Function of interleukin-6 as an inhibitor of meiotic DNA synthesis in the rat seminiferous epithelium

ELSEVIER

Moleculru and Cellular Endocrinology 108 (1995) 193-198

Function of interleukin-6 as an inhibitor of meiotic DNA synthesis in the rat seminiferous epithelium Harri Hakovirtaaa*, Viqar Syedb, Bernard JCgoub, Martti Parvinena %stitute of Biomedicine, Department of Anatomy, Universityof Turku, FIN-20520 Turku, Finland bCERM, INSERM CJF 91-04, Universite’de Rennes 1, Campus de Beaulieu, 35042 Rennes Cedex, Bretagne, France

Received 15 August 1994; accepted 21 December 1994

Abstract Interleukin-6 bioactivity (IL-6) has been shown to be present in Sertoli cells. To further characterize the IL-6 in the seminiferous epithelium, the IL-6 like-antigen was detected, stage-specific basal distribution of IL-6-like bioactivity and its regulation by FSH, CAMP and TPA was characterized in isolated, rat seminiferous tubule segments. In addition, the effects of human recombinant IL-6 on stage-specific DNA synthesis was investigated. Both monoclonal and polyclonal antibodies recognized Mr 22 and 23 kDa of IL-6 like immunoreactivity in the seminiferous epithelium. The basal IL-6 production showed high levels during stages XIII-XIV-I-V, low during VII and VIII. FSH stimulated IL-6 production at nearly all stages and most significantly at stage VII of the cycle. Human recombinant IL-6 dose-dependently inhibited the onset of meiotic DNA synthesis of preleptotene spermatocytes, and a minor inhibition was found on advanced (A3-type B) spermatogonia. These results support the hypothesis that IL-6 is a stage-specific paracrine regulator of the seminiferous epithelium exerting a specific inhibitory action on meiotic DNA synthesis. Keywords: Rat; Testis; Seminiferous epithelium; Sertoli cells; Follicle stimulating hormone; Interleukin-6; DNA synthesis

1. Introduction Interleukin-6 (IL-6) is a member of the cytokine family first described as interferon-/?2 (Haegeman et al., 1986). It is produced by many different cell types, such as

fibroblasts, monocytes, endothelial cells, as well as Tcells, and it is an important regulator of host defence responses (van Snick, 1990). In addition, several endocrine organs have been shown to produce IL-6 (Iwamoto et al., 1991; Spangelo et al., 1991; Gorospe et al., 1992). An IL6-like factor was recently described in the testis, where it originates from Leydig (Boockfor et al., 1994) and Sertoli cells (Syed et al., 1993). Its secretion was markedly stimulated by lipopolysaccharide and latex beads, known stimulators of monocyte/macrophage IL-6 production, and the IL-6 bioactivity could be neutralized by a specific monoclonal antibody. From testicular tissue components, residual bodies and cytoplasts from elongated spermatids had a similar stimulatory action. The levels of IL-6

showed variation at different stages of the cycle of the seminiferous epithelium; stages II-VI and VII-VIII showed the highest and lowest values, respectively, and the secretion by Sertoli cells was stimulated by FSH and CAMP. To find out the possible functional role of IL-6 in the seminiferous epithelium, we analysed the effects of human recombinant IL-6 on stage-specific DNA synthesis of rat seminiferous tubular segments in vitro, characterized the IL-6 like immunoreactivity with monoclonal and polyclonal antibody, and compared the earlier results (Syed et al., 1993) with detailed analysis of IL-6 secretion and its regulation by follicle stimulating hormone, dibutyryl cyclic AMP and an unspecific activator of protein kinase C, phorbol ester 12-0-tetradecanoylphorbol-13acetate (TPA). 2. Materials and methods 2. I. Animals and chemicals

*Corresponding 73.52.

author, Tel.: +358 21 633 7351; Fax: +358 21 633

Sprague-Dawley rats (2-3 months old) were used as experimental animals. They were housed two per cage in

