Abstract
Gastric parietal cells synthesize and secrete estradiol-17β (E2) into gastric veins joining the portal vein, and a large amount of gastric E2 first binds to its receptors in the liver. However, the role of the gastric E2 is not entirely clear during postnatal development. The objective of this study was to reveal the onset of aromatase and other steroid-synthesizing enzymes in the gastric mucosa; to determine the period of rising E2 levels in the portal vein; and to further understand the relationship between gastric E2 and liver estrogen receptor α (ERα). The immunoblot bands and the immunohistochemistry of gastric mucosa revealed that aromatase protein began to express itself at 20 days and then increased in the levels of aromatase protein from 20 days onward. Expression of mRNAs for gastric aromatase (Cyp19a1) and other steroid-synthesizing enzymes, 17α-Hydroxylase (Cyp17a1) and 17β-hydroxysteroid dehydrogenase (HSD17b3), also increased similar to the increment of aromatase protein. Portal venous E2 levels were elevated after 20 days and increased remarkably between 23 and 30 days, similar to gastric aromatase mRNA levels. The E2 level was approximately three times higher at 40 days than that at 20 days. The liver weight and Esr1 levels began to increase after 20 days and the increment was positively correlated with the change of portal venous E2 levels. These findings suggest that some changes may occur around 20 days to regulate the gastric E2 synthesis and secretion.
Introduction
Estrogen is an important factor to regulate the postnatal sexual maturation in the rat. For half a century, it has been reported that the steroid hormone evokes functions of uterine tissue, the brain, and the pituitary during postnatal development (Ramirez & Sawyer 1965, Smith & Davidson 1968, Baker & Kragt 1969, Döhler & Wuttke 1975, Andrews et al. 1981, Döcke et al. 1984). The enhanced expression of estrogenic effects during postnatal maturation processes could be due to a change in estrogen target tissues in response to the amount of plasma estrogen. However, it has been shown that the estrogen target tissues are maturated by an increased plasma estrogen that is secreted from uncertain tissues.
In the metabolic pathway of steroidogenesis, progesterone is converted into estradiol-17β (E2) via androgens, enzymes, 17α-hydroxylase, 17β-hydroxysteroid dehydrogenase (17β-HSD), and aromatase. Though the aromatase activity in testicular Sertoli cells and granulose cells of the ovary is higher than other tissues in adult rats (Dorrington & Armstrong 1975, Erickson & Hsueh 1978, Fukuda et al. 1979), the specific activity of aromatization in testis and ovary is very low in neonatal and immature rats. The activity in the hypothalamus is several times higher than that in any other tissue including the testis and ovaries of fetal rats (George & Ojeda 1982). The fact that neural E2 can stimulate the growth and balancing of hypothalamic neurons suggests that locally formed estrogen may be involved not only in sexual differentiation but also in more general aspects of brain development (Toran-Allerand 1976, Arai & Matsumoto 1978, George & Ojeda 1982), but may not be enough to secrete into the systemic circulation to maturate estrogen target tissues. It has been reported that the gastric parietal cells aromatize androgens to estrogens by the aromatase and that the specific aromatase activity in gastric mucosa is corresponding to that in the ovary in adult rats (Ueyama et al. 2002). Gastric parietal cells secrete a large amount of estrogens into the portal vein and then about 90% of the gastric estrogen is bound to estrogen receptors (ERs) in the liver. Therefore, the amount of gastric estrogen flow into the systemic circulation is dependent on the liver function (Kobayashi et al. 2013). It is widely accepted that estrogens are rendered biologically inactive when they are bound to plasma estrogen binding protein or α-fetoprotein in fetal and neonatal animals (Michel et al. 1974, Radanyi et al. 1977). The amount of main plasma estrogen binding protein, a α-fetoprotein being produced from yolk sac and liver, is highest at the end of fetal periods and exponentially declines in concentration during a few days of postnatal life (Masseyeff et al. 1975, Germain et al. 1978). In this study, it was hypothesized that the stomach might be a main organ to produce E2 after diminishing plasma estrogen binding proteins and that the gastric E2 might have an important role for liver function during postnatal development.
The research described in this article was designed to define the role of gastric estrogen regulating serum estrogen levels during postnatal development of male rats. The results of this study suggest that gastric E2 and liver function are important factors for the serum estrogen levels in the systemic circulation during the postnatal development of rats.
