABP (/) feeder layer, spermatids cocultivated in direct contact with MSC-rABP (/) feeder layer in the presence of 50 ng/ml testosterone (Sigma) and spermatids cocultivated in direct contact with MSC-rABP (/) feeder layer in the presence of 50 ng/ml 17b-estradiol (Sigma). Each culture was carried out for 72 h at 34 8C in a humidified atmosphere of 95% air, 5% CO2, then processed for analysis. In preliminary experiments, CHO-rABP (/) feeder layers were also included in order to fulfil the compar-
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isons but they were found to be unable to sustain spermatid viability and to survive without serum and at 34 8C. 2.3.5. Sampling After 72 h, total cell number, adherence and viability cell counts were evaluated for each condition. Media were collected and stored at /20 8C until immunoblot analysis of rat ABP. Isolated spermatids cultured on the microporous membrane (without cell /cell contact condition) and spermatids in cell /cell contact with the three different feeder layer types were processed to RNA extraction. 2.4. Cell viability Viability was checked using the trypan blue exclusion test. In cocultures without contact, spermatids were collected from the apical chamber, pelleted and counted. In cell /cell contact cocultures, attached (cells recovered by trypsinization, 5 min in 0.5 ml of 0.125% trypsin (Sigma) at 34 8C) and unattached viable spermatids were scored. In the resulting mixed suspension spermatid counts were performed under phase-contrast microscopy according to morphological criteria. 2.5. Immunoblot analysis of medium ABP content 2.5.1. Collected media Media collected from the different culture types were concentrated first by dialysis against polyethylene glycol using the 6000 /8000 Spectra-POR dialysis membrane (Spectrum Medical Industries, Inc., CA, USA), second by centrifugation at 2200 /g at 4 8C for 3 /5 h on 5000 Da exclusion membrane (Ultrafree R-MC Centrifugal Filters Units, Millipore) leading to concentration factor of 80/100 fold. After dilution of the concentrate in PBS, 1 ml was blotted onto Hybond C-Extra membrane (0.45 mm, Amersham, Life Sciences, Les Ulis, France) and subjected to immunoblot analysis. 2.5.2. Immunoblot analysis Non-specific membrane binding was blocked by incubation in 0.05 M Tris-HCl buffer containing 0.15 NaCl (pH 5.3), 5% casein and 3% gelatin (Sigma) under agitation for 4 h at room temperature. Membranes were then subjected to immunodetection using a rabbit polyclonal antiserum against human SHBG kindly provided by C. Grenot (1:1000 in 0.05% Tris-HCl) under agitation overnight at 4 8C, then a 1 h 45 min treatment under agitation at room temperature with Horse radish peroxidase-labelled anti-rabbit IgG (Santa Cruz biotechnology, Tebu, Le Perray en Yvelines, France) at 1:8000 in 0.05% Tris-HCl buffer. The detection of the signal was performed by incubating the membrane in a chemiluminescent substrate solution
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(Luminol/H2O2, 1:0.1 (vol/vol), Covalab, Lyon, France) directly on the Kodak Digital Science 1DTM screen (KDS1D, Kodak, Rochester, NY, USA). The signal intensity was measured by the KDS1D quantification software. On each membrane, five pure hSHBG protein samples of defined increasing concentration ranging from 22.22 fmoles up to 111.11 fmoles were treated at the same time in order to calculate the concentration of immunopositive ABP/SHBG content in the samples. The specificity of the primary antibody was checked by omitting it and by preincubating the membrane with excess of the pure protein for 1 h before the primary antibody. These controls were always negative. Samples coming from diluted adult rat testicular fluid were also treated to check for rat/mouse ABP specificity and used as positive controls. Concentrated initial somatic culture media, initial coculture and 3day-spermatid monoculture media were analyzed on each membrane. 2.5.3. Analyzed samples ABP content was measured (1) in the medium from four independent cultures of the three feeder layer types cultivated in the presence of 5% FCS at 37 8C; (2) in the medium from four independent cultures of the three feeder layer types cultivated without FCS at 34 8C; (3) in the medium from four independent cultures of the eight different spermatid coculture conditions; (4) in the medium from four independent spermatid monocultures. 2.6. RT-PCR analysis of Tnp1 gene expression 2.6.1. RNA preparation Total RNA was extracted from all spermatid cocultures, spermatid monocultures and freshly isolated spermatids, and as control, from the feeder layer cultured at 34 8C using Tri Reagent as suggested by the manufacturer (Sigma). RNA quantity was assessed by optical density measurement at 260 nm using the Beckman DU-64 Spectrophotometer. 2.6.2. RT-PCR protocol cDNA were obtained from 150 ng total RNA by reverse transcription at 50 8C for 45 min using Avian Myeloblastosis Virus reverse transcriptase (0.4 U/ml) and then amplified using Accu Taq LA DNA polymerase (0.05 U/ml) in a final volume of 50 ml containing MgCl2 (3 mM), dNTPs (0.2 mM each), RNase inhibitor (0.8 U/ml) and specific primers (0.4 mM for TP1 and 0.1 mM for b-actin) by heating at 94 8C for 3 min, followed by 30 cycles of 1 min at 94 8C, 1 min at 59 8C, 1 min at 68 8C, and a final extension at 68 8C for 5 min (all products from Enhanced Avian RT-PCR kit, Sigma) in a PCR thermocycler (PCR Express, Hybaid). The selected primers were:
/ Rat TP1 primers: 26/50; 200 /176, size product: 175 bp. / Rat b-actin primers: 784/913; 1447 /1422, size product: 661 bp. PCR analysis was carried out from the logarithmic phase of amplification for the two genes. As control, sterile water was added instead of RNA in the RT-PCR mix and submitted to the RT-PCR reaction. RNA samples were also subjected to PCR amplification without reverse transcription to check for contaminating DNA. b-actin was used as control of the quality of the RNAs. We also used cyclophillin A which is constitutively expressed in testis as another internal control (data not shown). For each culture condition, the RT-PCR was performed in duplicate on RNA samples coming from three independent cultures. Five microliters of RT-PCR products were electrophoresed in parallel with DNA size marker (PCR low ladder, Sigma) on 1% agarose gel (Sigma) with 0.01% bromide ethidium (Bio-Rad, Marnes-la-Coquette, France) for 1 h 20 at 100 V and photographed under UV light using Gel Doc, 1000 UV fluorescent system (Bio-Rad, Ivry sur Seine, France). The presence of TP1 transcripts was also checked in freshly isolated adult rat spermatids and from adult rat testis as positive controls and in the three somatic feeder layers: MSC-rABP (/); MSC-rABP (/); CHO-rABP (/) as negative controls. 2.6.3. Semi quantitative RT-PCR product analysis Quantitative analysis of TP1 cDNA band intensity was performed by densitometry on pictures of the separation gel using the Gel Doc 1000 UV Fluorescent System and its quantitation software (Bio-Rad). The TP1 specific signal, obtained from RT-PCR in duplicate from three independent cultures of each condition, was normalized to the ß-actin signal. 2.7. cDNA sequencing In order to assess the specificity of Tnp1 gene expression, PCR products were separated from Ethidium bromide and agarose by centrifugation on New GenElute Minus EtBr Spin Columns (Supelco, Sigma) at 12000 /g for 10 min and sequenced using ABI PRISM Dye Terminator Sequencing Ready Reaction reagents (Perkin /Elmer, Roissy, France) according to manufacturer indications. The DNA sequences obtained after acrylamide separation and 373 A Applied Biosystems sequencer analysis (Perkin/Elmer) were checked by the BLAST search program and screened for homology against the Genbank EMBL. The percentage of identity with the expected sequence was 95% for TP1 transcripts.
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2.8. Statistical analysis All quantitative data (mRNA levels and ABP contents in the coculture medium) are presented as mean9/ S.E.M. Statistical comparisons referred to Student’s ttest for paired data when significant differences were detected by using the one-way analysis of variance (ANOVA). P 5/0.05 was considered statistically significant (StatMed).
3. Results 3.1. Spermatid viability index As shown in Table 1, direct contacts with MSC-rABP (/) or MSC-rABP (/) were beneficial to spermatid survival and there was no significant difference whether ABP was produced or not. The absence of contact reduced, but did not abolish, the positive effect exerted by the MSC feeder layer which, here also, was shown not to be dependent on the presence of ABP. Since, as demonstrated in preliminary experiments, cocultures performed with CHO-rABP (/) cell line could not maintain an acceptable spermatid viability index (data not shown), they were excluded from the experimental design. Regarding to CHO-rABP (/) cells, they were able to sustain partially spermatid survival but only when direct cell /cell contacts were allowed. Thus, whatever the feeder layer used (MSC-rABP (/), Table 1 Spermatid viability following 3 days coculture with the different feeder layers with or without cell /cell contact using the trypan blue exclusion test
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MSC-rABP (/), or CHO-rABP (/)), all coculture conditions allowing cell /cell contact enhanced significantly rat spermatid viability compared with rat spermatid monocultures.
