Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683-8504, Japan
1 To whom correspondence should be addressed. Email: souichi{at}grape.med.tottri-u.ac.jp
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Abstract |
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Key words: glycoprotein 130/interleukin-6/soluble interleukin-6 receptor/sperm motility/peritoneal fluid
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Introduction |
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The volume of peritoneal fluid (PF) is significantly elevated in infertile women with endometriosis compared to those without endometriosis (Harada et al., 1997a). PF is reported to be associated with infertility in several ways, e.g. inhibiting sperm motion (Drudy et al., 1994
; Aeby et al., 1996
), sperm acrosome reaction (Arumugam, 1994
), mouse embryo growth (Prough et al., 1990
), sperm binding to the zona pellucida (Coddington et al., 1992
), and ciliary action in the human Fallopian tube (Lyons et al., 2002
). Since the PF of infertile women with endometriosis contains abundant cytokines and growth factors, these factors may exert those detrimental effects on the reproductive process and possibly contribute to endometriosis-associated infertility (Harada et al., 2001
).
Interleukin-6 (IL-6), a pleiotropic cytokine produced by a variety of cell types, plays a pivotal role as a mediator of numerous physiological and pathogenic processes (Tagoh et al., 1989). It has also been suggested that IL-6 has important functions in reproductive physiology, including the regulation of ovarian steroid production, folliculogenesis, and early events related to implantation (Gorospe et al., 1992
; Ray et al., 1997
). IL-6 exerts these effects to bind cognate membrane receptor complexes, consisting of an 80 kDa ligand binding subunit [glycoprotein (gp) 80: interleukin-6 receptor; IL-6R] and a 130 kDa signal transducing protein (gp130). IL-6R binds IL-6 with low affinity and must associate with gp130 for high affinity binding and signal transduction to occur (Jacobs et al., 1992
). A 55 kDa soluble form of IL-6R (soluble IL-6 receptor; sIL-6R), which is proteolytically cleaved from IL-6R, exists in body fluid. It can enhance the bioactivity of IL-6 by direct binding of the IL-6/sIL-6R complex to gp130 (Rose-John et al., 1994).
We previously reported that levels of IL-6 in PF were significantly higher in infertile women with endometriosis and that its soluble receptor also exists in PF (Harada et al., 1997a). Although the main source of IL-6 had been considered to be macrophages, our previous study demonstrated that IL-6 is produced in endometriotic stromal cells at levels similar to that in macrophages (Tsudo et al., 2000
).
Accordingly, we hypothesize that IL-6 and sIL-6R may be responsible for the reported adverse effects on reproductive processes of PF from patients with endometriosis. In this study, we focused on the effects of IL-6 and sIL-6R on in vitro sperm motility. In addition, we also examined the expressions of IL-6, IL-6R and gp130 in sperm.
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Materials and methods |
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Effects of IL-6 and sIL-6R on sperm motility
All 20 sperm samples were diluted with serum-free HTF medium with various concentrations of IL-6 (010 ng/ml, recombinant human IL-6; Genzyme, USA) with or without sIL-6R (50 ng/ml; recombinant human IL-6 soluble receptor; R&D Systems, USA). The final concentration of sperm was adjusted to 5x106/ml, and 0.5 ml aliquots placed in 6 ml culture tubes (Falcon; Becton Dickinson UK Ltd). A monoclonal antibody against IL-6R (5 µg/ml; monoclonal anti-human IL-6 receptor antibody; Genzyme/Techne, USA) was used to neutralize the specific effects of IL-6 and sIL-6R. Each sample was incubated at 37°C under a humid atmosphere of 5% CO2 in air. A part of the aliquots was evaluated by computer-assisted sperm motion analysis (CASA; version 10 HTM-IVOS, Hamilton Thorne Research, USA) system at 24 h. At the same time, the sperm motility on the control samples was also assessed.
RTPCR
Total RNA was extracted from the sperm by the guanidium thiocyanate method according to the manufacturer's instructions (Isogen; Nippon Gene Co. Ltd, Japan). Reverse transcription of RNA from sperm into cDNA and PCR amplification were performed using the Gene Amp RNA PCR Core Kit (Perkin Elmer, USA). The reverse transcription of RNA to cDNA was performed with 2.5 IU/ml of MuLV reverse transcriptase as follows: 10 min at 30°C, 20 min at 47C, 5 min at 99°C and 5 min at 4°C. PCR amplification was carried out in 25 mmol/l of a MgCl2 solution (2 mmol/l), 10 x PCR buffer II (1x), DI water (65.5 ml) containing 0.1% DEPE, and Ampli Taq DNA polymerase (2.5 U/100 ml). Samples were amplified for 32 cycles of denaturation (30 s at 94°C), annealing (30 s at 60°C), synthesis (1.5 min at 72°C), and primer extension of 5 min at 75°C after each cycle.
