ABSTRACT
Active oxygen derived from gp91phox is critical for gestation. However, no reports have evaluated the relationship between reactive oxygen species (ROS) and the number of births in a given pregnancy. In this study, we examined the influence of ROS produced by gp91phox activity on the number of births using C57BL/6j (control) and gp91phox-knockout (gp91phox−/-) mice. The number of births in gp91phox−/- mice was found to be lower than that in control mice. We observed sequential increases in gp91phox, ROS, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), caspase-1, and interleukin-18 (IL-18), followed by increased expression of mucin1 (MUC1), in control mice. However, none of these markers were upregulated in gp91phox−/- mice. In addition, in control mice administered IL-18 or MUC1 inhibitors, the number of births decreased to a number similar to that of gp91phox−/- mice. These results suggest that ROS derived from gp91phox activity altered the inflammatory system and produced IL-18, which subsequently increased the expression of MUC1, thereby modulating fetal development.
Abbreviations: IL-1 β: interleukin-1β; IL-18: interleukin-18; NLRP3: nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; IgA: immunoglobulin A; MUC1: mucin1
Introduction
The production of reactive oxygen species (ROS) induces oxidative stress in living organisms, resulting in major adverse effects. Excessive ROS can also affect reproduction. Sperm are easily influenced by aggressive free radicals and are prone to DNA damage [Bronson Citation2016], which may induce blastogenic lesions after fertilization. In the female body, ROS are necessary. During ovulation after the production of ovarian follicles, ROS are associated with the ovarian follicle rhexis [Sugino Citation2005; Takiguchi et al. Citation2004; Sugino et al. Citation2000]. If conception does not occur, yellow-body atrophia is needed to permit subsequent cycles of ovarian follicle growth, and ROS plays a critical role in this process [Sugino and Okuda Citation2007; Sugino Citation2006]. However, while ROS helps maintain homeostasis in the ovaries, if ROS levels become too high, adverse effects can occur. For example, excessive ROS during ovulation reduces the substantia of the ovum and cause fertilization lesions [Tamura et al. Citation2008a]. Moreover, excessive ROS during metaphase of the luteal phase can cause luteal dysfunction [Tamura et al. Citation2008b]. In addition, ROS adversely affects the uterine mucous membrane [Yuan et al. Citation2014]. Thus, ROS generally disrupts the gestation process.
ROS are produced by cell membrane gp91phox NADPH oxidase (Nox2). Gp91phox is a subunit of the enzymatic complex and binds with p22phox [Sumimoto Citation2008]. We previously measured the number of births and weights of offspring in gp91phox-knockout (gp91phox−/-) mice and found that these mice tended to have smaller litters than control mice [Hiramoto et al. Citation2016b]. In addition, silencing of the gp91phox gene suppressed the expression of insulin-like growth factor-1 (IGF-1) through a ROS-dependent mechanism, resulting in low birth weight offspring. However, the mechanisms through which ROS decreased the number of births was unclear.
Therefore, in this study, we aimed to clarify the role of ROS in gestation by examining the mechanism through which reduced levels of ROS decreased the number of births using gp91phox−/- mice.
Results
Litter size and expression of gp91phox in the uterus of graviditas gp91phox−/- mice
The number of births in gp91phox−/- mice was lower than that in wild-type mice (). The expression of gp91phox in the uterus was increased during graviditas (days 6 and 15) in C57BL/6j (control) mice (). In contrast, in gp91phox−/- mice, gp91phox expression was nearly absent (). Similarly, the plasma ROS levels were increased in graviditas C57BL/6j mice compared with that in nonpregnant control mice (). Notably, however, ROS levels were unchanged in graviditas gp91phox−/- mice compared with that in nonpregnant control mice (). In addition, the expression of gp91phox and ROS throughout gestation peaked at day 6 and then decreased until day 15, after which the expression did not change (data not shown). shows the values for days 6 and 15.
Figure 1. Effects of pregnancy on the disappearance of embryo and expression of gp91phox. The number of births and embryos (A), expression of gp91phox in the uterus (B), and the plasma levels of reactive oxygen species (ROS) (C) in graviditas C57BL/6j (control) and gp91phox−/- mice. Uterus samples were extracted on day 6 and 15 of gestation. The values are presented as the means ± SD of data from six animals. *p<0.05.