3.2. Basal ABP secretion in the medium of the different feeder layers Using a quantitative immunoassay with a polyclonal anti-hSHBG antiserum which crossreacted with rat testicular ABP, we were able to quantify the level of immunopositive ABP secreted in the medium. As shown in Table 2, MSC-rABP (/) cells secreted the highest level of ABP (754.659/73.21 fmol/ml), while the non testicular cell line CHO-rABP (/) provided a 4.4 fold lower content (171.479/28.16 vs. 754.659/73.21 fmol/ml) when cultured at 37 8C and in the presence of FCS (Table 2, A). As expected from previous results (Ducray et al., 1998), no detectable immunopositive rABP was found in the culture medium of untransfected mouse Sertoli cells MSC-rABP (/). In the conditions selected for adult spermatid cocultures (reduced temperature, 34 8C and absence of FCS), the ABP content in transfected cell line media was diminished (Table 2, B). Again, MSC-rABP (/) cell line secreted more rABP than CHO-rABP (/) cells (114.039/1.19 vs. 75.829/19.53 fmol/ml). Nevertheless, these three feeder layers were efficient to produce ABP thus giving the possibility of a comparative study of the effects of the presence or the absence of ABP on cocultivated spermatids.
Culture conditions
Spermatid viability (%)
Table 2 Rat ABP detection in the medium collected from feeder cell cultures
Spermatid cocultures (3 days ) With contact/MSC-rABP () With contact/MSC-rABP () With contact/CHO-rABP () Without contact/MSC-rABP () Without contact/MSC-rABP () Without contact/CHO-rABP () Spermatid cocultures (3 days)
84.6692.83 80.5892.01 66.5394.10 68.3691.00 72.1690.86 55.3692.93 52.5092.93
Cell cultures
ABP content (fmol/ml)
A MSC-rABP () MSC-rABP () CHO-rABP ()
ND 754.65973.21a 171.47928.16c
B MSC-rABP () MSC-rABP () CHO-rABP ()
ND 114.0391.19b 75.82919.53
In cell /cell contact cocultures, adherent and non-adherent viable spermatids were counted in the removed media and in the mixed suspension obtained after trypsinization. In coculture without contact, spermatids were collected in the apical chamber, pelleted and counted. Spermatids from 3-day monocultures were also checked for viability. Results are expressed as percentage of the initial number of spermatids used for coculture and as the mean9S.E.M. of two counts realized on four independent cultures of each condition (n 4). The values were compared using the Student’s t -test. Differences were all significant at P 5 0.05 except for MSC-rABP () compared with MSC-rABP () in coculture with contact, for CHO-rABP () with contact compared with MSC-rABP (), to MSC-rABP () and to CHO-rABP () without contact, and for CHO-rABP () without contact compared with the spermatid monoculture.
(A) Feeder cell cultures were performed at 37 8C and supplemented with 5% of FCS. (B) Feeder cell cultures were performed at 34 8C in the absence of FCS. Media were collected after 3 days and rABP content was assessed by immunoquantitation using a rabbit polyclonal antiserum against human SHBG (1:1000) and densitometric analysis. Results are expressed as fmol/ml and as the mean9S.E.M., n 4. ABP was not detectable (ND) in MSC-rABP (). Differences in ABP content are significant between MSC-rABP () and CHO-rABP () at 37 8C. a, P 5 0.001 and at 34 8C; b, P 5 0.005; differences are significant between MSC-rABP () and CHO-rABP () at 37 vs. 34 8C, a, P 5 0.001; c, P 5 0.005.
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3.3. Rat Tnp1 gene expression A 175 bp cDNA band, corresponding to the amplification of rat Tnp1 gene, checked by sequencing (95% identity), was detected in all spermatid fractions. No Tnp1 gene expression was found in any of the three somatic feeder layer. As illustrated on Fig. 1, when cultured alone, spermatids showed a marked fall of Tnp1 gene expression. When cocultivated, spermatids showed varying transcript concentrations related to the presence or the absence of rABP, to the presence of cell /cell contacts and to the nature of the feeder layer. MSC-rABP (/) or CHO -rABP (/) in direct contact with spermatids strongly raised the level of TP1 transcripts by a factor 5 and by a factor 2, respectively, compared with the level of TP1 transcripts in spermatid monoculture. In this coculture mode, MSC-rABP (/) induced a highly increased level (/3.6) of TP1 mRNA compared with that obtained in the presence of MSC-rABP (/).
Moreover, TP1 mRNA level was enhanced by a factor 1.2 compared with the in vivo initial level. CHO-rABP (/) were clearly less effective than MSC-rABP (/). In spermatid cocultures avoiding cell /cell contact with the feeder layer cells, MSC-rABP (/) were unable to maintain the TP1 transcript concentration over that observed in spermatid monocultures or in cocultures with MSC-rABP (/). This level was 3.7-fold decreased compared with cell /cell contact cocultures with the same feeder layer. CHO-rABP (/) exhibited an adverse effect since the amount of TP1 transcript showed a 5fold reduction compared with spermatid monoculture. 3.4. Consequences of testosterone and b-estradiol adjunction regarding to rABP effects on spermatid Tnp1 mRNA level Since the most substantial effects of rABP were obtained when spermatids were cultivated in direct contacts with MSC-rABP (/) cells, both testosterone and 17b-estradiol adjunction experiments were conducted in these conditions (Fig. 1, column 7, 8). 3.4.1. Testosterone Testosterone adjunction in the culture medium led to cancellation of the up-regulation of Tnp1 gene expression elicited by the coculture of spermatids with MSCrABP (/) cells, thus resulting in a comparable level of TP1 mRNA to that observed initially, in vivo (freshly isolated spermatids). 3.4.2. 17 b-Estradiol 17 b-Estradiol adjunction in the medium induced a dramatic 91% fall of TP1 transcript number, that reached the very low value observed in spermatid monocultures. Indeed, b-estradiol showed an adverse effect on Tnp1 gene expression up-regulation elicited by coculture of spermatids with MSC-rABP (/). 3.5. Incidence of the various coculture conditions on ABP concentration in the medium
Fig. 1. Effects of the different culture conditions on rat TP1 mRNA following semi-quantitative RT-PCR analysis. Total RNAs from each culture condition were analyzed by RT-PCR for the expression of rat TP1 and rat b-actin (used as internal control of amplification and for TP1 signal intensity normalization). Values are the mean (columns)9/ S.E.M. (bars) of TP1/ß-actin ratio for six separate RT-PCR reactions. The values were compared using the Students’s t -test. Differences were significant at P 5/0.05 in column 0 vs. 3, in column 1 vs. 9, in column 4 vs. 6; P 5/0.005 in column 2 vs. 4, in column 3 vs. 8, in column 5 vs. 9; P 5/0.001 in column 0 vs. 1, 2, 3, 4, 5, 6, 7 and 9, in column 1 vs. 3, 4, 7 and 8, in column 2 vs. 3, 7, 8 and 9, in column 3 vs. 4, 5, 6, 7 and 9, in column 7 vs. 4, 5, 6 and 8, in column 8 vs. 4, 5, 6 and 9, in column 9 vs. 4 and 6.
As expected, in the media of MSC-rABP (/) no immunoreactive ABP was detected even in the presence of spermatids. 3.5.1. Effects of spermatid-feeder layer cell contacts Three different conditions were investigated: feeder layer cells alone, feeder layer cells and spermatids in direct contact, feeder layer cells and spermatids without contact (Fig. 2, columns 1/6). Concerning CHO-rABP (/) feeder layer cells, no significant differences have been showed between the three conditions, suggesting that no cross talk could take place between spermatids and CHO cells.
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4. Discussion
Fig. 2. Effects of the culture conditions on ABP secretion by the different rABP-transfected feeder layers. All cultures were realized at 34 8C in the same medium described in Section 2. In 1 and 4, the feeder layer cells were cultivated without spermatids. In 2, 5, 7 and 8, spermatids were directly loaded on the feeder layer cells. In 3 and 6, spermatids were deposited in the apical chamber of culture dish without cell /cell contact with the feeder layer cells. After 3 days, all media were collected and ABP content was assayed by immunoquantitation using a rabbit polyclonal antiserum against human SHBG (1:1000). Data are expressed as fmol/ml and as mean (columns)9/ S.E.M. (bars), n/4. P 5/0.05 in column 1 vs. 2, 4 and 6; P 5/0.005 in column 2 vs. 4, in column 4 vs. 7, in column 6 vs. 7; P 5/0.001 in column 1 vs. 3, 5 and 8, in column 2 vs. 3, 7 and 8, in column 4 vs. 8, in column 5 vs. 7 and 8, in column 6 vs. 8.