For PCR analysis, previously described specific primers for human IL-6R and human gp130 were used: IL-6R (sense): 5'-CATTGCCATTGTTCTGAGGTTC-3' and IL-6R (anti-sense): 5'-AGTAGTCTGTATTGCTGATGTC-3'; gp130 (sense): 5'-ACAGATGAAGGTGGGAAGGAT-3' and gp130 (anti-sense): 5'-AGATGACATGCATGAAGACCC-3' (Yamazaki et al., 1996; Yoshioka et al., 1999
). The distances between primers, including the primers, were 251 and 423 bp respectively. PCR products were resolved on 2% agarose gel with a small molecular weight DNA marker (x174 digested with Hae III).
The PCR products were transferred to a nylon membrane (Sarton, Germany) using a vacuum blotter with 0.4 mol/l NaOH and 1 mol/l NaCl. The DNA on the membrane was fixed with a UV cross-linker. The membranes were hybridized with internal probes of IL-6R (5'-ACAAGCATGCATCCG-3') or gp130 (5'-CCAGATCCTTCAAAG-3'). The biotin-labelled product on the membrane was detected with a Smilight kit (Sumitomo Metal Industry Co., Japan). The membrane was treated with streptavidinalkaline phosphatase, followed by chemiluminescence detection. The membrane was exposed to X-ray film for 15 min at room temperature (Iwabe et al., 1998).
Western blot analysis
IL-6, IL-6R and gp130 protein expressions in sperm were examined by means of western blot analysis as previously described in detail (Yoshida et al., 2002). Briefly, the frozen sperm pellets were lysed with lysis buffer containing 50 mmol/l TrisHCl (pH 7.6), 150 mmol/l NaCl, 0.1% SDS, 1 mmol/l dithiothreitol, and 1x Complete Protease Inhibitor Cocktail (Boehringer Mannheim, Germany). The lysate was centrifuged and supernatant was prepared. Protein concentration of the supernatant was measured by Bradford assay. Eighty micrograms of protein samples were resolved by electrophoresis on a 420% gradient polyacrylamide gel. These separated protein samples were then electroblotted onto a nitrocellulose membrane before blocking in 2.5% skimmed milk for 60 min. The membrane was then probed with anti-IL-6R (1:1000) or anti-gp130 (1:1000) antibodies, (both from Genzyme/Techne) at 4°C for 12 h followed by a peroxidase-conjugated secondary antibody (1:1000). Protein bands were visualized using an enhanced chemiluminescence system (Amersham, UK). The protein band size was determined using a Full Range Rainbow (Amersham) molecular weight marker. In addition, the total protein from endometrial stromal cells, which express both IL-6R and gp130 as previously described (Yoshioka et al., 1999
), was used as a positive control.
Statistical analysis
Results were analysed using two-way analysis of variance, followed by Student's paired sample t-test in order to compare the values obtained with various treatments versus the values of control. The data were expressed as means±SEM. P<0.05 was accepted as statistically significant.
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Results |
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Discussion |
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For most cytokines (such as IL-2 and TNF), soluble receptors have antagonistic activity, precluding their ligand binding to specific membrane receptors. In contrast, sIL-6R is unique in exerting an agonistic action with its ligand, resulting in subsequent signal transduction. Because gp130 is expressed ubiquitously on various cells, IL-6 can exert its biological effect in the presence of sIL-6R, even though effector cells lack IL-6R on their plasma membrane, as demonstrated by negative control of thyroid function in cultured human thyroid follicles (Yamazaki et al., 1996
). The IL-6/sIL-6R complex associates with the constitutively expressed gp130 on IL-6R-negative cells to induce homodimerization of gp130, leading to a cellular response. Thus, our findings suggest an underlying mechanism in which IL-6 increases in the PF of patients with endometriosis, combines with sIL-6R that also exists in PF, and the combined IL-6/sIL-6R associate with the gp130 expressed on IL-6R-negative sperm. Consequently, combined IL-6/sIL-6R may exert adverse effects on sperm motility.
