IL-7 and IL-8 inhibit gamete interaction in the zona-free hamster egg sperm penetration assay

Current thought in reproductive endocrinology suggests hat endometriosis-associated subfertility may be the result of an adverse influence of activated immunocompetent cells on fertilization and embryo development. Inflammatory ediators such as interleukin-1 and tumour necrosis actor have been implicated in the pathophysiology of this process. The purpose of this study was to assess the effect of two recently characterized cytokines, interleukin-7 (IL-7 ) and interleukin-8 (IL-8), on gamete interaction in the perm penetration assay (SPA). Donor sperm were preincubated or 4 h with 0.5, 5, 50, or 500 ng ml-1 of human ecombinant IL-7 or IL-8. Sperm penetration was determined by an experienced gametologist by the presence of decondensed sperm heads or pronuclei formation. A dose-dependent inhibition of gamete interaction was observed following coincubation with either IL-7 or IL-8. These data offer the possibility that IL-7 and IL-8 may play a role in the pathogenesis of immunocompetent cell-associated subfertility.


Introduction
Research over the last decade has led to a blurring of the distinctions between endocrinology, immunology, and reproduction. Inflammatory mediators which were presumed to function solely as regulators of immunocompetent cell function have been found to have wide ranging activities as growth factors and paracrine regulators of organ function. Monokines and lymphokines have been shown to modulate ovarian steroidogenesis in vitro and have been hypothesized to contribute to various aspects of ovarian and endometrial homeostasis. 1'2 Activated peritoneal exudative cells have been demonstrated to inhibit early reproductive events in a rodent model. 3 These data have been offered in support of a hypothesis suggesting that alterations of the peritoneal inflammatory environment are responsible for impaired fertility in patients suffering from endometriosis and unexplained infertility. 4 In this report we describe experiments characterizing the effect of interleukin-7 (IL-7, pre-B-cell growth factor) and interleukin-8 (IL-8, macrophage-derived chemotactic factor) on gamete interaction in the hamster egg sperm penetration assay (SPA).

Materials and Methods
Sperm preparation: Semen specimens were obtained on five separate occasions from proven donors serving as fertile controls for the SPA performed (C) 1992 Rapid Communications of Oxford Ltd. in our clinical laboratory. Semen samples were allowed to liquify for 30-60 min at 37C. Concentration and motility were assessed using a Makler chamber. The sample was divided into 0.5 ml aliquots and layered with 3.0 ml of Biggers, Whitten, Whittingham (BWW) media for overnight incubation.
Sperm penetration assay: Sexually mature female golden hamsters (6-8 weeks old) were subjected to ovarian hyperstimulation with intraperitoneal (i.p.) injections of 40 IU of pregnant mare's serum gonadotropins (PMSG, Sigma Chemical Company, St Louis, MO) on cycle day one followed by 40 IU i.p. of human chorionic gonadotropins (hCG, Sigma) 52-56 h later. Sixteen hours after hCG administration, hamsters were sacrificed by cervical dislocation for oocyte collection. The cumulus cell mass was dissipated with 0.1% hyaluronidase (Sigma). Zonae pellucidae were removed by treatment with 0.1% trypsin (Sigma). Eggs were rinsed twice and transferred to wells containing sperm suspensions. Sperm samples were adjusted to a final concentration of 1 106 spermatozoa/ml under the following conditions: neat BWW media, IL-7 or IL-8 at concentrations of 0.5, 5, 50, and 500ngm1-1.
Following a 4 h incubation, oocytes were mounted and read. Penetration of hamster eggs was determined by counting swollen sperm heads and associated tails in cytoplasm.
Cytokines: Recombinant human IL-7 was provided by the Immunex Corporation, Seattle, WA. Human recombinate IL-8 was provided as a gift by Dr. In higher doses of IL-8 (50-500ngml-1), vacuole formation characteristic of oocyte activation was observed in the cytoplasm of some eggs. These eggs were never found to have products of fertilization (i.e. swollen sperm head or pronuclei with accompanying mid-piece and tail) in their cytoplasm. lymphocytes and activated macrophages as well as elevated levels of certain inflammatory mediators. [4][5][6] In vitro and in vivo gamete interaction is inhibited in the presence of peritoneal fluid obtained from endometriosis patients. 7'8 Studies by Hill and coworkers have demonstrated certain inflammatory peptides to have detrimental effects on measures of early reproductive performance in vitro. In their study, gamete interaction in the sperm penetration assay was markedly inhibited by gamma interferon and tumour necrosis factor in physiological concentrations. 9 Human granulocyte macrophage colony stimulating factor (GM-CSF), B-cell growth factor (BCGF), and interleukin-2 (IL-2) had no effect on gamete interaction or were inhibitory only in the highest concentrations tested. In vitro mouse embryo development was inhibited by gamma interferon, colony stimulating factor, and BCGF. IL-2 had no effect on mouse embryo development; tumour necrosis factor and IL-1 were embryo toxic only in high concentrations. Although aberrant humoral and cellular immune function have been implicated in the pathogenesis of infertility in endometriosis patients, the identity of the pathological efl:ector(s) has not been adequately elucidated. While levels of peritoneal fluid inflammatory mediators (e.g., interleukin-1 (IL-1), tumour necrosis factor (TNF), and complement factor-3 (C3)) are more frequently elevated in endometriosis patients as compared to fertile controls and patients suffering from chronic pelvic adhesive disease, 40-60% of endometriosis patients test negative for the cytokine of interest in most reports, s'<l This observation has led investigators to study additional humoral and/or cellular factors as contributors to the infertility observed in these patients.
The data obtained in this study permit us to add IL-7 and IL-8 to the list of inflammatory mediators known to exert a deleterious influence on gamete performance. Preincubation with as little as 0.5 ng m1-1 of IL-7 or IL-8 produced a dramatic inhibition of penetration in the SPA. The increased incidence of oocyte activation observed suggests that the reduced penetration rate is the result of adverse influence on both sperm function and egg metabolism.
Although these data are provocative, as with most inflammatory mediators, it is difficult to assess the relevance of IL-7 and IL-8-induced inhibition of gamete interaction to the pathogenesis of immunocompetent cell-mediated subfertility. The dynamics of most inflammatory peptides (including IL-7 and IL-8) in peritoneal fluid and the reproductive tract have not been elucidated. At present IL-8 is thought to act primarily as a chernoattractant and activator of rnacrophages to sites of inflammation. 12 The physiological role of IL-7 is less clear. IL-7 is known to stimulate the proliferation of bone marrow-derived pre-B-cells 13 and to serve as a growth factor for T-cells. 14 There are no data describing tumour cell lysis or modulation of nonimmunocompetent cell metabolism by IL-8. The data obtained in this study offer the possibility that IL-7 and/or IL-8 may play a role in the pathogenesis of endometriosis-associated subfertility and unexplained infertility in these individuals. Additional studies characterizing the presence of IL-7 and IL-8 in peritoneal fluid and its relationship to the inhibition of in vitro gamete interaction and other inflammatory mediators such as TNF and IL-1 are in progress.