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Lipopolysaccharide induces alterations in ovaries and serum level of progesterone and 17β-estradiol in the mouse

2010; Elsevier BV; Volume: 95; Issue: 4 Linguagem: Inglês

10.1016/j.fertnstert.2010.08.046

1556-5653

Varkha Agrawal, Mukesh K. Jaiswal, Yogesh Jaiswal,

Endometriosis Research and Treatment

Our objective was to investigate the effect of gram-negative bacterial infection on the ovaries and serum level of P4 and 17β-E2 during the preimplantation days of pregnancy in the mouse. We found that lipopolysaccharide alters the serum level of P4 and E2 during the preimplantation days of pregnancy and elevates the E2/P4 ratio, which may keep the uterus nonreceptive during the preimplantation days of pregnancy and also not prepare the developing blastocysts for implantation in the mouse. A large infiltration of macrophages in the corpora lutea and appearance of graafian follicles from day 3.5 of pregnancy because of lipopolysaccharide treatment, which reflect a gram-negative bacterial infection, may be responsible for ovarian dysfunction and altered P4 and E2 level in serum. Our objective was to investigate the effect of gram-negative bacterial infection on the ovaries and serum level of P4 and 17β-E2 during the preimplantation days of pregnancy in the mouse. We found that lipopolysaccharide alters the serum level of P4 and E2 during the preimplantation days of pregnancy and elevates the E2/P4 ratio, which may keep the uterus nonreceptive during the preimplantation days of pregnancy and also not prepare the developing blastocysts for implantation in the mouse. A large infiltration of macrophages in the corpora lutea and appearance of graafian follicles from day 3.5 of pregnancy because of lipopolysaccharide treatment, which reflect a gram-negative bacterial infection, may be responsible for ovarian dysfunction and altered P4 and E2 level in serum. A fundamental feature of implantation is synchronized development of the embryo to the blastocyst stage and differentiation of the uterus to a receptive state (1Paria B.C. Reese J. Das S.K. Dey S.K. Deciphering the cross-talk of implantation: advances and challenges.Science. 2002; 296: 2185-2188Crossref PubMed Scopus (425) Google Scholar, 2Paria B.C. Limb H. Das S.K. Reese J. Dey S.K. Molecular signaling in uterine receptivity for implantation.Semin Cell Dev Biol. 2000; 11: 67-76Crossref PubMed Scopus (119) Google Scholar). The transition of a receptive to a nonreceptive uterus leads to failure of blastocyst implantation. Progesterone (P4) regulates the uterine receptivity for blastocyst attachment and coordinates uterine and embryonic interactions (3Lee K. Jeong J. Tsai M.J. Tsai S. Lydon J.P. DeMayo F.J. Molecular mechanisms involved in progesterone receptor regulation of uterine function.J Steroid Biochem Mol Biol. 2006; 102: 41-50Crossref PubMed Scopus (44) Google Scholar). Estrogen is essential for on-time uterine receptivity (4Paria B.C. Huet-Hudson Y.M. Dey S.K. Blastocyst’s state of activity determines the “window” of implantation in the receptive mouse uterus.Proc Natl Acad Sci U S A. 1993; 90: 10159-10162Crossref PubMed Scopus (431) Google Scholar) and activates the dormant blastocysts to make them adhesion competent for implantation (5Hamatani T. 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CUB-Réa Network: Current epidemiology of septic shock.Am J Respir Crit Care Med. 2003; 168: 165-172Crossref PubMed Scopus (532) Google Scholar), and is associated with adverse developmental outcome including embryonic resorption and preterm delivery (7Deb K. Chaturvedi M.M. Jaiswal Y.K. Comprehending the role of LPS in Gram-negative bacterial vaginosis: ogling into the causes of unfulfilled child-wish.Arch Gynecol Obstet. 2004; 270: 133-146Crossref PubMed Scopus (46) Google Scholar, 15Deb K. Chaturvedi M.M. Jaiswal Y.K. Gram-negative bacterial endotoxin induced infertility: a bird’s eye view.Gynecol Obstet Invest. 2004; 57: 224-232Crossref PubMed Scopus (30) Google Scholar). We established a gram-negative bacterial infection model in the mouse in which the intraperitoneal injection of LPS (250 μg/kg body weight on day 0.5 of pregnancy) causes embryonic cell death (16Jaiswal Y.K. Chaturvedi M.M. Deb K. Effect of bacterial endotoxins on superovulated mouse embryos in vivo: is CSF-1 involved in endotoxininduced pregnancy loss?.Infect Dis Obstet Gynecol. 