Furthermore, our data corroborate previous reports demonstrating that IL-6 levels in seminal plasma or cervical mucus were significantly higher in infertile patients compared with the fertile control patients, and that its levels in seminal plasma negatively correlated with sperm motility (Gruschwitz et al., 1996; Naz and Butler, 1996
). Since the seminal plasma contains sIL-6R (Dousset et al., 1997
), increased IL-6 in seminal plasma and cervical mucus associate with sIL-6R, and then the binding of IL-6/sIL-6R complex to gp130 presented in sperm may result in the impairment of sperm function.
For the association of sperm motility and seminal plasma IL-6 concentrations, some authors reported different results, suggesting that IL-6 levels in seminal plasma did not correlate with sperm motility (Dousset et al., 1997; Kocak et al., 2002
; Friebe et al., 2003
). The reasons for the controversy are not evident but may reflect the effects of other factors contained in seminal plasma. Seminal plasma contains various factors that stimulate or maintain sperm motility, such as carnitine (Gurbuz et al., 2003
), as well as inhibitory factors. The balance of those factors could regulate sperm motions in the seminal plasma. We speculate that the effects of the factors other than IL-6 may be responsible for the controversy.
Seminal plasma reportedly contains similar levels of IL-6 with PF (Eggert-Kruse et al., 2001). In contrast, concentrations of IL-6 were much higher in cervical mucus of infertile women than in PF of patients with endometriosis (Naz and Butler, 1996
). Indeed it seems that the sperm contact with cervical mucus may be the most significant in vivo. The exposure to elevated IL-6 levels in PF of patients with endometriosis and in the Fallopian tubes subsequent to contact with cervical mucus may further impair the sperm motility. Additionally it is speculated that the seminal plasma, which contains the protective factors of sperm motility, has been removed from the fallopian tubes, and then IL-6 contained in PF diffusing down the fallopian tubes may affect sperm motions at lower concentrations than cervical mucus.
In the present study, we observed an inhibition of sperm motility at a concentration of 0.1 ng/ml of IL-6. The median IL-6 concentration in PF of patients with endometriosis (measured in our previous study) was lower than levels used in the present study (Harada et al., 1997a
). Thus, this study has only a limited ability to determine the cause of endometriosis-associated infertility for all patients. However, in general, half of endometriosis patients are reported to be infertile. The inhibition of sperm motility by IL-6 may be involved in the infertility of at least some patients with endometriosis who have highly elevated levels of IL-6 in PF.
PF diffusing into the tubal and endometrial environment may affect sperm and their interaction with the oocyte and embryo development. Many authors have demonstrated that the PF of patients with endometriosis has detrimental effects on several steps in the reproductive process. On the other hand, various substances, including interleukins, were reported to increase in the PF of infertile women with endometriosis (Ramey et al., 1993; Harada et al., 2001). Recently, some of these substances are believed to be a cause of endometriosis-associated infertility. In fact, substances such as IL-1 (Sueldo et al., 1990
), tumour necrosis factor-
(Eisermann et al., 1989
), endometrial placental protein 14 (Oehninger et al., 1995
), antibodies to transferrin and
2-HS glycoprotein (Pillai et al., 1998
) were reported to adversely affect sperm motility and function and/or embryo development.
We previously reported that IL-6 was increased in the PF of patients with endometriosis and correlated with the extent of active endometriotic lesions (Harada et al., 1997a). We also demonstrated that IL-6 was produced by endometriotic stromal cells at levels similar to those of peritoneal macrophages from patients with endometriosis (Tsudo et al., 2000
). One study demonstrated that the addition of IL-6 to culture medium reduced the blastocyst formation rate of mouse embryos, suggesting that increased IL-6 in the PF of endometriosis patients may contribute to infertility (Harada et al., 1997b
).
Thus, the concept that IL-6 may contribute to endometriosis-associated infertility has been conjectural so far. The present study supports earlier studies that demonstrated the inhibitory effects of PF of patients with endometriosis on sperm motility (Aeby et al., 1996) and functions (Coddington et al., 1992
; Arumugam, 1994
), and it supports the contribution of IL-6 to endometriosis-associated infertility by adversely affecting sperm motility. From our series of studies on IL-6, we conclude that IL-6 may contribute to the pathogenesis of endometriosis-associated infertility.