Materials and methods
Animals
Wistar male rats (Japan SLC, Inc., Shizuoka, Japan) from 15 to 40 days of age were used. All animals were housed in air-conditioned quarters with a 12 h light:12 h darkness cycle (light 0700 to 1900 h) and given a standard pellet diet and tap water ad libitum. All animals were killed at 0800 h at each day under sodium pentobarbital anesthesia (40 mg/kg, Abbott Co Ltd). Blood was taken from the portal vein and abdominal artery. The liver and gastric mucosae were then removed; each animal group consisted of four rats. All procedures were performed in accordance with the institutional guidelines and approved by the animal research ethics committee at Yamagata University.
Western blotting
Gastric mucosa were homogenized in a lysis buffer containing a protease inhibitor (1 mM phenylmethylsulphonyl fluoride, Sigma), and the protein concentration was estimated using the Bromopyrogallol Red-molybdenum (VI) complex protein determination kit (Wako Pure Chem, Osaka, Japan). For immunoblotting, 20 μg total protein were separated on 10% SDS–PAGE. Protein samples were transferred onto PVDF membranes, blocked with 4% non-fat dry milk in TBST, and incubated with the primary antibody against aromatase (1:100 000, AbD Serotec, Oxford, UK) and β-actin (1:10 000, Santa Cruz Biotechnology, Inc.). Followed by reaction with anti-mouse IgG coupled to peroxidase (Nichirei, Tokyo, Japan), blots were revealed with ImmunoStar LD (Wako Pure Chem). Average exposure time was 1 min.
Immunohistochemistry
For immunohistochemical staining, the stomach was fixed overnight at 4 °C in Bouin's solution without acetic acid. Tissues were then dehydrated in a graded ethanol series and embedded in Paraplast embedding media (Sigma). Serial sections at a thickness of 5 μm were prepared. Sections at every 50 μm interval were mounted onto glass slides. The sections were immunostained by the peroxidase-labeled antibody method using antibodies against the aromatase (1:10 000, AbD Serotec), and stomach sections from adult male rats were used as positive specimens. The immunostained areas were then expressed in terms of mm2/mm2.
Estimation of mRNA levels by real-time RT-PCR
Total RNAs were prepared from the gastric mucosa and the liver by an RNA Isolation Kit (RNAspin mini, GE Healthcare UK Ltd, Buckinghamshire, UK), which included a genomic DNA digestive step by DNase I treatment. Expression of steroid-synthesizing enzyme mRNAs in the stomach and of Esr1 in the liver was determined by real-time RT-PCR. Total RNA (0.5 μg) was converted into cDNA by reverse transcription using a High-Capacity RNA-to-cDNA Master Mix (Applied Biosystems). All primers at a length of 20 bps were designed in-house following sequences based on the nucleotide sequences of the rat and were intron-spanning. Primer sequences used were as follows: cytochrome P450 aromatase (EC: 1.14.14.1, Cyp19a1, GenBank accession no.
Real-time PCR was performed in the Applied Biosystems 7300 Fast Real-Time PCR System with a Power SYBR Green PCR Master Mix according to the manufacturer's protocol. PCR conditions were 10 min at 95 °C, with 40 cycles of denaturation for 15 s at 95 °C and an annealing–extension for 60 s at 60 °C. According to the computational method of Livak & Schmitten (2001), a relative amount of each gene in each animal including β-actin was calculated based on a threshold of cycles and compared with that found in the controls. To confirm the absence of contamination of genomic DNA in total RNAs, PCR was carried out without the RT step and none of the total RNAs could detect PCR products of either primer set within 40 PCR cycles.
Measurement of serum E2 concentrations
Serum E2 concentrations were measured using an E2 EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA). The assay is based on the competition between free E2 and an estradiol tracer, which is estradiol linked to an acetylcholinesterase molecule at a limited number of estradiol-specific rabbit antiserum binding sites. Briefly, plasma samples and antiserum to E2 were incubated overnight at room temperature in wells of an anti-rabbit IgG-coated plate. Subsequently, the E2 linked to acetylcholinesterase was added and kept for 1 h at room temperature. After washing the plate, Ellman's reagent, which contains the substrate to acetylcholinesterase, was added to develop the color for 1.5 h in the dark. The amount of reaction product was measured at 405 nm with the detection range of the system being 0.3–3000 pg/ml E2. To eliminate nonspecific binding, plasma samples were pre-absorbed by dextran-coated charcoal and measured at the same time.