2006; https://doi.org/10.1155/IDOG/2006/32050Crossref PubMed Scopus (15) Google Scholar) and leads to implantation failure on day 5.5 of pregnancy (17Deb K. Chaturvedi M.M. Jaiswal Y.K. A “minimum dose” of LPS required for implantation failure: assessment of its effect on the maternal reproductive organs and IL-1α expression in mouse.Reproduction. 2004; 128: 87-97Crossref PubMed Scopus (50) Google Scholar). Lipopolysaccharide causes an antifertility effect by altering expression of tumor necrosis factor-α (18Deb K. Chaturvedi M.M. Jaiswal Y.K. The role of TNF-α in Gram-negative bacterial LPS induced implantation failure.Reprod Med Biol. 2005; 4: 79-88Google Scholar), interleukin-1 (17Deb K. Chaturvedi M.M. Jaiswal Y.K. A “minimum dose” of LPS required for implantation failure: assessment of its effect on the maternal reproductive organs and IL-1α expression in mouse.Reproduction. 2004; 128: 87-97Crossref PubMed Scopus (50) Google Scholar, 19Deb K. Chaturvedi M.M. Jaiswal Y.K. Gram-negative bacterial LPS induced poor uterine receptivity and implantation failure in mouse: alterations in IL-1β, expression in the preimplantation embryo and uterine horns.Infect Dis Obstet Gynecol. 2005; 13: 125-133Crossref PubMed Scopus (31) Google Scholar), and colony-stimulating factor (16Jaiswal Y.K. Chaturvedi M.M. Deb K. Effect of bacterial endotoxins on superovulated mouse embryos in vivo: is CSF-1 involved in endotoxininduced pregnancy loss?.Infect Dis Obstet Gynecol. 2006; https://doi.org/10.1155/IDOG/2006/32050Crossref PubMed Scopus (15) Google Scholar). Disturbance in embryonic and uterine secretion of these cytokines may trigger DNA damage in embryos (20Jaiswal Y.K. Jaiswal M.K. Agrawal V. Chaturvedi M.M. Bacterial endotoxin (LPS) induced DNA damage in preimplanting embryonic and uterine cells inhibits implantation.Fertil Steril. 2008; 91: 2095-2103Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 21Jaiswal Y.K. Jaiswal M.K. Agrawal V. Chaturvedi M.M. Deb K. Maternal Gram-negative bacterial infection induced apoptosis of the implanting blastocyst.Journal of the Turkish-German Gynecological Association. 2007; 8: 190-193Google Scholar) and uterine cells (21Jaiswal Y.K. Jaiswal M.K. Agrawal V. Chaturvedi M.M. Deb K. Maternal Gram-negative bacterial infection induced apoptosis of the implanting blastocyst.Journal of the Turkish-German Gynecological Association. 2007; 8: 190-193Google Scholar) during preimplantation days and leads to implantation failure in the mouse. Lipopolysaccharide stimulates macrophages to secrete cytokines, which inhibit ovarian functions (22Sancho-Tello M. Tash J.S. Roby K.F. Terranova P.F. Effects of lipopolysaccharide on ovarian function in the pregnant mare serum gonadotropin-treated immature rat.Endocrine. 1993; 1: 503-511Google Scholar). The aim of this work was to unravel the effect of gram-negative bacterial infection on follicular development and corpora lutea (CL) in ovaries and serum level of P4 and 17β-Estradiol (E2) during preimplantation days of pregnancy in the mouse. This study was conducted in accordance with institutional ethics committee guidelines for the care and use of animals in research. Park strain females (6–8 weeks, 20–21 g) in proestrus (selected by measuring vaginal electrical resistance [23Agrawal V. Jaiswal M.K. Chaturvedi M.M. Tiwari D.C. Jaiswal Y.K. Lipopolysaccharide alters the vaginal electrical resistance in cycling and pregnant mice.Am J Reprod Immunol. 2009; 61: 158-166Crossref PubMed Scopus (13) Google Scholar]) were impregnated naturally and checked on the following day for the presence of a vaginal plug. The morning of plugging was considered day 0.5 of pregnancy. Conditions of gram-negative bacterial infection were induced by injecting LPS (Salmonella enterica serotype minnesota Re 595; Sigma, St. Louis, MO) (250 μg/kg body weight, intraperitoneally) on day 0.5 of pregnancy (16Jaiswal Y.K. Chaturvedi M.M. Deb K. Effect of bacterial endotoxins on superovulated mouse embryos in vivo: is CSF-1 involved in endotoxininduced pregnancy loss?.Infect Dis Obstet Gynecol. 