In addition, of the molecular targets evaluated in this study, only gp91phox and IGF-1 showed differential expression between gp91phox−/- mice and C57BL6j mice. Therefore, gp91phox−/- mice were assumed to be viable.
Expression of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), caspase-1, interleukin (IL)-1β, and IL-18 in graviditas gp91phox−/- mice
Since gp91phox expression peaked on day 6 in wild-type mice (), we used day 6 of gestation as a reference point in subsequent studies. In wild-type graviditas C57BL/6j mice, we observed strong expression of NLRP3 and caspase-1 in the uterus compared with those in nonpregnant C57BL/6j mice. However, in gp91phox−/- mice, this difference was not observed between nonpregnant control mice and gestating mice (). Similarly, IL-1β and IL-18 levels were increased in graviditas C57BL/6j mice compared with that in nonpregnant control mice; in contrast, the levels of these two cytokines were virtually unchanged in graviditas gp91phox−/- mice compared with that in nonpregnant control mice ().
Figure 2. Analysis of inflammatory substances and mucin1 (MUC1) in gp91phox−/- mice. Expression of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), and caspase-1 in the uterus (A) and the plasma levels of interleukin (IL)-1β and IL-18 (B) in graviditas C57BL/6j and gp91phox−/- mice. Expression of MUC1 in the uterus in graviditas C57BL/6j and gp91phox−/- mice (C). The uterus is believed to strongly express MUC1, which protects the embryo. The expression of MUC1 in pregnant gp91phox−/- mice was lower than in pregnancy wild type mice. In subsequent examinations, we used the samples from day 6 of gestation based on the results in . The values are presented as the means ± SD of data from six animals. *p<0.05.
Expression of mucin 1 (MUC1) in the uterus of graviditas gp91phox−/- mice
MUC1 is a mucin core protein that is predominantly expressed in the uterus. In C57BL/6j mice, the expression of MUC1 in the uterus increased during gestation. However, surprisingly, no marked changes in the expression of MUC1 were observed during gestation in gp91phox−/- mice. In addition, no differences were observed among C57BL/6j control mice, gp91phox−/- control mice, and gp91phox−/- gestating mice ().
Effects of a caspase-1 inhibitor in graviditas gp91phox−/- and C57BL/6j mice
We then evaluated the number of births, plasma IL-1β, and IL-18 levels, and expression of MUC1 in the uterus during gestation. In gestating C57BL/6j mice, the expression levels of IL-1β, IL-18, and MUC1 decreased following administration of a caspase-1 inhibitor. In contrast, no changes were observed following caspase-1 inhibitor administration in gestating gp91phox−/- mice ().
Figure 3. Effects of caspase-1 inhibitor on gp91phox−/- mice. Effects of caspase-1 inhibition on the number of births, plasma levels of IL-1β and IL-18, and expression of caspase-1 and MUC1 in the uterus in graviditas C57BL/6j and gp91phox−/- mice. These values were only high in non-treated pregnant wild type mice. The values are presented as the means ± SD of data from six animals. *p<0.05.
Effects of an IL-1β inhibitor in graviditas gp91phox−/- and C57BL/6j mice
In gestating gp91phox−/- mice, the number of births was low (), even following administration of an IL-1β inhibitor (). In addition, in gestating C57BL/6j mice, the increases in IL1β, IL-18, and MUC1 expression did not change following administration of an IL-1β inhibitor ().
Figure 4. Effects of an IL-1β inhibitor on gp91phox−/- mice. Effects of IL-1β inhibition on the number of births, plasma levels of IL-1β and IL-18, and expression of MUC1 in the uterus in graviditas C57BL/6j and gp91phox−/- mice. These values did not change in non-treated and treated pregnant wild type mice. The values are presented as the means ± SD of data from six animals. *p<0.05.
Effects of IL-18 binding protein (IL-18BP) administration in graviditas gp91phox−/- and C57BL/6j mice
n the C57BL/6j mice, the number of births decreased to a number similar to that of gp91phox−/- mice following treatment with IL-18BP. In gestating C57BL/6j mice, the levels of IL-18 and IL-1β remained high following administration of IL-18BP; conversely, the levels of IL-18 and IL-1β remained low in gestating gp91phox–/- mice. In contrast, in gestating C57BL/6j mice, the expression of MUC1 in the uterus was markedly inhibited by administration of IL-18BP. Furthermore, in gestating gp91phox−/- mice, the expression of MUC1 did not change with IL-18BP administration ().