Statistical analysis
The data were statistically analyzed by one-way ANOVA followed by the Bonferroni/Dunn test and linear regression, as appropriate, using StatView software (Hulinks, Inc., Tokyo, Japan). The P value was set at either <0.05 or <0.01.
Results
Western blots of aromatase in gastric mucosa
To detect aromatase, western blot analysis was performed, and the gastric mucosa showed a single band with a molecular mass of 55 kDa, which matched the molecular mass of aromatase (Fig. 1). The immunoblot bands revealed that aromatase expression started at 20 days, and the levels of gastric aromatase protein increased with the advancing age of animals. The expression of aromatase protein reached a plateau at 30 days and maintained high levels onward.
Western blot analysis of gastric aromatase protein during the postnatal developmental rat. Lysates of gastric mucosa at 15-, 20-, 23-, 25-, 27-, 30-, 35-, and 40-day rats were loaded on SDS–PAGE, and aromatase (upper lane) and β-actin (lower lane) were detected by immunoblotting. The experiments were conducted by loading equal amounts of gastric mucosal proteins in each lane. Aromatase protein began to increase after 20 days stepwise.
Citation: Journal of Endocrinology 218, 1; 10.1530/JOE-13-0074
Change of the population of stomach cells immunostained with antibodies to aromatase
Light micrographs of immunostained sections at various ages are shown in Fig. 2A, B, C, D, E, F, G, H, and I. None of stomach cells in gastric mucosa were immunostained with aromatase antibody at 15 days (Fig. 2A), and a few cells appeared to be slightly stained at 20 days (Fig. 2B and C). The number of aromatase cells and their staining intensities were increased with advancing ages after 20 days (Fig. 2D, E, F, G, H, and I). Furthermore, it was observed that the height of mucosal epithelium was thickened twice from 15 to 40 days. Figure 2J shows the change in the population of aromatase cells in gastric mucosa, and its value is expressed in terms of mm2/mm2. The number of aromatase cells started increasing around 20 days, and it greatly increased until day 40 in accordance with the increment in epithelial thickness and stomach size.
Light photomicrographs of developmental gastric mucosa. Stomach sections at 15 (A), 20 (B, C is the denoted area of B), 23 (D), 25 (E), 27 (F), 30 (G), 35 (H), and 40 (I) days were immunostained with antibodies to aromatase. A few numbers of cells were slightly stained at 20 days (B and C, arrowheads). The number of immunostained cells and their immunostainabilities were increased after 23 days, and the immunostained area reached a plateau from 30 days onward (J). Scale bars in A, B, D, E, F, G, H, and I indicate 200 μm, and the scale bar in C indicates 50 μm. Values show the immunostained area (mm2/mm2) and the standard error of over 30 sections in each group.
Citation: Journal of Endocrinology 218, 1; 10.1530/JOE-13-0074
Expression of steroid metabolic enzyme mRNAs in the gastric mucosa
Gene expression of mucosal enzyme mRNAs, which encode three metabolic enzymes from progesterone to E2, was determined by a real-time PCR method from 15 to 40 days (Fig. 3). Comparing to the expression of Cyp17a1 at 15 days, it increased significantly at 20 days and then gradually increased toward the peripubertal period at 40 days (Fig. 3A). The expressions of HSD17b3 and Cyp19a1 were low during the first 3 weeks of life but then started to increase significantly at 23 days keeping their elevation until 40 days (Fig. 3B and C respectively). The expressions of Cyp17a1, HSD17b3, and Cyp19a1 at 40 days were 25, 10, and 20 times higher than those at 15 days respectively (P<0.01). The age-dependent increase in Cyp19a1 expression was consistent with the results of western blot and immunohistochemistry, and the expression of Cyp19a1 was positively correlated with that of Cyp17a1 mRNA (P<0.0001, R2=0.723) and HSD17b3 (P<0.0001, R2=0.824). Therefore, E2 synthesis from progesterone increased age dependently after 23 days.