Concerning MSC-rABP (/) feeder layer cells, the establishment of direct contacts with spermatids led to clear changes in ABP level. Indeed, ABP content was about a factor of 1.4 higher than in the medium without spermatids (162.869/32.99 vs. 114.039/1.19 fmol/ml). When spermatids were not in contact with MSC-rABP (/), ABP level was decreased by about one half of the cell /cell contact level (62.159/11.90 vs. 162.869/32.99 fmol/ml). Paradoxically, this level was also below that obtained in complete absence of spermatids. 3.5.2. Sex-steroid hormone adjunction Since coculture of spermatids in direct contacts with MSC-rABP (/) cells was proved the most efficient condition regarding to ABP delivery in the medium, investigations of the consequences of testosterone and 17ß-estradiol adjunction on ABP level were performed in this situation only (Fig. 2, column 7, 8). Clearly, the adjunction of 50 ng/ml testosterone or 50 ng/ml of 17ßestradiol in the coculture medium for 3 days did not lead to significant changes in the level of secreted ABP.
Spermiogenesis provides a valuable opportunity to explore the putative role of ABP on germ cell maturation. ABP concentrates in the apical part of the Sertoli cells at stages VII/IX in the adult male rat, which makes it temporally and spatially available to regulate spermiogenic events (Attramadal et al., 1981). Studies on mutant restricted rats (Hre), and hypophysectomized pregnenolone-injected rats showed a positive correlation between sperm fertilizing ability and the levels of ABP (Anthony et al., 1984). Indirect experiments from Huang et al. (1991) clearly established that ABP status and the maintenance of spermiogenesis were linked. Roberts and Zirkin (1993) proposed that ABP could be involved in the failure of advanced spermatid production by inhibition of Sertoli cell testosterone action, in vitro. Finally, in transgenic 11shbg mice, high levels of SHBG mRNA were accumulated in Sertoli cell luminal cytoplasm close to step 9/12 spermatids (Ja¨nne et al., 1998). The coculture system used here clearly demonstrated its efficiency in sustaining rat spermatid survival as well as in producing a detectable concentration of rat ABP in the medium during the whole experimental procedure. That mouse Sertoli-derived cell lines, secreting or not ABP, were beneficial to germ cells is not surprising since Sertoli cell factors are able to maintain spermatid viability (Cameron et al., 1991) and cocultures with TM4 cells improved germ cell survival (De Felici and Dolci, 1991). This was not the case for CHO cells since we did not succeed in coculturing germ cells with this cell line at 34 8C and without serum. Unexpectedly, rABP-transfected CHO cells revealed, on the contrary, their capacity to grow at 34 8C without fetal calf serum and to support spermatid survival. It is not clear for the moment whether ABP accumulation within the cytoplasm of transfected CHO cells could contribute to such an adaptation. This intriguing observation warrants further study. Concerning the evaluation of the putative role of ABP during spermiogenesis, the Tnp 1 gene encoding the TP1 (Kistler et al., 1987, 1975) was chosen to serve as marker of spermatid physiology. In the rat, Tnp1 gene exhibits the highest level of expression (Mali et al., 1989) when the ABP secretion peak is maximal, from the step VIII up to X of the seminiferous epithelium cycle (Parvinen et al., 1986). TP1 transcripts are stored in round spermatid cytoplasm from step VII/VIII and then translated with a delay from the step XI of the seminiferous cycle (Marret et al., 1998). When Tnp1 is not expressed in human testis, an important failure of spermatogenesis characterized by a round spermatid arrest, is observed (Steger et al., 1999). In Tnp1/ mice fertility is reduced (60% of males were infertile). In addition, an abnormal pattern of chromatin condensation and a severe reduction of sperm motility are observed (Yu et al., 2000).
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However, regulation factors of Tnp1 gene, both at transcriptional and translational levels, remain poorly understood. Our results clearly show that monocultures for 3 days in a serum-, FSH-, steroid- and ABP- free medium were unable to maintain rat spermatid TP1 mRNA at a level higher than approximately 25% of the initial value (freshly isolated spermatids */column 0, Fig. 1). By contrast, spermatid coculture in contact with CHO or Sertoli cells producing recombinant rat ABP, were able to rescue and, even, to upregulate Tnp1 gene expression. The different coculture conditions demonstrated TP1 mRNA level resulting from the presence of rABP in the culture medium to be also dependent on the nature of the feeder layer cells and on the adjunction of sex steroids in the medium. First of all, it must be stressed that MSC-rABP (/) feeder layer whether or not in direct contact with spermatids, was not able to rescue TP1 mRNA level in spermatids (Fig. 1, columns 2, 5) although its efficiency in maintaining spermatid survival was demonstrated (Table 1). Thus, the fall of TP1 mRNA level observed in spermatids cannot be held as a consequence of poor survival at 3 days but should be related to the absence of some specific factor in the spermatid environment. From all situations tested, the highest Tnp1 expression was observed when two conditions were combined: presence of rABP and direct contacts between spermatids and feeder layer cells, whatever their origin (column 3,4, Fig. 1). Yet, Tnp1 expression was twice and a half higher in the case of MSC- rABP (/) contact than CHO- rABP (/) one. Sequence comparisons showed ABP to be related to the C-terminal globular (G) domains of laminin and other extracellular matrix and regulatory proteins such as protein S, Gas6 and agrin (Joseph, 1997). Germ cells could possess corresponding receptors as for example spermatids having laminin receptors (Fulcher et al., 1993). Thus ABP may possibly act as an extracellular ligand at the cell /cell interface eliciting signal responses in spermatids, leading to Tnp1 gene transcription stimulation. This hypothesis is supported by the finding of a higher adhesion index of germ cells when plated on ABP-producing Sertoli cells (manuscript in preparation) and by the formation of multiple cytoplasm ramifications by TM4 cells after rABP transfection, compared with untransfected ones (Ducray et al., 1998), which could expand the surface of the membrane available for MSC- rABP (/)/spermatid interactions. The two latter phenomena were not observed in the case of CHO-rABP (/) feeder layers. Most likely spermatid/MSC-rABP (/) direct contacts better mimic the in vivo situation allowing juxtacrine communications to occur. Such a situation could be responsible for a higher concentration of ABP at the Sertoli/germ cell interface than in the coculture medium, or for an enhanced uptake of the
binding protein by spermatids. This is supported by the earlier demonstration of a rapid transfer of labelledABP from Sertoli cells to germ cells (Ge´rard et al., 1994; Ge´rard, 1995). This is also consistent with the presence of large amounts of SHBG mRNA within the Sertoli cell cytoplasm close to step 9 /12 spermatids from transgenic mice expressing the human shbg gene (Ja¨nne et al., 1998) and accumulation of immunopositive rat ABP in apical Sertoli cell cytoplasm along the plasma membrane of elongating spermatids (Attramadal et al. 1981; Gerard, data not shown). A quite different way of thinking is to look at the effective concentration of ABP in the culture medium owing to the variations observed in relation to coculture conditions. As shown in the Section 3, by far the highest concentration of immunoreactive ABP was found when spermatids were cocultivated in contact with MSCrABP (/). This is not surprising since positive regulation of germ cells on ABP secretion by Sertoli cells is well documented (Galdieri et al., 1984; Le Magueresse and Je´gou, 1988) as are the negative effects of germ cells depletion (Bardin et al., 1993). In our experiments, the highest Tnp1 gene expression was correlated with the highest ABP concentration. Conversely, coculture conditions leading to low ABP concentrations in the medium were also the conditions of low expression, thus suggesting a possible dose-dependent effect of ABP. Although these first sets of experiments were only focussed on the search for a putative role of ABP on spermiogenesis and not to disclose the mechanisms involved, two hypothesis could be proposed regarding to the control of TP1 mRNA level by ABP, which could serve as guidelines for further experiments. Even in the absence of sex steroids, SHBG, the plasmatic equivalent of ABP, is able to considerably increase intracellular cAMP level in different target cells in vitro (Queipo et al., 1998; Fortunati et al., 1999). Since Tnp1 gene in a variety of species, including man, contains cAMPresponse elements (CREs) in the promoter region (Oliva and Dixon, 1991; Kistler et al., 1994). ABP in elevating cAMP level in spermatids, might stimulate Tnp1 gene expression. Alternatively, TP1 mRNAs are stored in a translationally repressed state for several days and then translate within elongating spermatids (Hecht, 1990; Morales et al., 1991; Eddy et al., 1993). If ABP can maintain repression or interfere with depression mechanisms, TP1 mRNA would have also be found at conserved level in spermatid after 3 days of culture, as was effectively observed in our experiments. Testosterone and estradiol are both ABP ligands, the highest affinity being for androgens. Adding testosterone or estradiol to the culture medium before plating spermatids on the feeder layer cells resulted in a statistically significant lower spermatid TP1 mRNA content, implying the liganded form of ABP to be less effective than the ligand-free one. Since ABP membrane-
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receptors have been shown to be present on germ cell lineage (Steinberger et al., 1984; Felden et al., 1992), this could suggest a similar mechanism as for SHBG, whose binding on human prostatic cell membranes RSHBG is prevented by prior liganding to sex steroid hormones (Hryb et al., 1990). Nevertheless, it must be stressed that adding testosterone led to a decreased spermatid TP1 mRNA content decreased not below the value exhibited by freshly isolated spermatids, thus representative of the intratesticular physiological level, while adding estradiol drastically reduced this content approximately to one tenth of the initial value. The discrepancy between the results obtained in adding one or the other sex hormone indicates that, on its own, sex hormone liganding of ABP cannot be here the cause, or at least the unique cause, of the fall of spermatid TP1 mRNA content compared with the level observed when using unliganted ABP. The highly different effects observed on spermatids lead to the conclusion of an action depending mainly on sex hormones themselves. It is generally accepted that androgen receptors are not present in spermatids (Sharpe, 1994). On the opposite, both ERbmRNA and ERb-protein are revealed in round spermatids from stage I/VIII of the seminiferous epithelium (Saunders et al., 1998; van Pelt et al., 1999). This could account for the striking differences observed between the effects of the two sex steroid hormones since, contrary to testosterone, estradiol would have operated directly on spermatids, thus interfering with Tnp1 gene ABP-effect. Based on TP1 mRNA content, our in vitro results evoked an antagonist effect of estradiol against ABP action on spermatids. Interestingly, in vivo, ERbmRNA and ERb-protein are at their highest level in spermatids when seminiferous ABP content is at its lowest. In summary, by using TP1 mRNA quantification as marker to follow up rat spermatid maturation cocultivated with somatic cells transfected or not with a rat ABP gene construct, this work clearly demonstrates that ABP itself could be involved, namely in the absence of sex-steroid hormone as well as in the absence of other Sertoli cell products, in spermiogenetic events related to Tnp1 gene expression. This substantiates previous findings from different groups supporting the idea that ABP would not have to be confined in the role of a sex-steroid hormone carrier, protecting them from degradation and controlling their bioavailability. In the same way Queipo et al. (1998) recently established that SHBG, the plasmatic equivalent of ABP, encoded by the same gene, is able to raise both hCG and cAMP concentrations in cultured human cytotrophoblasts, in the absence of sex-steroid ligand. Moreover, Fortunati et al. (1999), demonstrated that SHBG, even without estradiol supply, caused a significant increase of cAMP in breast cancer cells MCF-7 maintained in serum-free medium.