Figure 5. Effects of an IL-18 inhibitor on gp91phox−/- mice. Effects of IL-18 binding protein on the number of births, plasma levels of IL-1β and IL-18, and expression of MUC1 in the uterus in graviditas C57BL/6j and gp91phox−/- mice. Only the MUC1 values were high in non-treatment pregnant wild type mice. The values are presented as the means ± SD of data from six animals. *p<0.05.
Effects of a MUC1 inhibitor in graviditas gp91phox−/- and C57BL/6j mice
Plasma IL-1β and IL-18 levels on day 6 of gestation were not affected by administration of a MUC1 inhibitor, and the levels in C57BL/6j mice remained higher than those in gp91phox−/- mice. However, in C57BL/6j mice, the number of births decreased to a number similar to that in gp91phox−/- mice ().
Figure 6. Effects of a MUC1 inhibitor on gp91phox−/- mice. Effects of MUC1 inhibition on the number of births, plasma levels of IL-1β and IL-18, and expression of MUC1 in the uterus in graviditas C57BL/6j and gp91phox−/- mice. These values did not change in non-treated and treated pregnant wild type mice. The values are presented as the means ± SD of data from six animals. *p<0.05.
Effects of uterine IgA expression in graviditas gp91phox−/- and C57BL/6j mice
To evaluate the relationship between immunity and gp91phox, we observed the uterine expression of IgA (). The expression of IgA was higher in gp91phox−/- mice than in C57BL/6j mice, with peak levels observed during gestation in gp91phox−/- mice. No marked differences in the expression of IgA were noted between gestating and nongestating C57BL/6j mice.
Figure 7. Effects of pregnancy on the expression of IgA in the uterus. The data reflect the results of typical experiments in six animals. The expression of immunoglobulin A (IgA) was increased in gp91phox−/- mice and further increased by gestation. Scale bar = 100 μm. The values are presented as the means ± SD of data from six animals. *p<0.05.
Discussion
In the present study, we examined the mechanism through which ROS decreases the number of births using gp91phox–/- mice. Gp91phox−/- mice showed lower uterine levels of NLRP3, caspase-1, and MUC1 and lower plasma levels of IL-1β and IL-18 than C57BL/6j mice during gestation. In addition, C57BL/6j mice administered an IL-1β inhibitor produced larger litters than similarly treated gp91phox−/- mice. However, in C57BL/6j mice administered either caspase-1 or IL-18 inhibitors, the number of births was not markedly different from those in gp91phox−/- mice.
Gp91phox NADPH oxidase plays a major role in biophylaxis owing to its bactericidal activities [Nauseef Citation2004; Cross and Segal Citation2004; Quinn and Gauss Citation2004]. In addition, this protein has been shown to be deeply involved in the functional regulation of the ovary and uterine mucous membrane [Sugino Citation2005; Sugino et al. Citation2000; Sugino and Okuda Citation2007], underscoring its involvement in reproduction.
We previously showed that the birth weights of offspring were lower in gp91phox−/- mice than in C57BL/6j mice [Hiramoto et al. Citation2016b]. We proposed the following mechanism for this reduction in body weight: ROS derived from gp91phox activates inflammatory NLRP3, which increases the expression of caspase-1; caspase-1 in turn increases the expression of IL-1β, and IL-1β is then believed to increase the expression of IGF-1, which subsequently increases birth weight.
In the present study, the litter size was smaller in gp91phox−/- mice than in C57BL/6j mice. ROS are known to be associated with ovulation [Shkolnik et al. Citation2011] and are involved in the ovarian follicle rhexis. In gp91phox−/- mice, in which little gp91phox-dependent ROS are generated, decreased ovulation occurs, subsequently reducing the number of births [Hiramoto et al. Citation2016b]. However, the observed reduction in the number of follicles in gp91phox−/- mice does not appear to influence fertilization [Brannstrom et al. Citation1995]. Alternatively, other reports have shown that the number of births decreased in the presence of excessive ROS [Al-Gubory et al. Citation2010; Fujii et al. Citation2005]. Therefore, moderate ROS are required for gestation. Notably, no studies have directly measured the amount of ROS necessary during gestation. The mice in our study showed ROS levels of approximately 50-100 pg/ml based on this examination. However, since the amount of ROS varies during the gestational period, the amount of ROS necessary at the time of gestation is not known.