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Acknowledgements |
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References |
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Arumugam K (1994) Endometriosis and infertility: raised iron concentration in the peritoneal fluid and its effect on the acrosome reaction. Hum Reprod 9, 11531157.[Abstract]
Coddington CC, Oehninger S, Cunningham DS, Hansen K, Sueldo CE and Hodgen GD (1992) Peritoneal fluid from patients with endometriosis decreases sperm binding to the zona pellucida in the hemizona assay: a preliminary report. Fertil Steril 57, 783786.[ISI][Medline]
Dousset B, Hussenet F, Daudin M, Bujan L, Foliguet B and Nabet P (1997) Seminal cytokine concentrations (IL-1beta, IL-2, IL-6, sR IL-2, sR IL-6), semen parameters and blood hormonal status in male infertility. Hum Reprod 12, 14761479.[Abstract]
Drudy L, Lewis SE, Barry-Kinsella C, Harrison RF and Thompson W (1994) The influence of peritoneal fluid from patients with minimal stage or treated endometriosis on sperm motility parameters using computer-assisted semen analysis. Hum Reprod 9, 24182423.[Abstract]
Eggert-Kruse W, Boit R, Rohr G, Aufenanger J, Hund M and Strowitzki T (2001) Relationship of seminal plasma interleukin (IL) -8 and IL-6 with semen quality. Hum Reprod 16, 517528.
Eisermann J, Register KB, Strickler RC and Collins JL (1989) The effect of tumor necrosis factor on human sperm motility in vitro. J Androl 10, 270274.
Fernandez-Shaw S, Hicks BR, Yudkin PL, Kennedy S, Barlow DH and Starkey PM (1993) Anti-endometrial and anti-endothelial auto-antibodies in women with endometriosis. Hum Reprod 8, 310315.[Abstract]
Friebe K, Bohring C, Skrzypek J and Krause W (2003) Levels of interleukin-6 and interleukin-8 in seminal fluid of men attending an andrological clinic. Andrologia 35, 126129.[Medline]
Gorospe WC, Hughes FM and Spangelo BL (1992) Interleukin-6: effects on and production by rat granulosa cells in vitro. Endocrinology 130, 17501752.[Abstract]
Gruschwitz MS, Brezinschek R and Brezinschek HP (1996) Cytokine levels in the seminal plasma of infertile males. J Androl 17, 158163.
Gurbuz B, Yalti S, Ficicioglu C and Zehir K (2003) Relationship between semen quality and seminal plasma total carnitine in infertile men. J Obstet Gynaecol 23, 653656.[CrossRef][Medline]
Haney AF (1991) The pathogenesis and etiology of endometriosis. In Thomas E and Rock J (eds) Modern Approaches to Endometriosis. Kluwer, Norwell, MA, pp. 320.
Harada T, Yoshioka H, Yoshida S, Iwabe T, Onohara Y, Tanikawa M and Terakawa N (1997a) Increased interleukin-6 levels in peritoneal fluid of infertile patients with active endometriosis. Am J Obstet Gynecol 176, 593597.[ISI][Medline]
Harada T, Taniguchi F, Iwabe T, Onohara M, Tanikawa M and Terakawa N (1997b) Cytokines in peritoneal fluid from women with active endometriosis. In Minaguchi H and Sugimoto O (eds) Endometriosis Today. Parthenon, UK, pp. 144148.