2006; https://doi.org/10.1155/IDOG/2006/32050Crossref PubMed Scopus (15) Google Scholar). Pregnant animals were divided into two groups; controls and treated animals received normal saline solution and LPS, respectively, on day 0.5 of pregnancy. Serum was collected from days 1.5 to 4.5 of pregnancy. Concentrations of P4 and E2 were measured by P4 ELISA kit (Demeditec Diagnostics GmbH, Kiel-Wellsee, Germany) and E2-sensitive ELISA kit (DRG International, Mountainside, NJ), respectively. Animals were killed from days 0.5 to 5.5 of pregnancy, ovaries were collected, and histopathologic examination was performed according to standard procedure (24McManus J.F.A. Mowry R.W. Staining methods histological and histochemical. Paul B. Hoeber, New York1960Google Scholar). Cross-sections of ovaries were checked for presence of macrophages (25Bagavandoss P. Wiggins R.C. Kunkel S.L. Remick D.G. Keyes P.L. Tumor necrosis factor production and accumulation of inflammatory cells in the corpus luteum of pseudopregnancy and pregnancy in rabbits.Biol Reprod. 1990; 42: 367-376Crossref PubMed Scopus (135) Google Scholar) and mitotic bodies (26Finn C.A. Pope M. Milligam S.R. Control of uterine stromal mitosis in relation to uterine sensitivity and decidualization in mice.J Reprod Fertil. 1995; 103: 153-158Crossref PubMed Scopus (21) Google Scholar). Results were analyzed by ANOVA with Duncan’s multiple-range tests for comparison of significance level (P) between both groups. A gradual increase in serum level of P4 was observed from days 1.5 (8.67 ± 0.77 ng/mL) to 3.5 (24.13 ± 2.35 ng/mL) of pregnancy, and the same higher level was maintained till the day of implantation in control animals (24.55 ± 1.46 ng/mL). However, in LPS-treated animals the serum level of P4 increased from day 1.5 (7.87 ± 0.71 ng/mL) to 2.5 (16.92 ± 0.58 ng/mL) of pregnancy followed by a significant decrease on day 3.5 (13.28 ± 1.04 ng/mL) (P<.01), and the same decreased level was maintained till the day of implantation (12.28 ± 0.76 ng/mL) (P<.01) as compared with respective controls (Fig. 1A ). Thus, the serum level of P4 decreased significantly during preimplantation days in LPS-treated animals. The serum level of E2 decreased gradually from day 1.5 to 4.5 (9.27 ± 1.24 to 4.66 ± 0.83 pg/mL) in control animals. Lipopolysaccharide treatment resulted in a significantly higher serum level of E2 on day 2.5 (16.31 ± 1.49 pg/mL) (P<.01) followed by a slight drop on day 3.5 (11.14 ± 2.13 pg/mL) and a significant increase on day 4.375 to 4.5 (15.29 ± 0.11 to 15.61 ± 1.84 pg/mL) (P<.01) of pregnancy as compared with the respective controls (Fig. 1B). Thus, the serum level of E2 increased significantly during preimplantation days in LPS-treated animals. Serum E2/P4 ratio was calculated and compared in both groups by using the measured E2 and P4 values from days 1.5 to 4.5 of pregnancy (Fig. 1C). A sharp decrease in serum E2/P4 ratio was observed from days 1.5 to 4.5 (1.23 ± 0.19 to 0.20 ± 0.03) of pregnancy in control animals. However, the altered serum level of E2 and P4 in LPS-treated animals significantly elevated the E2/P4 ratio from day 2.5 to 4.5 of pregnancy (0.94 ± 0.04 to 1.25 ± 0.18) as compared with their respective controls (P<.05) (Fig. 1C). Thus, the LPS treatment significantly elevates the serum E2/P4 ratio during preimplantation days. Ovarian cross-sections were studied for luteinization of CL (Fig. 1D), macrophage infiltration (Fig. 1E), and mitotic body count in CL (Fig. 1F). No difference was observed between ovarian cross-sections from days 0.5 to 2.5 of pregnancy between both groups. Ovarian cross-sections from LPS-treated animals show shrunken CL cells with morphologically indistinct boundaries from days 3.5 to 5.5 of pregnancy (Fig. 1D). In control, CL cells were vacuolated with clear and distinct cell boundaries (Fig. 1D). The infiltration of macrophages was increased slightly on day 3.5 followed by a significant increase from days 4.5 to 5.5 of pregnancy (P<.01) in LPS-treated animals (Fig. 1E). The regression of CL in LPS-treated animals explicates the low serum level of P4 caused by LPS treatment. No differences in mitotic body count were found in both groups (Fig. 1F). The pattern of follicular development was observed in ovarian cross-sections from both groups that show the appearance of graafian follicles (Fig. 1D) in LPS-treated animals from days 3.5 to 5.5 of pregnancy, but not in controls. This explains the high serum level of E2 caused by LPS treatment. Our study reports that the serum level of P4 decreased and E2 increased during the preimplantation days of pregnancy in a gram-negative bacterial infection model. We also present evidence that these altered levels of ovarian steroids may be due to LPS-induced alterations in ovarian tissues. The results demonstrating that LPS treatment decreases the serum level of P4 during preimplantation days in the mouse are in agreement with a previous report that LPS injection on day 15 of pregnancy decreases the serum level of P4 in rats, where the source of P4 is the placenta (27Goyeneche A.A. Deis R.P. Gibori G. Telleria C.M. Progesterone promotes survival of the rat corpus luteum in the absence of cognate receptors.Biol Reprod. 