In our previous study, we investigated the relationship between the birth weights of offspring and the expression of IL-1β. Our results showed that IL-1β played an important role in the observed decrease in weight. Gp91phox−/- mice exhibit reduced birth weights of offspring compared with that of C57BL/6j mice [Hiramoto et al. Citation2016b]. Thus, we evaluated whether IL-1β may be related to the number of births by administration of an IL-1β inhibitor. Notably, treatment with the IL-1β inhibitor resulted in increased offspring numbers in C57BL/6j mice compared with that in gp91phox−/- mice, suggesting that IL-1β was unrelated to the number of births.
Gp91phox activates NLRP3, increases the secretion of IL-1β, and promotes the secretion of IL-18 [Brydges et al. Citation2013]. IL-18 is a cytokine of the IL-1 family [Sims and Smith Citation2010] that increases the production of mucin [Metwally et al. Citation2011; Ishii et al. Citation2005]. Mucin is found in the epithelium and helps protect cells against extracellular insults [Rose Citation1992]. In the present study, MUC1 expression in the uterus was lower in gp91phox−/- mice than in wild-type mice (). ROS originating from gp91phox induced the expression of MUC1, which protects the uterus and subsequently ensures fetal survival. When gestating C57BL/6j mice were treated with a MUC1 inhibitor, the number of births decreased (), indicating that MUC1 was necessary for fetal survival. In addition, MUC1 is also involved in the differentiation of the epidermis [Braga et al. Citation1992], and its expression changes throughout gestation [Braga and Gendler Citation1993]. The differentiation of the epidermis in the uterus may therefore affect fetal survival. However, details regarding the precise role of MUC1 in fetal survival are unclear, and further studies are required.
In the present study, IgA expression during gestation was increased in gp91phox−/- mice compared with that in wild-type mice (). ROS may disrupt IgA- producing cells in tissues [Nagayama et al. Citation2014]. However, although the secretion of IgA was decreased in gp91phox−/- mice, the low-oxidative-stress conditions facilitated the generation of IgA-producing cells. A fetus is considered foreign material, and IgA attachment to a fetus (or fertilized ovum) may therefore reduce the number of viable fetuses. Furthermore, MUC1 expression activates macrophages, which induce production of cyclooxygenase-2 and prostaglandin E2 (PGE2) [Inaba et al. Citation2003]. PGE2 inhibits immunity and apoptosis and weakens the mother’s immunological aggression against fetuses. In gp91phox−/- mice, low expression of MUC1 failed to prevent such immunological aggression, which likely reduced the number of viable fetuses. However, the relationship between fetuses and the immune system in these mice is unclear, and further studies are required to fully elucidate the immune mechanisms associated with fetal survival in these mice.
In this study, gp91phox-derived ROS increased the production of caspase-1 by NLRP3 inflammasomes, which subsequently induced the generation of IL-18. This IL-18 expression is believed to have induced MUC1 expression in the uterus (), facilitating fetal survival. These results showed that the presence of moderate amounts of gp91phox-derived ROS were necessary for gestation. However, the relationships between gestation and ROS have not been fully elucidated, and further studies are needed to confirm our findings, particularly through administration of food containing ROS-inducing agents to gp91phox−/- mice.
Figure 8. The mechanism of inflammasome activation and MUC1 expression in graviditas mice.
Materials and methods
Animals
Female C57BL/6j mice (SLC, Hamamatsu, Japan) and C57BL/6j gp91phox−/- mice (Jackson Laboratories, Bar Harbor, ME, USA) were used. The rearing conditions of mice were previously described [Hiramoto et al. Citation2016a]. We allocated six pregnant mice per group and recorded the number of births for each animal. This study was conducted in accordance with the Official Guide for the Care and Use of Laboratory Animals of Suzuka University of Medical Science (approval number: 34). All of the surgeries were performed under pentobarbital anesthesia, and all efforts were made to minimize suffering.