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a controlled environment at 2l’C with a 14 h light and 10 h darkness cycle having free access to water and food. Rats were killed with COZ. Recombinant human interleukin-6 (rhum-IL-6) was a generous gift from Dr. Stephen D. Wolpe (Genetics Institute, Cambridge, MA). The unit of its specific activity (5.1 x IO5 U/mg) was defined as a half maximal stimulation in the TlO proliferation assay. FSH (Fertinorm) was obtained from Serono (Aubonne, Switzerland), (Bu)+AMP and TPA from Sigma Chemical Co. (St. Louis, MO), and 3-isobutyl-lmethyl-xanthin (MIX) from Aldrich (Steinheim, Germany). Fetal calf serum, L-glutamine, L-arginine, 2mercaptoethanol, hypoxanthine, thymidine and gentamytin were obtained from Gibco-BRL (Cergy Pontoise, France). Monoclonal mouse anti-human recombinant IL6 IgG, was obtained from Genzyme, Cytokine Research Products, Cambridge, USA. Cross reactivity was tested by the producer for IL-l, IL-2, IL-3, IL-5 IL-7 GM-CSF or G-CSF. Polyclonal rabbit anti-human IL-6 IgG SO%/IgM 20% was obtained from Genzyme, Cytokine Research Products, Cambridge, MA, USA. Cross reactivity was tested by the producer for IL-l (a or /l), TNF (cr or p), IFN (a or /?) and GM-CSF. 2.2. Microdissection of the seminiferous tubule segments and tissue cultures Stages I, V, VIIa and VIII-IX of the seminiferous epithelial cycle were selected since they contain cells at representative phases of mitotic and meiotic DNA synthesis (Parvinen et al., 1991). During culture times of 24, 48 and 72 h, these stages differentiate through most of the stages of the cycle. Two millimeter seminiferous tubule segments were isolated under a trans-illuminating stereomicroscope in a continuous laminar flow for in vitro analyses of DNA synthesis. Stages were identified as described earlier (Parvinen and Vanha-Perttula, 1972). The tubule segments were transferred onto 96-well culture plates in 10~1 of PBS and incubated at 34°C for 24, 48 and 72 h in lOO@ of Ham’s Fl2/Dulbecco’s MEM (Gibco BRL, Paisley, UK) supplemented with 0.1% BSA (Sigma), G-penicillin 60 mg/l (Sigma) and streptomycin 500 mg/l (Sigma) in a humidified atmosphere containing 5 % CO,? in air. IL-6 was added in concentrations of 10, 50 and 100 U/ml. Tubules were pulselabelled during the last 4 h of the culture by adding 20 kBq of [methyl-3H[thymidine (TRA 120, 185 Gbq/ mmol, Amersham, UK). 2.3. Culture harvesting and assessment of DNA synthesis The cultures were harvested on filter discs (Whatman 934-AH) with a continuous flow of distilled water for 1 min. A scintillation wax (MeltiLexTMA 1450-441, Wallac Oy, Turku, Finland) was melted on the filters and the radioactivity was measured by a flat bed liquid scintillation counter equipped with two parallel detectors (1450

Microbeta, Wallac Oy, Turku, Finland). Three separate experiments were performed each with six replicate samples. 2.4. Autoradiography The labelled seminiferous tubules were carefully squashed between microscope slides and coverslips and frozen in liquid nitrogen. The coverslips were removed and the still frozen squash preparations were fixed in ethanol/glacial acetic acid (3: 1, v/v) for 30 min and airdried. The slides were dipped in Kodak NTB-3 nuclear track emulsion (Eastman Kodak Co., Rochester, NY), exposed for 2 days, developed and stained with hematoxylin. 2.5. Immunoblotting Seminiferous tubule segments (segments were pooled to the total length of 4 cm) were dissolved in lOO@ of 2X Laemmli solution and boiled for 15 min. After electrophoresis in 15% SDS-polyacrylamide gels, the proteins were transferred for 1 h at 250 mA onto a nitrocellulose (NC) membrane. The membrane was blocked with TBST for 30 min and incubated for 1 h at room temperature with either lOpg/ml of monoclonal mouse anti-human recombinant IL-6 (Genzyme 1618-01, Cytokine Research Products, Cambridge, USA) or lO,@ml of polyclonal rabbit anti-human IL-6 (Genzyme LP-716, Cytokine Research Products, Cambridge, USA). Controls were incubated in the blocking buffer. After incubation with the primary antibody, the membrane was washed again for 30 min with blocking buffer and incubated for 30 min with either rabbit anti-mouse IgG peroxidase conjugated secondary antibody (1:200, Dakopatts P 260) or swine anti-rabbit IgG peroxidase conjugated secondary antibody (I :200, Dakopatts P 217). Controls were run in parallel with the primary antibody treated samples in secondary antibody buffer. The membrane was then washed in room temperature five times in the blocking buffer with a total washing time of 2 h. Immunoreactivity was detected by peroxidase reaction using diaminobenzidine as a substrate for 10 min. Experiments were performed twice from three separate animals. Molecular weight markers (14-94 kDa, Pharmacia 17-0446-01, USA) were run parallel to the samples, transferred, cut out of the NC membrane, and stained with Ponceau S. 2.6. Preparation of samples for IL-6 bioassay Seminiferous tubules were microdissected according to their trans-illumination pattern and ten pools (5 X 2 mm segments = 1 cm) were collected from following stages: I, II-III, IV-V, VI, VIIab, VIIcd, VIII, IX-XI, XII and XIII-XIV (Parvinen and Ruokonen, 1982). The pools were incubated at 34“C for 20 h in 500~1 of culture media containing either FSH (100 rig/ml), MIX (0.1 mM), (Bu)zcAMP (0.2 mM), TPA (100 nM) or plain medium in a humidified atmosphere containing 5% CO* in air. Tu-