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A direct action of ABP on germ cells may have functional consequences of importance within the testis, especially on seminiferous epithelium cycle control as well as at critical periods such as spermatogenesis onset, which is associated with changes both in intratesticular ABP level and in apical/basal direction of its secretion by Sertoli cells (Gunsalus et al., 1980). Yet ABP must be held as only one of the multile factors integrated in spermatogenesis control, thus rendering delineation fairly complicated to establish from in vivo studies. Among the factors suspected interfering with ABP, two of them seemed to clearly emerge from our in vitro results concerning TP1 mRNA. The first one consists in the expected modulation of ABP effects by sex-steroid hormones. The question open, however, as to whether sex steroids are acting through allosteric changes of the protein in a same manner as demonstrated by Rosner et al. (1999) for SHBG, or in modifying the synthesis of factors involved in target cell sensitivity to ABP. The crucial effect of estradiol on spermatid TP1 mRNA is in favor of the latter hypothesis. The second one consists in the requirement of direct membrane/membrane contacts between spermatids and ABP producing cells from testicular (MSC-rABP (/) and, to a lesser extent, from non testicular (CHO-rABP (/)) origin, to obtain ABP effects on Tnp1 gene activation. This points out that spatial relationships between germ cells and somatic cells, within the seminiferous tubules, are probably of utmost importance for spermatogenesis progression and have to be carefully investigated when attempting to achieve spermatogenesis in vitro.
Acknowledgements We are grateful to G.L. Hammond (The University of Western Ontario, LRCC Cancer Research Laboratory, London, Ont., Canada) for the gift of the plasmid pRc/ CMV- rat Androgen-Binding Protein (ABP), to Dr C. Grenot (INSERM U 329, Lyon, France) for providing pure human SHBG and polyclonal anti-SHBG. The authors also thank Professor C. Branlant, Dr A. Mougin for Gel doc facilities (UMR 7567 ‘Maturation des ARN et Enzymologie mole´culaire’, UHP, Nancy, France), Professor J.L. Olivier (Laboratoire de Biochimie, Hoˆpital central, CHU, Nancy, France) for Kodak image analysis facilities, M. Adam, B. Cunin, R. Verdun for technical assistance. This work was supported by FARO.
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Effects of androgen-binding protein (ABP) on spermatid Tnp1 gene expression in vitro Julie Della-Maria a, Anne Gerard a,*, Patricia Franck b, Hubert Gerard a a
EA 3442 ‘Ge´ne´tique, Signalisation, Diffe´renciation’, De´partement de Cytologie, Histologie et Biologie du De´veloppement, Faculte´ de Me´decine, Universite´ Henri Poincare´ de Nancy, 9, avenue de la Foreˆt de Haye, BP 184, 54505 Vandoeuvre-le`s-Nancy Cedex, France b Laboratoire de Biochimie, Hoˆpital Central, CHU, 54000 Nancy, France
Abstract In vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur via a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat spermatids. Using semi-quantitative RT-PCR, the expression of the Tnp 1 gene encoding the Transition Protein 1, involved in the histone to protamine replacement during spermatid nuclear transformation, was analyzed. Our results provide clear evidence that Sertoli cell-derived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones. To our knowledge this is the first evidence of an effect of ABP itself on male germ cells. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rat androgen-binding protein (Sertoli cell ABP); Spermiogenesis; Tnp1 gene expression
1. Introduction Rat Androgen-binding Protein (rABP) is a major 90 kDa testicular glycoprotein encoded by the abp /shbg gene and produced by the Sertoli cells. ABP is known to bind, transport and concentrate sex steroids as well as to protect them from catabolism in the testicular fluids (Hagena¨s et al., 1975; Bardin et al., 1981; Joseph, 1994). In human testis, however, beside the testicular transcript encoding the entire steroid-binding protein, there are several differentially spliced transcripts encoding truncated proteins that are unable to bind steroids (Hammond et al., 1989; Joseph et al., 1996; Sullivan et al., 1993). This raises the question of potential functions for testicular ABP other than regulating the bioaviability of steroids. ABP/SHBG membrane binding sites have been demonstrated on spermatogenic cells in several animal species (Steinberger et al., 1984; Felden et al., 1992;
* Corresponding author. Tel.: /33-3-8359-2893; fax: /33-3-83592883 E-mail address: [email protected] (A. Gerard).
Porto et al., 1992; Bedjou et al., 1996). Internalized radio-labeled ABP and gold-labeled SHBG have been detected by electron microscopy within spermatogenic cells (Ge´rard et al., 1994). It was thus proposed that ABP could act as a paracrine factor produced by Sertoli cells and be involved in germ cell maturation. But, until now, demonstration of the physiological effect of ABP binding and/or internalization on spermatogenesis is thoroughly lacking. In this paper, in vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur through a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat germ cells. Paracrine/juxtacrine conditions were obtained by using bicameral chambers allowing Sertoli cells and germ cells to be cocultivated in direct contact or not. In an additional set of experiments, we investigated whether steroid adjunction (testosterone or 17b-estradiol) could modify the effects of ABP in these cocultures.
0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 2 ) 0 0 3 7 6 - 3
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Spermatids were chosen as target germ cells to test ABP action since we previously demonstrated specific binding and internalization of ABP within spermatids, as well as accumulation of cytoplasmic Sertoli ABP close to elongating spermatids (Ge´rard et al., 1994). The expression of the Tnp 1 gene encoding the Transition Protein 1 (TP1, Kistler et al., 1975, 1987), concerned in the histone-protamine exchange during the spermatid nuclear transformation, was selected as a marker to follow this critical event during spermiogenesis. Our results provided clear evidence that Sertoli cellderived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones.
2. Materials and methods
medium. The plasmid pRc/CMV-rat ABP cDNA was inserted into CHO cells using the polyethylenimine (PEI, Exgen 500, Euromedex, Strasbourg, France) transfection methodology previously used for mouse Sertoli cells (Ducray et al., 1998). Neomycin-resistant clones were selected using G418 containing culture medium during 20 days and checked for the presence of recombinant rat ABP in the culture medium by probing with a rabbit polyclonal antibody raised against rat testicular ABP or human SHBG. Transfected CHO cells producing recombinant rat ABP in the culture medium were pooled for use in the subsequent experiments and designated CHO-rABP (/). Untransfected CHO cells were also tested and designated CHO-rABP (/). Vector-only transfected Mouse Sertoli cells and CHO cells were checked for the presence of recombinant rABP and were always found negative.