2003; 68: 151-158Crossref PubMed Scopus (59) Google Scholar). Our results representing regression of CL and increased infiltration of macrophages in CL near implantation days explain the observed low serum level of P4 in LPS-treated animals. The CL maintain the uterine endometrium for blastocyst implantation by secreting adequate amounts of P4 during early pregnancy. Progesterone acts as immunomodulator at the maternal-fetal interface and plays a role in the survival of the fetus (28Peltier M.R. Immunology of term and preterm labor.Reprod Biol Endocrinol. 2003; 1: 122Crossref PubMed Scopus (202) Google Scholar). Lipopolysaccharide treatment alters the expression of tumor necrosis factor-α (18Deb K. Chaturvedi M.M. Jaiswal Y.K. The role of TNF-α in Gram-negative bacterial LPS induced implantation failure.Reprod Med Biol. 2005; 4: 79-88Google Scholar), interleukin-1 (17Deb K. Chaturvedi M.M. Jaiswal Y.K. A “minimum dose” of LPS required for implantation failure: assessment of its effect on the maternal reproductive organs and IL-1α expression in mouse.Reproduction. 2004; 128: 87-97Crossref PubMed Scopus (50) Google Scholar, 19Deb K. Chaturvedi M.M. Jaiswal Y.K. Gram-negative bacterial LPS induced poor uterine receptivity and implantation failure in mouse: alterations in IL-1β, expression in the preimplantation embryo and uterine horns.Infect Dis Obstet Gynecol. 2005; 13: 125-133Crossref PubMed Scopus (31) Google Scholar), and colony-stimulating factor-1 (16Jaiswal Y.K. Chaturvedi M.M. Deb K. Effect of bacterial endotoxins on superovulated mouse embryos in vivo: is CSF-1 involved in endotoxininduced pregnancy loss?.Infect Dis Obstet Gynecol. 2006; https://doi.org/10.1155/IDOG/2006/32050Crossref PubMed Scopus (15) Google Scholar) and inhibits the implantation of the blastocyst (17Deb K. Chaturvedi M.M. Jaiswal Y.K. A “minimum dose” of LPS required for implantation failure: assessment of its effect on the maternal reproductive organs and IL-1α expression in mouse.Reproduction. 2004; 128: 87-97Crossref PubMed Scopus (50) Google Scholar), as reported previously. Cytokine-rich environments cause the generation of superoxide radicals in CL (29Sugino N. Telleria C.M. Gibori G. Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase in the rat corpus luteum: induction of manganese superoxide dismutase messenger ribonucleic acid by inflammatory cytokines.Biol Reprod. 1998; 59: 208-215Crossref PubMed Scopus (61) Google Scholar) and its regression, which may be responsible for the low level of P4 during preimplantation days in LPS-treated animals. In primates, P4 concentration remains elevated until the delivery of the placenta and removal and decrease of P4 causes labor (28Peltier M.R. Immunology of term and preterm labor.Reprod Biol Endocrinol. 2003; 1: 122Crossref PubMed Scopus (202) Google Scholar, 30Bosu W.T.K. Johansson E.D.B. Gemzell C. Peripheral plasma levels of oesterogens, progesterone and 17alpha-hydroxyprogesterone during gestation in the rhesus monkey.Acta Endocrinol (Copenh). 1973; 74: 348-360PubMed Google Scholar, 31Illingworth D.V. Challis J.R.G. Ackland N. Burton A.M. Heap R.B. Perry J.S. Parturition in the guinea-pig; plasma level of steroid hormones, steroid-binding proteins, and oxytocin, and the effect of corticosteroids, prostaglandins and adrenocorticotrophin.J Endocrinol. 1974; 63: 557-570Crossref PubMed Scopus (31) Google Scholar). It may be postulated that elevated levels of P4 during preimplantation days are involved in survival of fetuses and the protective effect of P4 may be reduced during gram-negative bacterial infection because of the observed low level of P4 in serum. Our results in the gram-negative bacterial infection model show that the serum level of E2 increased during preimplantation days. It may be postulated that the receptive state of the uterus is altered at the molecular level in the presence of a higher E2 and lower P4 level in serum, leading to implantation failure in LPS-treated animals. This postulation is supported by evidence that higher estrogen levels promptly transform the uterus to a refractory state (32Ma W. Song H. Das S.K. Paria B.C. Dey S.K. Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation.Proc Natl Acad Sci U S A. 2003; 100: 2963-2968Crossref PubMed Scopus (421) Google Scholar, 33Simon C. Domínguez F. Valbuena D. Pellicer A. The role of estrogen in uterine receptivity and blastocyst implantation.Trends Endocrinol Metab. 2003; 14: 197-199Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Uterine gene expression contributing to blastocyst implantation is maintained at a lower E2 dose, whereas gene expression promptly becomes aberrant at higher E2 levels (32Ma W. Song H. Das S.K. Paria B.C. Dey S.K. Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation.Proc Natl Acad Sci U S A. 2003; 100: 2963-2968Crossref PubMed Scopus (421) Google Scholar). In LPS-treated animals, the observed high E2 and low P4 level in serum during preimplantation days may transform the uterine receptivity to a refractory state that results in unsuccessful pregnancy in the mouse. In the present study we have shown the presence of graafian follicles in LPS-treated animals from days 3.5 to 5.5 of pregnancy. This may support the observed high serum level of E2 in LPS-treated animals during preimplantation days of pregnancy, because granulosa cells of graafian follicles produce E2. The presence of graafian follicles in LPS-treated animals from day 3.5 of pregnancy also correlated with our previous observation that LPS-treated animals start a normal reproductive cycle after implantation failure (23Agrawal V. Jaiswal M.K. Chaturvedi M.M. Tiwari D.C. Jaiswal Y.K. Lipopolysaccharide alters the vaginal electrical resistance in cycling and pregnant mice.Am J Reprod Immunol. 2009; 61: 158-166Crossref PubMed Scopus (13) Google Scholar). A distinct E2/P4 ratio is required during preimplantation days to maintain the uterine receptivity and to help developing blastocysts in reaching an implantation-competent state. The observed significantly higher E2/P4 ratio in LPS-treated animals may keep the uterus nonreceptive during implantation and not prepare the developing blastocysts for implantation. This altered hormone ratio may be the probable cause of early pregnancy loss in response to gram-negative bacterial infections. Two different studies reported that decreased P4/estrogens ratios influence the pattern of early embryonic development and implantation by decreased numbers of normal blastocysts in the rat (34Lapolt P.S. Day J.R. Lu J.K.H. Effects of estradiol and progesterone on early embryonic development in aging rats.Biol Reprod. 1990; 43: 843-850Crossref PubMed Scopus (9) Google Scholar, 35Day JR, Lapolt PS, Lu JK. Plasma patterns of prolactin, progesterone, and estradiol during early pregnancy in aging rats: relation to embryonic development. Biol Reprod 44:786–790.Google Scholar). Lipopolysaccharide treatment induces the high number of abnormal embryos during preimplantation days, as reported previously (20Jaiswal Y.K. Jaiswal M.K. Agrawal V. Chaturvedi M.M. Bacterial endotoxin (LPS) induced DNA damage in preimplanting embryonic and uterine cells inhibits implantation.Fertil Steril. 2008; 91: 2095-2103Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 21Jaiswal Y.K. Jaiswal M.K. Agrawal V. Chaturvedi M.M. Deb K. Maternal Gram-negative bacterial infection induced apoptosis of the implanting blastocyst.Journal of the Turkish-German Gynecological Association. 2007; 8: 190-193Google Scholar). This observation suggests that a significantly elevated serum E2/P4 ratio may lead to the recovery of a higher number of abnormal embryos during preimplantation days of pregnancy in LPS-treated animals. This finding strongly suggests that LPS-induced ovarian dysfunction alters the E2/P4, which disturbs the required cross-talk between the implanting embryo and endometrium. The mechanism of early pregnancy loss during gram-negative bacterial infection is elucidated by this finding, which suggests that P4 and E2 are two of the chief players in the decisive event of life, that is, implantation.