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bule segments were removed by centrifugation, and the media were stored at -80°C. 2.7. Bioassay of IL-6 All samples were assayed in the same assay to avoid interassay variations. IL-6 bioactivity was quantified using the specific IL-6-dependent mouse-mouse hybrid cell line, 7TDI (van Snick et al., 1986), as described by Syed et al. (1993). The results were expressed as unit per ml of tubule culture medium. 2.8. Data processing and statistical analyses Statistical analyses were performed with analysis of variance and Student’s t-test. Effects of rhum-IL-6 on stage-specific DNA synthesis were assayed in three different experiments containing six replicate samples. Both basal production and hormonal regulation of stagespecific IL-6 production were assayed in two different experiments containing three replicate samples.

VIIa->VIIb

VIII-IX->IX

I-71

3. Results 3.1. Effects of IL-6 on stage-specific DNA synthesis Effects of rhum-IL-6 on stage-specific DNA synthesis were observed by using a in vitro measurement of thymidine incorporation in the cultured tubule segments of seminiferous epithelium. During 3 days in culture, the chosen stages develop through most of the stages of the seminiferous epithelium as described earlier (Parvinen et al., 1991). Recombinant-human-IL-6 had no significant effect on stage-specific thymidine incorporation in tubule segments cultured for 24 or 48 h (Fig. lA,B). However, significant (P < 0.01) inhibition of thymidine incorporation was observed at stage VIIa cultured for 72 h, with all concentrations studied (developed to stage VIII during 72 h culture) (Fig. 1C). Autoradiography from the labelled stage VIIa tubule segments cultured in the corresponding culture conditions showed decreased grain accumulation on preleptotene spermatocytes cultured in the presence of IL6 compared to the tubule segments cultured in the control medium (Fig. 2). Other cell types within tubules did not show grain accumulation. A slight but significant (P <:0.05) inhibition was also observed at stages VIII-IX (developed to stage XII during 72 h in culture) and I (developed to stage V during 72 h in culture) cultured for 72 h, but it did not show any clear dose-response. 3.2. Regulation of the stage-specific IL-6 production and IL-6 like antigen in the seminiferous epithelium

A detailed analyses of basal IL-6 production and hormonal regulation at stages I, II-III, IV-V, VI, VIIab, VIIcd, VIII, IX-XI, XII and XIII-XIV of the cycle were investigated. The basal IL-6 production was highest at stages II-III of the cycle, and high levels at stages XIII-

v->VII

VIIa->VIII

VIII-IX->XII

I->v

Fig. 1. The 13H]thymidine incorporation (counts per minute (cpm), mean f SEM, normalized to the 2 mm of tubule) at stages V, VIIa, VIII-IX and I of seminiferous epithelium cultured for (A) 24, (B) 48 and (C) 72 h with 0, 10,SO and 100 U/m1 of rhum-IL-6. The arrows at the horizontal axis show the progression of stages of the seminiferous epitheliai cycle during the culture. Statistical signifkances: ? c 0.01 and bP < 0.05 compared with controls. Effects of rhum-IL-6 on stagespecific DNA synthesis were assayed in three different experiments containing six replicate samples. For each bar n = 18.