2.1. Somatic cell lines 2.1.1. Sertoli cell lines The cell line derived from 15-day-old prepubertal Balb/c murine Sertoli cells, TM4 (Mather, 1980) was obtained from ECACC (European Collection of Animal Cell Cultures, Valbonne, France) and cultured at 37 8C, in Ham’s F12-Dulbecco’s Modified Eagle’s medium (DMEM, 1:1, vol/vol) supplemented with 2 mM Lglutamine, 10 IU/ml penicillin, 10 mg/ml streptomycin, 10 mg/ml insulin, 5 mg/ml transferrin and 5 ng/ml sodium selenite and 5% fetal calf serum (FCS) (all Sigma products, L’Isle d’Abeau, France), without FSH and testosterone supplementation. This Mouse Sertoli cell line was hereafter designated MSC-rABP (/) owing to the absence of rat ABP production. This cell line was stably transfected with a rat ABP construct, the plasmid pRc/CMV-containing the cloned 1.6 Kpb rat Androgen-Binding Protein (ABP) cDNA inserted into a Hind III/Xba I-digested pRc/CMV for eukaryotic expression, the neonmycin and ampicillin resistance genes for selection in mammalian cells and Escherichia Coli, respectively (kindly provided by G.L. Hammond). Neomycin-resistant clones selected by adding to the culture medium 200 mg Geneticin/ml for 3 weeks (G418, Life Technologies, Cergy Pontoise, France) were shown to produce recombinant rat ABP as already reported (Ducray et al., 1998). Neomycinresistant clones were pooled for use in the subsequent experiments and designated MSC-rABP (/). 2.1.2. CHO cell lines (Chinese Hamster ovary) Chinese Hamster ovary (CHO) cells were used to determine the effects of somatic, non testicular, cell lines. CHO cells were obtained from ECACC and maintained in culture at 37 8C, in the same culture
2.2. Adult rat spermatid preparation Germ cells were isolated from 60-day-old Lewis rat testes according to the enzymo-mechanic method previously described (Ge´rard et al., 1996) combining 1 mg/ ml collagenase (Roche, Meylan, France) and 25 U/ml hyaluronidase (Choay, Gentilly, France) in Ham’s F12DMEM (1:1, vol/vol) supplemented with 2 mM LGlutamine, 10 IU/ml pencillin, 10 mg/ml streptomycin, (Sigma products) at 32 8C for 30 min under mechanical agitation. Germ cell suspension was washed in Ham’s F12-DMEM (1:1, vol/vol) supplemented with 2 mM Lglutamine, 10 IU/ml penicillin, 10 mg/ml steptomycin, 0.2% bovine serum albumin (BSA, Roche), 20 mg/ml soybean trypsin inhibitor and 10 mg/ml of DNase I (Roche). Sertoli cell and spermatozoa were removed by a 2-step-filtration through a nylon 0.45 mm filter (Millipore, Molsheim, France) and sterile absorbent cotton wool. Germ cells were then fractionated by centrifugation through a 30/60% Percoll density gradient (Sigma) in Ham’s F12-DMEM (1:1, vol/vol) at 325/g for 2 h. The highly enriched fractions containing spermatids were pooled and used for coculture experiments after washing in the supplemented culture medium. The purity of the spermatid fractions was analyzed by phase-contrast microscopy. The proportion of round and elongated spermatids was evaluated using a hematimeter and was in a range of 54.869/1.12 and 34.789/ 2.92%, respectively (contaminating spermatocytes and residual bodies were 5.89/2.92 and 4.559/2.25%, respectively; n/4). Cell viability was checked using the trypan blue exclusion test and was found to be 93.839/1.73% (n /4).
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2.3. Cell cultures 2.3.1. Establishment of the feeder cell monolayers MSC-rABP (/), MSC-rABP (/), and CHO-rABP (/) cells were plated in 10-cm2 bicameral culture chambers (Nunc, Noisy-le Grand, France) at the density of 6 /104 cells per cm2 for Sertoli cells and 8 /104 cells per cm2 for CHO cells in 2 ml of the somatic culture medium cited above, for 24 h, at 37 8C in a humidified atmosphere of 95% air and 5% CO2. After 24 h, the feeder layer reached 80% confluency. 2.3.2. Adult rat spermatid*/feeder cell cocultures After 24 h, adherent feeder monolayers were renewed with 2 ml serum-free coculture medium containing Ham’s F12-DMEM (1:1, vol/vol) supplemented with 2 mM L-glutamine, 10 IU/ml penicillin, 10 mg/ml streptomycin, 10 mg/ml insulin, 5 mg/ml transferrin and 5 ng/ml sodium selenite and 0.65% DL-lactic acid and 1% sodium pyruvate (all Sigma products) and grown at a reduced temperature of 34 8C. Freshly fractionated spermatids were then added to the three somatic layers according to two different modes: either 1 ml containing 3 /106 spermatids was loaded on the somatic monolayer leading to direct germ cell-somatic cell contacts, or one ml containing 3 /106 spermatids was loaded onto the microporous membrane of the apical chamber of the bicameral culture dish (0.22 mm Millicell, Millipore) so that no cell /cell contact could be established between spermatids and the somatic cell monolayers growing in the basal chamber. 2.3.3. Adult rat spermatid monocultures For comparison purpose, 3/106 freshly isolated spermatids were cultured without any feeder layer in 3 ml of the same medium as used for cocultures and in the same conditions. 2.3.4. Protocols A total of four independent cultures were grown in each of the nine following different conditions: spermatids alone, spermatids cocultivated in direct contact with MSC-rABP (/) or MSC-rABP (/) or CHO-rABP (/) feeder layer, spermatids cocultivated without contact with MSC-rABP (/) or MSC-rABP (/) or CHO-rABP (/) feeder layer, spermatids cocultivated in direct contact with MSC-rABP (/) feeder layer in the presence of 50 ng/ml testosterone (Sigma) and spermatids cocultivated in direct contact with MSC-rABP (/) feeder layer in the presence of 50 ng/ml 17b-estradiol (Sigma). Each culture was carried out for 72 h at 34 8C in a humidified atmosphere of 95% air, 5% CO2, then processed for analysis. In preliminary experiments, CHO-rABP (/) feeder layers were also included in order to fulfil the compar-
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isons but they were found to be unable to sustain spermatid viability and to survive without serum and at 34 8C. 2.3.5. Sampling After 72 h, total cell number, adherence and viability cell counts were evaluated for each condition. Media were collected and stored at /20 8C until immunoblot analysis of rat ABP. Isolated spermatids cultured on the microporous membrane (without cell /cell contact condition) and spermatids in cell /cell contact with the three different feeder layer types were processed to RNA extraction. 2.4. Cell viability Viability was checked using the trypan blue exclusion test. In cocultures without contact, spermatids were collected from the apical chamber, pelleted and counted. In cell /cell contact cocultures, attached (cells recovered by trypsinization, 5 min in 0.5 ml of 0.125% trypsin (Sigma) at 34 8C) and unattached viable spermatids were scored. In the resulting mixed suspension spermatid counts were performed under phase-contrast microscopy according to morphological criteria. 2.5. Immunoblot analysis of medium ABP content 2.5.1. Collected media Media collected from the different culture types were concentrated first by dialysis against polyethylene glycol using the 6000 /8000 Spectra-POR dialysis membrane (Spectrum Medical Industries, Inc., CA, USA), second by centrifugation at 2200 /g at 4 8C for 3 /5 h on 5000 Da exclusion membrane (Ultrafree R-MC Centrifugal Filters Units, Millipore) leading to concentration factor of 80/100 fold. After dilution of the concentrate in PBS, 1 ml was blotted onto Hybond C-Extra membrane (0.45 mm, Amersham, Life Sciences, Les Ulis, France) and subjected to immunoblot analysis. 2.5.2. Immunoblot analysis Non-specific membrane binding was blocked by incubation in 0.05 M Tris-HCl buffer containing 0.15 NaCl (pH 5.3), 5% casein and 3% gelatin (Sigma) under agitation for 4 h at room temperature. Membranes were then subjected to immunodetection using a rabbit polyclonal antiserum against human SHBG kindly provided by C. Grenot (1:1000 in 0.05% Tris-HCl) under agitation overnight at 4 8C, then a 1 h 45 min treatment under agitation at room temperature with Horse radish peroxidase-labelled anti-rabbit IgG (Santa Cruz biotechnology, Tebu, Le Perray en Yvelines, France) at 1:8000 in 0.05% Tris-HCl buffer. The detection of the signal was performed by incubating the membrane in a chemiluminescent substrate solution
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(Luminol/H2O2, 1:0.1 (vol/vol), Covalab, Lyon, France) directly on the Kodak Digital Science 1DTM screen (KDS1D, Kodak, Rochester, NY, USA). The signal intensity was measured by the KDS1D quantification software. On each membrane, five pure hSHBG protein samples of defined increasing concentration ranging from 22.22 fmoles up to 111.11 fmoles were treated at the same time in order to calculate the concentration of immunopositive ABP/SHBG content in the samples. The specificity of the primary antibody was checked by omitting it and by preincubating the membrane with excess of the pure protein for 1 h before the primary antibody. These controls were always negative. Samples coming from diluted adult rat testicular fluid were also treated to check for rat/mouse ABP specificity and used as positive controls. Concentrated initial somatic culture media, initial coculture and 3day-spermatid monoculture media were analyzed on each membrane. 2.5.3. Analyzed samples ABP content was measured (1) in the medium from four independent cultures of the three feeder layer types cultivated in the presence of 5% FCS at 37 8C; (2) in the medium from four independent cultures of the three feeder layer types cultivated without FCS at 34 8C; (3) in the medium from four independent cultures of the eight different spermatid coculture conditions; (4) in the medium from four independent spermatid monocultures. 2.6. RT-PCR analysis of Tnp1 gene expression 2.6.1. RNA preparation Total RNA was extracted from all spermatid cocultures, spermatid monocultures and freshly isolated spermatids, and as control, from the feeder layer cultured at 34 8C using Tri Reagent as suggested by the manufacturer (Sigma). RNA quantity was assessed by optical density measurement at 260 nm using the Beckman DU-64 Spectrophotometer. 2.6.2. RT-PCR protocol cDNA were obtained from 150 ng total RNA by reverse transcription at 50 8C for 45 min using Avian Myeloblastosis Virus reverse transcriptase (0.4 U/ml) and then amplified using Accu Taq LA DNA polymerase (0.05 U/ml) in a final volume of 50 ml containing MgCl2 (3 mM), dNTPs (0.2 mM each), RNase inhibitor (0.8 U/ml) and specific primers (0.4 mM for TP1 and 0.1 mM for b-actin) by heating at 94 8C for 3 min, followed by 30 cycles of 1 min at 94 8C, 1 min at 59 8C, 1 min at 68 8C, and a final extension at 68 8C for 5 min (all products from Enhanced Avian RT-PCR kit, Sigma) in a PCR thermocycler (PCR Express, Hybaid). The selected primers were:
/ Rat TP1 primers: 26/50; 200 /176, size product: 175 bp. / Rat b-actin primers: 784/913; 1447 /1422, size product: 661 bp. PCR analysis was carried out from the logarithmic phase of amplification for the two genes. As control, sterile water was added instead of RNA in the RT-PCR mix and submitted to the RT-PCR reaction. RNA samples were also subjected to PCR amplification without reverse transcription to check for contaminating DNA. b-actin was used as control of the quality of the RNAs. We also used cyclophillin A which is constitutively expressed in testis as another internal control (data not shown). For each culture condition, the RT-PCR was performed in duplicate on RNA samples coming from three independent cultures. Five microliters of RT-PCR products were electrophoresed in parallel with DNA size marker (PCR low ladder, Sigma) on 1% agarose gel (Sigma) with 0.01% bromide ethidium (Bio-Rad, Marnes-la-Coquette, France) for 1 h 20 at 100 V and photographed under UV light using Gel Doc, 1000 UV fluorescent system (Bio-Rad, Ivry sur Seine, France). The presence of TP1 transcripts was also checked in freshly isolated adult rat spermatids and from adult rat testis as positive controls and in the three somatic feeder layers: MSC-rABP (/); MSC-rABP (/); CHO-rABP (/) as negative controls. 2.6.3. Semi quantitative RT-PCR product analysis Quantitative analysis of TP1 cDNA band intensity was performed by densitometry on pictures of the separation gel using the Gel Doc 1000 UV Fluorescent System and its quantitation software (Bio-Rad). The TP1 specific signal, obtained from RT-PCR in duplicate from three independent cultures of each condition, was normalized to the ß-actin signal. 2.7. cDNA sequencing In order to assess the specificity of Tnp1 gene expression, PCR products were separated from Ethidium bromide and agarose by centrifugation on New GenElute Minus EtBr Spin Columns (Supelco, Sigma) at 12000 /g for 10 min and sequenced using ABI PRISM Dye Terminator Sequencing Ready Reaction reagents (Perkin /Elmer, Roissy, France) according to manufacturer indications. The DNA sequences obtained after acrylamide separation and 373 A Applied Biosystems sequencer analysis (Perkin/Elmer) were checked by the BLAST search program and screened for homology against the Genbank EMBL. The percentage of identity with the expected sequence was 95% for TP1 transcripts.
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2.8. Statistical analysis All quantitative data (mRNA levels and ABP contents in the coculture medium) are presented as mean9/ S.E.M. Statistical comparisons referred to Student’s ttest for paired data when significant differences were detected by using the one-way analysis of variance (ANOVA). P 5/0.05 was considered statistically significant (StatMed).
3. Results 3.1. Spermatid viability index As shown in Table 1, direct contacts with MSC-rABP (/) or MSC-rABP (/) were beneficial to spermatid survival and there was no significant difference whether ABP was produced or not. The absence of contact reduced, but did not abolish, the positive effect exerted by the MSC feeder layer which, here also, was shown not to be dependent on the presence of ABP. Since, as demonstrated in preliminary experiments, cocultures performed with CHO-rABP (/) cell line could not maintain an acceptable spermatid viability index (data not shown), they were excluded from the experimental design. Regarding to CHO-rABP (/) cells, they were able to sustain partially spermatid survival but only when direct cell /cell contacts were allowed. Thus, whatever the feeder layer used (MSC-rABP (/), Table 1 Spermatid viability following 3 days coculture with the different feeder layers with or without cell /cell contact using the trypan blue exclusion test
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MSC-rABP (/), or CHO-rABP (/)), all coculture conditions allowing cell /cell contact enhanced significantly rat spermatid viability compared with rat spermatid monocultures.
3.2. Basal ABP secretion in the medium of the different feeder layers Using a quantitative immunoassay with a polyclonal anti-hSHBG antiserum which crossreacted with rat testicular ABP, we were able to quantify the level of immunopositive ABP secreted in the medium. As shown in Table 2, MSC-rABP (/) cells secreted the highest level of ABP (754.659/73.21 fmol/ml), while the non testicular cell line CHO-rABP (/) provided a 4.4 fold lower content (171.479/28.16 vs. 754.659/73.21 fmol/ml) when cultured at 37 8C and in the presence of FCS (Table 2, A). As expected from previous results (Ducray et al., 1998), no detectable immunopositive rABP was found in the culture medium of untransfected mouse Sertoli cells MSC-rABP (/). In the conditions selected for adult spermatid cocultures (reduced temperature, 34 8C and absence of FCS), the ABP content in transfected cell line media was diminished (Table 2, B). Again, MSC-rABP (/) cell line secreted more rABP than CHO-rABP (/) cells (114.039/1.19 vs. 75.829/19.53 fmol/ml). Nevertheless, these three feeder layers were efficient to produce ABP thus giving the possibility of a comparative study of the effects of the presence or the absence of ABP on cocultivated spermatids.
Culture conditions
Spermatid viability (%)
Table 2 Rat ABP detection in the medium collected from feeder cell cultures
Spermatid cocultures (3 days ) With contact/MSC-rABP () With contact/MSC-rABP () With contact/CHO-rABP () Without contact/MSC-rABP () Without contact/MSC-rABP () Without contact/CHO-rABP () Spermatid cocultures (3 days)
84.6692.83 80.5892.01 66.5394.10 68.3691.00 72.1690.86 55.3692.93 52.5092.93
Cell cultures
ABP content (fmol/ml)
A MSC-rABP () MSC-rABP () CHO-rABP ()
ND 754.65973.21a 171.47928.16c
B MSC-rABP () MSC-rABP () CHO-rABP ()
ND 114.0391.19b 75.82919.53
In cell /cell contact cocultures, adherent and non-adherent viable spermatids were counted in the removed media and in the mixed suspension obtained after trypsinization. In coculture without contact, spermatids were collected in the apical chamber, pelleted and counted. Spermatids from 3-day monocultures were also checked for viability. Results are expressed as percentage of the initial number of spermatids used for coculture and as the mean9S.E.M. of two counts realized on four independent cultures of each condition (n 4). The values were compared using the Student’s t -test. Differences were all significant at P 5 0.05 except for MSC-rABP () compared with MSC-rABP () in coculture with contact, for CHO-rABP () with contact compared with MSC-rABP (), to MSC-rABP () and to CHO-rABP () without contact, and for CHO-rABP () without contact compared with the spermatid monoculture.
(A) Feeder cell cultures were performed at 37 8C and supplemented with 5% of FCS. (B) Feeder cell cultures were performed at 34 8C in the absence of FCS. Media were collected after 3 days and rABP content was assessed by immunoquantitation using a rabbit polyclonal antiserum against human SHBG (1:1000) and densitometric analysis. Results are expressed as fmol/ml and as the mean9S.E.M., n 4. ABP was not detectable (ND) in MSC-rABP (). Differences in ABP content are significant between MSC-rABP () and CHO-rABP () at 37 8C. a, P 5 0.001 and at 34 8C; b, P 5 0.005; differences are significant between MSC-rABP () and CHO-rABP () at 37 vs. 34 8C, a, P 5 0.001; c, P 5 0.005.
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3.3. Rat Tnp1 gene expression A 175 bp cDNA band, corresponding to the amplification of rat Tnp1 gene, checked by sequencing (95% identity), was detected in all spermatid fractions. No Tnp1 gene expression was found in any of the three somatic feeder layer. As illustrated on Fig. 1, when cultured alone, spermatids showed a marked fall of Tnp1 gene expression. When cocultivated, spermatids showed varying transcript concentrations related to the presence or the absence of rABP, to the presence of cell /cell contacts and to the nature of the feeder layer. MSC-rABP (/) or CHO -rABP (/) in direct contact with spermatids strongly raised the level of TP1 transcripts by a factor 5 and by a factor 2, respectively, compared with the level of TP1 transcripts in spermatid monoculture. In this coculture mode, MSC-rABP (/) induced a highly increased level (/3.6) of TP1 mRNA compared with that obtained in the presence of MSC-rABP (/).