Harada T, Iwabe T and Terakawa N (2001) Role of cytokines in endometriosis. Fertil Steril 76, 110.[CrossRef][ISI][Medline]
Harlow CR, Cahill DJ, Maile LA, Talbot WM, Mears J, Wardle PG and Hull MG (1996) Reduced perovulatory granulosa cell steroidogenesis in women with endometriosis. J Clin Endocrinol Metab 81, 426429.[Abstract]
Hirschowitz JS, Soler NG and Wortsman J (1978) The galactorrhea-endometriosis syndrome. Lancet 1(8070), 896898.[Medline]
Huleihel M, Lunenfeld E, Horowitz S, Levy A, Potashnik G, Mazor M and Glezerman M (2000) Involvement of serum and lipopolysaccharide in the production of interleukin-1- and interleukin-6-like molecules by human sperm cells. Am J Reprod Immunol 43, 4146.[ISI][Medline]
Iwabe T, Harada T, Tsudo T, Tanikawa M, Onohara Y and Teralawa N (1998) Pathogenic significance of increased levels of interleukin-8 in peritoneal fluid of patients with endometriosis. Fertil Steril 69, 924930.[CrossRef][ISI][Medline]
Jacobs AL, Sehgal PB, Julian J and Carson DD (1992) Secretion and hormonal regulation of interleukin-6 production by mouse uterine stromal and polarized epithelial cells cultured in vitro. Endocrinology 131, 10371045.[Abstract]
Kocak I, Yenisey C, Dundar M, Okyay P and Serter M (2002) Relationship between seminal plasma interleukin-6 and tumor necrosis factor alpha levels with semen parameters in fertile and infertile men. Urol Res 30, 263267.[CrossRef][Medline]
Lyons RA, Djahanbakhch O, Saridogan E, Naftalin AA, Mahmood T, Weekes A and Chenoy R (2002) Peritoneal fluid, endometriosis, and ciliary beat frequency in the human fallopian tube. Lancet 360, 12211222.[CrossRef][Medline]
Mio Y, Toda T, Harada T and Terakawa N (1992) Luteinized unruptured follicle in the early stage of endometriosis as a cause of unexplained infertility. Am J Obstet Gynecol 167, 271273.[Medline]
Naz RK and Butler A (1996) Interleukin-6 and -8 levels in sera and cervical mucus of fertile, idiopathic infertile women: implication in infertility. Am J Reprod Immunol 35, 534540.[Medline]
Oehninger S, Coddington CC, Hodgen GD and Seppala M (1995) Factors affecting fertilization: endometrial placental protein 14 reduces the capacity of human spermatozoa to bind to the human zona pellucida. Fertil Steril 63, 377383.[ISI][Medline]
Pillai S, Rust PF and Howard L (1998) Effects of antibodies to transferrin and alpha 2HS-glycoprotein on in vitro sperm motion: implications in infertility associated with endometriosis. Am J Reprod Immunol 39, 235242.[CrossRef][Medline]
Prough SG, Aksel S, Gilmore SM and Yeoman RR (1990) Peritoneal fluid fractions from patients with endometriosis on mouse embryo growth. Fertil Steril 54, 783786.[ISI][Medline]
Ramey JW and Archer DF (1993) Peritoneal fluid: its relevance to the development of endometriosis. Fertil Steril 60, 114.[ISI][Medline]
Ray P, Ghosh SK, Zhang DH and Ray A (1997) Repression of interleukin-6 gene expression by 17-estradiol: inhibition of the DNA-binding activity of the transcription factors NF-IL6 and NF-
B by the estrogen receptor. FEBS Lett 409, 7985.[CrossRef][ISI][Medline]
Rose-John S and Heinrich PC (1994) Soluble receptors for cytokines and growth factors: generation and biological function. Biochem J 300, 281290.[ISI][Medline]
Saito M, Yoshida K, Hibi M, Taga T and Kishimoto T (1992) Molecular cloning of a murine receptor-associated signal transducer, gp130, and its regulated expression in vivo. J Immunol 148, 40664071.
Sueldo CE, Kelly E, Montoro L, Subias E, Baccaro M, Swanson JA, Steinleitner A and Lambert H (1990) Effect of interleukin-1 on gamete interaction and mouse embryo development. J Reprod Med 35, 868872.[Medline]
Tagoh H, Nishimoto N, Ogata A and Yoshizaki K (1989) Multiplicity in the production and the function of IL-6. Clin Immunol 21, 12251241.
Tsudo T, Harada T, Iwabe T, Tanikawa M, Nagano Y, Ito M, Taniguchi F and Terakawa N (2000) Altered gene expression and secretion of interleukin-6 in stromal cells derived from endometriotic tissues. Fertil Steril 73, 205211.[CrossRef][ISI][Medline]
Yamazaki K, Yamada E, Kanaji Y, Shizume K, Wang DS, Maruo N and Obara T (1996) Interleukin-6 inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles. Endocrinology 137, 48574863.[Abstract]
Yoshida S, Harada T, Iwabe T, Taniguchi F, Fujii A, Sakamoto Y, Yamauchi N, Shiota G and Terakawa N (2002) Induction of hepatocyte growth factor in stromal cells by tumor-derived basic fibroblast growth factor enhances growth and invasion of endometrial cancer. J Clin Endocrinol Metab 87, 23762383.
Yoshioka H, Harada T, Iwabe T, Nagano Y, Taniguchi F, Tanikawa M and Terakawa N (1999) Menstrual cycle-specific inhibition in the proliferation of endometrial stromal cells by interleukin-6 and its soluble receptor. Am J Obstet Gynecol 180, 10881094.[ISI][Medline]
Submitted on January 5, 2004; accepted on April 22, 2004.