XIV, I and IV-V of the cycle (Fig. 3). The lowest level was found at stage VIIab, and low levels were found throughout stages VII and VIII of the cycle. During stages IX-XII of the cycle, rising values were found, and at stage VI, the IL-6 level declined rapidly. The variation was significant (P c O.OOl), and the values of stage VII were clearly (P c 0.01) below those of all other stages. FSH had a slight stimulatory effect on IL-6 at most stages of the cycle, and particularly at stage VII (P < 0.001) (Fig. 3A). Stimulation by (Bu)$AMP was not as effi-

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1993), acting either on mitotic (SBder et al., 1992) or meiotic (Parvinen et al., 1992) or both types of DNA synthesis (Parvinen et al., 1991; Hakovirta et al., 1993; Hakovirta et al., 1994). The effects of IL-6 on stagespecific DNA synthesis in the rat seminiferous epithelium together with the observations of the detection of IL-6 like antigen and the hormonal regulation of interleukin-6like factor in the seminiferous epithelium, suggests that IL-6 is a paracrine regulator of spermatogenesis regulating the germ cell proliferation during the cycle of seminiferous epithelium. Earlier observations of the 8

IA

n Basal

b

u.m IV-V Vl

I

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IX-XI

XII XIII-XIV

8 ,

8

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Fig. 2. Autoradiography showing the [%Q’thymidine incorporation during the last 4 h of the culture from the seminiferous tubules from stage VIIa of the cycle cultured for 72 h: (A) in the absence of IL-6 preleptotene spermatocytes (Pl) show intense grain accumulation; (B) in culture medium containing 100 U/ml of mum-IL-6 grain accumulation cannot be detected. Scale 1160pm.

I

Basal

m CAMP

h

I

11.m

IV-V

VI

Web

VIIc-d

VIII

IX-XI

XII XIII-XIV

I

II-III

IV-V

VI

V&-b

VlIc-d

VU1

IX-XI

XII XIII-XIV

8

stimulations were only observed at stages I, IV-V and XIII-XIV of the cycle (Fig. 3B). The unspecific activator of protein kinase C, TPA, significantly inhibited the production of IL-6 in the seminiferous tubules at stages IV-VIII, of the cycle. At other stages of the cycle TPA had no effect on IL-6 production (Fig. 3C). IL-6 like immunoreactivity was detected on 15% SDSpage. Both monoclonal and polyclonal antibodies cross reacted with 22 and 23 kDa protein (Fig. 4). No immunoreactivity could be detected in samples incubated with secondary antibody (data not shown).

cient;

significant

4. Discussion Several growth factors have been suggested to be paracrine regulators in the testis and in the seminiferous epithelium (BellvC and Zheng, 1989; Skinner, 1991; JCgou

6

0

Fig. 3. The basal IL-6 production (X 10v3 units/ml/20 h per 10 mm of tubule) at different stages of the cycle of the seminiferous epithelium and the effects of (A) FSH, (B) (Bu)zcAMP and (C) TPA on IL-6 production at defined stages of the cycle. Statistical significances: ap < 0.001, bP < 0.01 and ‘F’ < 0.05 compared with controls. The hormonal regulation of stage-specific IL-Glike-bioactivity production was assayed in two different experiments containing three replicate samples. For each barn = 6.

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kDa AB

14Fig. 4. IL-6 like antigen in the seminiferous epithelium. Samples run on 15% SDS-page. Line A, IL-6 like antigen detected by mouse anti human IL-6 monoclonal antibody. Line B, IL-6 like antigen detected by rabbit anti human IL-6 polyclonal antibody. Arrows on the left side of the panel indicate molecular weight marker run parallel to the samples and arrows on the right side of the panel indicate the IL-6 like immunoreactivity of Mr 22 and 23 kDa.

growth factor effects have shown that they are mainly stimulative to the developing germ cells (Piillgnen et al., 1989; Mather et al., 1990). Only inhibin was shown to decrease the numbers of type-A3, In, type-B spermatogonia in vivo (van Dissel-Emiliani et al., 1989) and to inhibit stage-specific DNA synthesis in vitro (Hakovirta et al., 1993), however in germ cell-Sertoli cell co-cultures, inhibin A had no effect on cell proliferation (Mather et al., 1990). IL-6 is a pleiotrophic cytokine involved in B cell differentiation (Hirano et al., 1990), proliferation and differentiation in T cells (Zilberstein et al., 1986), inhibition of cell growth of certain myeloid leukemic cell lines and induction of their differentiation to macrophages (May et al., 1986) and inhibition of granulosa cell proliferation