Moreover, TP1 mRNA level was enhanced by a factor 1.2 compared with the in vivo initial level. CHO-rABP (/) were clearly less effective than MSC-rABP (/). In spermatid cocultures avoiding cell /cell contact with the feeder layer cells, MSC-rABP (/) were unable to maintain the TP1 transcript concentration over that observed in spermatid monocultures or in cocultures with MSC-rABP (/). This level was 3.7-fold decreased compared with cell /cell contact cocultures with the same feeder layer. CHO-rABP (/) exhibited an adverse effect since the amount of TP1 transcript showed a 5fold reduction compared with spermatid monoculture. 3.4. Consequences of testosterone and b-estradiol adjunction regarding to rABP effects on spermatid Tnp1 mRNA level Since the most substantial effects of rABP were obtained when spermatids were cultivated in direct contacts with MSC-rABP (/) cells, both testosterone and 17b-estradiol adjunction experiments were conducted in these conditions (Fig. 1, column 7, 8). 3.4.1. Testosterone Testosterone adjunction in the culture medium led to cancellation of the up-regulation of Tnp1 gene expression elicited by the coculture of spermatids with MSCrABP (/) cells, thus resulting in a comparable level of TP1 mRNA to that observed initially, in vivo (freshly isolated spermatids). 3.4.2. 17 b-Estradiol 17 b-Estradiol adjunction in the medium induced a dramatic 91% fall of TP1 transcript number, that reached the very low value observed in spermatid monocultures. Indeed, b-estradiol showed an adverse effect on Tnp1 gene expression up-regulation elicited by coculture of spermatids with MSC-rABP (/). 3.5. Incidence of the various coculture conditions on ABP concentration in the medium
Fig. 1. Effects of the different culture conditions on rat TP1 mRNA following semi-quantitative RT-PCR analysis. Total RNAs from each culture condition were analyzed by RT-PCR for the expression of rat TP1 and rat b-actin (used as internal control of amplification and for TP1 signal intensity normalization). Values are the mean (columns)9/ S.E.M. (bars) of TP1/ß-actin ratio for six separate RT-PCR reactions. The values were compared using the Students’s t -test. Differences were significant at P 5/0.05 in column 0 vs. 3, in column 1 vs. 9, in column 4 vs. 6; P 5/0.005 in column 2 vs. 4, in column 3 vs. 8, in column 5 vs. 9; P 5/0.001 in column 0 vs. 1, 2, 3, 4, 5, 6, 7 and 9, in column 1 vs. 3, 4, 7 and 8, in column 2 vs. 3, 7, 8 and 9, in column 3 vs. 4, 5, 6, 7 and 9, in column 7 vs. 4, 5, 6 and 8, in column 8 vs. 4, 5, 6 and 9, in column 9 vs. 4 and 6.
As expected, in the media of MSC-rABP (/) no immunoreactive ABP was detected even in the presence of spermatids. 3.5.1. Effects of spermatid-feeder layer cell contacts Three different conditions were investigated: feeder layer cells alone, feeder layer cells and spermatids in direct contact, feeder layer cells and spermatids without contact (Fig. 2, columns 1/6). Concerning CHO-rABP (/) feeder layer cells, no significant differences have been showed between the three conditions, suggesting that no cross talk could take place between spermatids and CHO cells.
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4. Discussion
Fig. 2. Effects of the culture conditions on ABP secretion by the different rABP-transfected feeder layers. All cultures were realized at 34 8C in the same medium described in Section 2. In 1 and 4, the feeder layer cells were cultivated without spermatids. In 2, 5, 7 and 8, spermatids were directly loaded on the feeder layer cells. In 3 and 6, spermatids were deposited in the apical chamber of culture dish without cell /cell contact with the feeder layer cells. After 3 days, all media were collected and ABP content was assayed by immunoquantitation using a rabbit polyclonal antiserum against human SHBG (1:1000). Data are expressed as fmol/ml and as mean (columns)9/ S.E.M. (bars), n/4. P 5/0.05 in column 1 vs. 2, 4 and 6; P 5/0.005 in column 2 vs. 4, in column 4 vs. 7, in column 6 vs. 7; P 5/0.001 in column 1 vs. 3, 5 and 8, in column 2 vs. 3, 7 and 8, in column 4 vs. 8, in column 5 vs. 7 and 8, in column 6 vs. 8.
Concerning MSC-rABP (/) feeder layer cells, the establishment of direct contacts with spermatids led to clear changes in ABP level. Indeed, ABP content was about a factor of 1.4 higher than in the medium without spermatids (162.869/32.99 vs. 114.039/1.19 fmol/ml). When spermatids were not in contact with MSC-rABP (/), ABP level was decreased by about one half of the cell /cell contact level (62.159/11.90 vs. 162.869/32.99 fmol/ml). Paradoxically, this level was also below that obtained in complete absence of spermatids. 3.5.2. Sex-steroid hormone adjunction Since coculture of spermatids in direct contacts with MSC-rABP (/) cells was proved the most efficient condition regarding to ABP delivery in the medium, investigations of the consequences of testosterone and 17ß-estradiol adjunction on ABP level were performed in this situation only (Fig. 2, column 7, 8). Clearly, the adjunction of 50 ng/ml testosterone or 50 ng/ml of 17ßestradiol in the coculture medium for 3 days did not lead to significant changes in the level of secreted ABP.
Spermiogenesis provides a valuable opportunity to explore the putative role of ABP on germ cell maturation. ABP concentrates in the apical part of the Sertoli cells at stages VII/IX in the adult male rat, which makes it temporally and spatially available to regulate spermiogenic events (Attramadal et al., 1981). Studies on mutant restricted rats (Hre), and hypophysectomized pregnenolone-injected rats showed a positive correlation between sperm fertilizing ability and the levels of ABP (Anthony et al., 1984). Indirect experiments from Huang et al. (1991) clearly established that ABP status and the maintenance of spermiogenesis were linked. Roberts and Zirkin (1993) proposed that ABP could be involved in the failure of advanced spermatid production by inhibition of Sertoli cell testosterone action, in vitro. Finally, in transgenic 11shbg mice, high levels of SHBG mRNA were accumulated in Sertoli cell luminal cytoplasm close to step 9/12 spermatids (Ja¨nne et al., 1998). The coculture system used here clearly demonstrated its efficiency in sustaining rat spermatid survival as well as in producing a detectable concentration of rat ABP in the medium during the whole experimental procedure. That mouse Sertoli-derived cell lines, secreting or not ABP, were beneficial to germ cells is not surprising since Sertoli cell factors are able to maintain spermatid viability (Cameron et al., 1991) and cocultures with TM4 cells improved germ cell survival (De Felici and Dolci, 1991). This was not the case for CHO cells since we did not succeed in coculturing germ cells with this cell line at 34 8C and without serum. Unexpectedly, rABP-transfected CHO cells revealed, on the contrary, their capacity to grow at 34 8C without fetal calf serum and to support spermatid survival. It is not clear for the moment whether ABP accumulation within the cytoplasm of transfected CHO cells could contribute to such an adaptation. This intriguing observation warrants further study. Concerning the evaluation of the putative role of ABP during spermiogenesis, the Tnp 1 gene encoding the TP1 (Kistler et al., 1987, 1975) was chosen to serve as marker of spermatid physiology. In the rat, Tnp1 gene exhibits the highest level of expression (Mali et al., 1989) when the ABP secretion peak is maximal, from the step VIII up to X of the seminiferous epithelium cycle (Parvinen et al., 1986). TP1 transcripts are stored in round spermatid cytoplasm from step VII/VIII and then translated with a delay from the step XI of the seminiferous cycle (Marret et al., 1998). When Tnp1 is not expressed in human testis, an important failure of spermatogenesis characterized by a round spermatid arrest, is observed (Steger et al., 1999). In Tnp1/ mice fertility is reduced (60% of males were infertile). In addition, an abnormal pattern of chromatin condensation and a severe reduction of sperm motility are observed (Yu et al., 2000).