Chemical treatment
Treatment with an interleukin-1β (IL-1β) receptor antagonist (IL-1RA)
An IL-1β receptor antagonist (IL-1RA, 10 mg/kg; ATGen Ltd., Gyeonggi-do, South Korea) in saline was injected intraperitoneally (i.p.) into the mice once a day throughout the graviditas period [Abbate et al. Citation2008]. Wild-type mice were treated with vehicle only (saline).
IL-18 binding protein (IL-18BP) treatment
We used IL-18BP isoform D (Novoprotein Scientific Inc., Summit, NJ, USA) to inhibit IL-18. IL-18BP (5 μg) in saline was injected i.p. into the mice once a day throughout the graviditas period [Yatsiv et al. Citation2002]. Wild-type mice were treated with vehicle only (saline).
Caspase-1 inhibitor treatment
Mice were treated with the caspase-1 inhibitor Ac-YVAD-CMK (10 mg/kg subcutaneously (s.c.); Calbiochem, La Jolla, CA, USA), once a day throughout the graviditas period [Murphey Citation2011]. Wild-type mice were treated with vehicle only (1:1 v/v saline/polyethylene glycol 300).
MUC1 inhibitor treatment
We used GO-201 trifluoroacetate salt (Sigma-Aldrich, St. Louis, MO, USA) as an inhibitor of MUC1. The mice were treated with GO-201 (30 mg/kg body weight i.p.), once a day throughout the graviditas period [Banerjee et al. Citation2012]. Wild-type mice were treated with vehicle only (saline).
Quantification of plasma IL-1β and IL-18 levels via enzyme-linked immunosorbent assays (ELISA)
Blood samples were obtained from the mice on day 6 of gestation. The plasma levels of IL-1β and IL-18 were determined using commercial ELISA kits (IL-1β; R&D Systems, Minneapolis, MN, USA; IL-18; Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) in accordance with the manufacturer’s instructions.
Measurement of plasma ROS concentration
Plasma ROS levels were determined using an OxiSelectTM In Vitro ROS/RNS Assay Kit (STA-347; Cell Biolabs, Inc., San Diego, CA, USA) in accordance with the manufacturer’s instructions.
Western blotting analysis
Uterine tissue samples were obtained from the mice on day 6 of gestation. The samples were homogenized in lysis buffer (Kurabo, Osaka, Japan) and centrifuged at 12,000 x g for 10 min. The details of the western blotting procedure were described previously [Hiramoto et al. Citation2016a]. Briefly, proteins were transferred to membranes, which were incubated with primary antibodies against NLRP3 (1:1000; Abnova, Taipei, Taiwan), caspase-1 (1:1000; Epitomics, Burlingame, CA, USA), gp91phox (1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), MUC1 (cell surface-associated; 1:1000; Bioss Inc., Woburn, MA, USA), or β-actin (1:5000; Sigma-Aldrich). The membranes were then treated with horseradish peroxidase-conjugated secondary antibodies (Life Technologies, Frederick, MD, USA). The images were acquired using a Multi-Gauge Software Program (Fujifilm, Greenwood, SC, USA).
Preparation and staining of the uterine tissue samples
Tissue samples were obtained from mice on day 6 of gestation. The specimens were fixed in phosphate-buffered paraformaldehyde (4%), embedded in frozen Tissu-Tek, OCT compound, and cut into 5 μm thick sections. After washing with phosphate-buffered saline, the thin sections were incubated with goat anti-mouse immunoglobulin A (IgA) polyclonal antibodies (1:100; ZYMED, San Francisco, CA, USA). The expression of IgA was evaluated immunohistochemically using a fluorescent microscope, as described previously [Yamamoto et al. Citation2004].
Statistical analysis
Data are reported as means ± standard deviations. For comparisons among groups, Student’s t-tests were applied. Differences with p values of less than 0.05 were considered statistically significant.
Declaration of interest
The authors declare that they have no conflicts of interest related to this article.
Additional information
Notes on contributors
Keiichi Hiramoto
Wrote the article and designed the study: KH; Analyzed and interpreted the data: KH, YY; Contributed the essential reagents and tools: EFS.
Yurika Yamate
Wrote the article and designed the study: KH; Analyzed and interpreted the data: KH, YY; Contributed the essential reagents and tools: EFS.
Eisuke F. Sato
Wrote the article and designed the study: KH; Analyzed and interpreted the data: KH, YY; Contributed the essential reagents and tools: EFS.
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