BASAL IL-6 SECRETION

I

II

Ill IV

1

(Alpizar and Spicer, 1994). Preleptotene spermatocytes inactive in DNA synthesis (meiotic G1 phase) at stage VIIa have been shown to proceed to the active meiotic DNA synthesis (stage VIII of the cycle) during 72 h in culture (Parvinen et al., 1991, 1992; SGder et al., 1992; Hakovirta et al., 1993; Hakovirta et al., 1994). In the present study, all the concentrations of IL-6 had an inhibiting effect on the activation of meiotic DNA synthesis (Fig. 1). IL-6 production in the physiological conditions is lowest at stages VII-VIII of the cycle (Fig. 3) and an increase in IL-6 production can be detected during stage IX-XI of the cycle (Fig. 3). Meiotic DNA synthesis is initiated during stages VII-VIII and terminated during stage IXXI of the cycle (Monesi, 1962; Clermont and Harvey, 1965); together with the present in vitro observations, this might suggest that the increased production of IL-6 during stages IX-XI of the cycle is associated with the termination of the meiotic replication in vivo. Therefore, in the testis, IL-6 seem to be a negative regulator of meiotic DNA synthesis and the present study is the first observation of a growth factor that has a potency to inhibit meiotic DNA synthesis in the seminiferous epithelium in vitro. The intracellular mechanisms leading to the inhibition of meiotic DNA synthesis is an interesting subject for further studies. In addition to the meiotic DNA synthesis, IL-6 slightly inhibited the DNA synthesis in stages, where the only DNA synthesizing cells are late type-A and type-B spermatogonia. This effect, however, was not clearly dosedependent and therefore, IL-6 seems to be a predominant negative regulator of meiotic DNA synthesis. The binding of FSH and the FSH-stimulated produc-

iEllelEIN RELATION TO PROPOSED TARGET CELLS 1

v

VI

VII

VIII

IX-XI

XII

XIII XlV

Vllar. m m

PRELEPTOTENE SPERMATOCYTES ADVANCED SPERMATOGONIA

Fig. 5. Summary and hypothesis about the distribution and action of IL-6 during rat spermatogenesis superimposed on the time-related stages of the rat seminiferous epithelium. The arrows indicate the progression of the IL-6-responsive stages during 3 days in vitro.

map of the

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tion of CAMP have been reported to be highest during stages XIII-V and lowest during stages VII and VIII of the cycle (Parvinen, 1982; Kangasniemi et al., 1990a,b). The distribution of basal IL-6 production was similar to these, however, there was a difference between FSH and CAMP stimulated IL-6 production, particularly during stages VII-VIII of the cycle, This may indicate that FSH action is mediated through other second messengers than CAMP during these stages. To investigate the role of protein kinase-C (PK-C) dependent pathways in the regulation of IL-6 production, tubule segments were cultured in the presence of PK-C activator TPA. A specific inhibition of IL-6 production was seen in stages IV-VIIab of the cycle. PK-C activity has been found to be highest during stages II-VI and lowest during stages VII-VIII of the cycle (Nikula et al., 1987), which is in accordance with the present findings. These observations suggest that the IL-6 production is upregulated by FSH and CAMP and downregulated by a PK-C dependent pathway. The presence of ILd-like antigen was detected in the seminiferous epithelium in M, 22 and 23 kDa. This is in accordance with the earlier observations demonstrating that IL-6 produced by the epidermal cells exhibits M, microheterogeneity within 21-28 kDa (Kirnbauer et al., 1989). Since both monoclonal and polyclonal antibodies detected the same molecular sizes, this further supports that IL-6 in the seminiferous epithelium occurs as 22 and 23 kDa protein. Fig. 5 summarizes the most important findings of this study showing the distribution of the basal secretion of IL-6-like bioactivity in relation to the stages of the cycle of the seminiferous epithelium. In preleptotene spermatocytes at stages VIII-IX of the cycle (inside the black box), the onset of meiosis takes place. Its type of action is different from any other factor investigated thus far. The physiologically low IL-6 activity at stage VII is obviously important for normal onset of meiotic DNA synthesis. Together these novel observations support the hypothesis that IL-6 is an important negative regulator of spermatogenesis with a primary target on meiotic DNA synthesis. Acknowledgements This study was supported in Finland by grants from The Academy of Finland, The Sigrid JusClius Foundation, Finnish Medical Foundation and in France by grants from INSERM, the Minist&re de la Recherche et la Technologie, and INRA (AIP Rkgulations intragonadiques). The authors want to thank Dr Pulkki for the antibodies.

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