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However, regulation factors of Tnp1 gene, both at transcriptional and translational levels, remain poorly understood. Our results clearly show that monocultures for 3 days in a serum-, FSH-, steroid- and ABP- free medium were unable to maintain rat spermatid TP1 mRNA at a level higher than approximately 25% of the initial value (freshly isolated spermatids */column 0, Fig. 1). By contrast, spermatid coculture in contact with CHO or Sertoli cells producing recombinant rat ABP, were able to rescue and, even, to upregulate Tnp1 gene expression. The different coculture conditions demonstrated TP1 mRNA level resulting from the presence of rABP in the culture medium to be also dependent on the nature of the feeder layer cells and on the adjunction of sex steroids in the medium. First of all, it must be stressed that MSC-rABP (/) feeder layer whether or not in direct contact with spermatids, was not able to rescue TP1 mRNA level in spermatids (Fig. 1, columns 2, 5) although its efficiency in maintaining spermatid survival was demonstrated (Table 1). Thus, the fall of TP1 mRNA level observed in spermatids cannot be held as a consequence of poor survival at 3 days but should be related to the absence of some specific factor in the spermatid environment. From all situations tested, the highest Tnp1 expression was observed when two conditions were combined: presence of rABP and direct contacts between spermatids and feeder layer cells, whatever their origin (column 3,4, Fig. 1). Yet, Tnp1 expression was twice and a half higher in the case of MSC- rABP (/) contact than CHO- rABP (/) one. Sequence comparisons showed ABP to be related to the C-terminal globular (G) domains of laminin and other extracellular matrix and regulatory proteins such as protein S, Gas6 and agrin (Joseph, 1997). Germ cells could possess corresponding receptors as for example spermatids having laminin receptors (Fulcher et al., 1993). Thus ABP may possibly act as an extracellular ligand at the cell /cell interface eliciting signal responses in spermatids, leading to Tnp1 gene transcription stimulation. This hypothesis is supported by the finding of a higher adhesion index of germ cells when plated on ABP-producing Sertoli cells (manuscript in preparation) and by the formation of multiple cytoplasm ramifications by TM4 cells after rABP transfection, compared with untransfected ones (Ducray et al., 1998), which could expand the surface of the membrane available for MSC- rABP (/)/spermatid interactions. The two latter phenomena were not observed in the case of CHO-rABP (/) feeder layers. Most likely spermatid/MSC-rABP (/) direct contacts better mimic the in vivo situation allowing juxtacrine communications to occur. Such a situation could be responsible for a higher concentration of ABP at the Sertoli/germ cell interface than in the coculture medium, or for an enhanced uptake of the
binding protein by spermatids. This is supported by the earlier demonstration of a rapid transfer of labelledABP from Sertoli cells to germ cells (Ge´rard et al., 1994; Ge´rard, 1995). This is also consistent with the presence of large amounts of SHBG mRNA within the Sertoli cell cytoplasm close to step 9 /12 spermatids from transgenic mice expressing the human shbg gene (Ja¨nne et al., 1998) and accumulation of immunopositive rat ABP in apical Sertoli cell cytoplasm along the plasma membrane of elongating spermatids (Attramadal et al. 1981; Gerard, data not shown). A quite different way of thinking is to look at the effective concentration of ABP in the culture medium owing to the variations observed in relation to coculture conditions. As shown in the Section 3, by far the highest concentration of immunoreactive ABP was found when spermatids were cocultivated in contact with MSCrABP (/). This is not surprising since positive regulation of germ cells on ABP secretion by Sertoli cells is well documented (Galdieri et al., 1984; Le Magueresse and Je´gou, 1988) as are the negative effects of germ cells depletion (Bardin et al., 1993). In our experiments, the highest Tnp1 gene expression was correlated with the highest ABP concentration. Conversely, coculture conditions leading to low ABP concentrations in the medium were also the conditions of low expression, thus suggesting a possible dose-dependent effect of ABP. Although these first sets of experiments were only focussed on the search for a putative role of ABP on spermiogenesis and not to disclose the mechanisms involved, two hypothesis could be proposed regarding to the control of TP1 mRNA level by ABP, which could serve as guidelines for further experiments. Even in the absence of sex steroids, SHBG, the plasmatic equivalent of ABP, is able to considerably increase intracellular cAMP level in different target cells in vitro (Queipo et al., 1998; Fortunati et al., 1999). Since Tnp1 gene in a variety of species, including man, contains cAMPresponse elements (CREs) in the promoter region (Oliva and Dixon, 1991; Kistler et al., 1994). ABP in elevating cAMP level in spermatids, might stimulate Tnp1 gene expression. Alternatively, TP1 mRNAs are stored in a translationally repressed state for several days and then translate within elongating spermatids (Hecht, 1990; Morales et al., 1991; Eddy et al., 1993). If ABP can maintain repression or interfere with depression mechanisms, TP1 mRNA would have also be found at conserved level in spermatid after 3 days of culture, as was effectively observed in our experiments. Testosterone and estradiol are both ABP ligands, the highest affinity being for androgens. Adding testosterone or estradiol to the culture medium before plating spermatids on the feeder layer cells resulted in a statistically significant lower spermatid TP1 mRNA content, implying the liganded form of ABP to be less effective than the ligand-free one. Since ABP membrane-
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receptors have been shown to be present on germ cell lineage (Steinberger et al., 1984; Felden et al., 1992), this could suggest a similar mechanism as for SHBG, whose binding on human prostatic cell membranes RSHBG is prevented by prior liganding to sex steroid hormones (Hryb et al., 1990). Nevertheless, it must be stressed that adding testosterone led to a decreased spermatid TP1 mRNA content decreased not below the value exhibited by freshly isolated spermatids, thus representative of the intratesticular physiological level, while adding estradiol drastically reduced this content approximately to one tenth of the initial value. The discrepancy between the results obtained in adding one or the other sex hormone indicates that, on its own, sex hormone liganding of ABP cannot be here the cause, or at least the unique cause, of the fall of spermatid TP1 mRNA content compared with the level observed when using unliganted ABP. The highly different effects observed on spermatids lead to the conclusion of an action depending mainly on sex hormones themselves. It is generally accepted that androgen receptors are not present in spermatids (Sharpe, 1994). On the opposite, both ERbmRNA and ERb-protein are revealed in round spermatids from stage I/VIII of the seminiferous epithelium (Saunders et al., 1998; van Pelt et al., 1999). This could account for the striking differences observed between the effects of the two sex steroid hormones since, contrary to testosterone, estradiol would have operated directly on spermatids, thus interfering with Tnp1 gene ABP-effect. Based on TP1 mRNA content, our in vitro results evoked an antagonist effect of estradiol against ABP action on spermatids. Interestingly, in vivo, ERbmRNA and ERb-protein are at their highest level in spermatids when seminiferous ABP content is at its lowest. In summary, by using TP1 mRNA quantification as marker to follow up rat spermatid maturation cocultivated with somatic cells transfected or not with a rat ABP gene construct, this work clearly demonstrates that ABP itself could be involved, namely in the absence of sex-steroid hormone as well as in the absence of other Sertoli cell products, in spermiogenetic events related to Tnp1 gene expression. This substantiates previous findings from different groups supporting the idea that ABP would not have to be confined in the role of a sex-steroid hormone carrier, protecting them from degradation and controlling their bioavailability. In the same way Queipo et al. (1998) recently established that SHBG, the plasmatic equivalent of ABP, encoded by the same gene, is able to raise both hCG and cAMP concentrations in cultured human cytotrophoblasts, in the absence of sex-steroid ligand. Moreover, Fortunati et al. (1999), demonstrated that SHBG, even without estradiol supply, caused a significant increase of cAMP in breast cancer cells MCF-7 maintained in serum-free medium.
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A direct action of ABP on germ cells may have functional consequences of importance within the testis, especially on seminiferous epithelium cycle control as well as at critical periods such as spermatogenesis onset, which is associated with changes both in intratesticular ABP level and in apical/basal direction of its secretion by Sertoli cells (Gunsalus et al., 1980). Yet ABP must be held as only one of the multile factors integrated in spermatogenesis control, thus rendering delineation fairly complicated to establish from in vivo studies. Among the factors suspected interfering with ABP, two of them seemed to clearly emerge from our in vitro results concerning TP1 mRNA. The first one consists in the expected modulation of ABP effects by sex-steroid hormones. The question open, however, as to whether sex steroids are acting through allosteric changes of the protein in a same manner as demonstrated by Rosner et al. (1999) for SHBG, or in modifying the synthesis of factors involved in target cell sensitivity to ABP. The crucial effect of estradiol on spermatid TP1 mRNA is in favor of the latter hypothesis. The second one consists in the requirement of direct membrane/membrane contacts between spermatids and ABP producing cells from testicular (MSC-rABP (/) and, to a lesser extent, from non testicular (CHO-rABP (/)) origin, to obtain ABP effects on Tnp1 gene activation. This points out that spatial relationships between germ cells and somatic cells, within the seminiferous tubules, are probably of utmost importance for spermatogenesis progression and have to be carefully investigated when attempting to achieve spermatogenesis in vitro.
Acknowledgements We are grateful to G.L. Hammond (The University of Western Ontario, LRCC Cancer Research Laboratory, London, Ont., Canada) for the gift of the plasmid pRc/ CMV- rat Androgen-Binding Protein (ABP), to Dr C. Grenot (INSERM U 329, Lyon, France) for providing pure human SHBG and polyclonal anti-SHBG. The authors also thank Professor C. Branlant, Dr A. Mougin for Gel doc facilities (UMR 7567 ‘Maturation des ARN et Enzymologie mole´culaire’, UHP, Nancy, France), Professor J.L. Olivier (Laboratoire de Biochimie, Hoˆpital central, CHU, Nancy, France) for Kodak image analysis facilities, M. Adam, B. Cunin, R. Verdun for technical assistance. This work was supported by FARO.
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