Gene expression level of aromatase (Cyp19a1), 17α-hydroxylase (CYP17a1), and 17β-HSD(3) (HSD17b3) in the gastric mucosa. Expression of estradiol-17β metabolic enzyme mRNAs was quantitatively evaluated by real-time PCR from 15 to 40 days rats. It was observed that the expression of Cyp17a1 (A), HSD17b3 (B), and Cyp19a1 (C) levels started to increase after 20–23 days, and positive correlations of Cyp19a1 levels with Cyp17a1 and HSD17b3 were clarified. Expression of Cyp19a1 normalized by β-actin. Each point shows the mean, and the bars represent the standard error of four animals. *P<0.05, **P<0.01 vs 15 days.
Citation: Journal of Endocrinology 218, 1; 10.1530/JOE-13-0074
Change of serum E2 levels in the portal vein and abdominal artery
The concentration of serum E2 in the portal vein and abdominal artery was measured chronologically, and the results are shown in Fig. 4. The concentrations of serum E2 in the portal vein and artery were 15–35 pg/ml between 15 and 23 days and then those in the portal vein started to elevate gradually from 25 days until 40 days. At 40 days, the value of serum E2 in the portal vein was 2.3 times higher than that at 23 days (P<0.05). On the other hand, the concentration of arterial E2 decreased form 23 to 27 days and kept constant levels at 10–27 pg/ml between 27 and 40 days. The regression analysis showed that the change of E2 levels in the portal vein was positively correlated with that of the Cyp19a1 expression (P<0.0001, R2=0.599); however, the change of arterial E2 levels was not correlative with that of the Cyp19a1 expression.
Change of estradiol-17β (E2) levels in portal vein and artery. The serum E2 levels were determined by enzyme immunoassay as shown in the Materials and method section. Portal venous E2 levels (closed circle) started to increase after 23 days and then continued to increase gradually thereafter. Arterial E2 (open circle) maintained the same level as portal venous E2 by 23 days and decreased to maintain the constant levels from 27 to 40 days. Changes in E2 levels in the portal vein showed positive correlations with gastric Cyp19a1 levels significantly. Each point shows the mean, and the bars represent the standard error of four animals. *P<0.05, **P<0.01 vs 15 days.
Citation: Journal of Endocrinology 218, 1; 10.1530/JOE-13-0074
Liver growth and expression of ERα mRNA in the liver
Liver weight increased with age from 15 to 40 days (Fig. 5A), and the increment of liver weight was positively correlated with that of serum E2 in the portal vein (P<0.0001, R2=0.426). Gene expression of liver Esr1 was measured by a real-time PCR method, and the result is shown in Fig. 5B. The expression of Esr1 increased gradually with age until 27 days and drastically increased until 40 days. The expression of Esr1 at 40 days was 46 times higher than that at 15 days. The age-dependent change of liver Esr1 was positively correlated with that of serum E2 in portal vein (P=0.0003, R2=0.373).
Change of the liver weight and the expression of liver Esr1. Liver weight was increased with advancing age of rats (A). The expression of Esr1 was measured by real-time PCR from 15 to 40 days, and Esr1 levels began to elevate at 20 days and increased after 30 days considerably (B). Changes of liver weight and Esr1 level showed positive correlation with portal venous estradiol-17β levels. The expression of Esr1 was normalized by β-actin expression. Each point shows the mean, and the bars represent the standard error of four animals. **P<0.01 vs 15 days.
Citation: Journal of Endocrinology 218, 1; 10.1530/JOE-13-0074
Discussion
Most estrogens are believed to be synthesized depending on the expression of aromatase in the ovary, uterus, and placenta in premenopausal females, and there are many reports on the extraovarian tissues and synthesized and secreted estrogens in adults (Nelson & Bulun 2001). In immature rats, the ovary in females (Meijs-Roelofs et al. 1973) and the adrenal gland in females and males (Baird et al. 1969, Weisz & Gunsalus 1973) have been postulated for a long time as the source of E2; however, it is difficult to explain the developmental change of serum E2 levels. Recently, it was reported that the stomach is one of the extraovarian tissues synthesized and secreted by E2, and the E2 levels in the portal vein are over ten times higher than those in the artery in normal conditions of adult male rats (Ueyama et al. 2002, 2004, Kobayashi et al. 2013). It is considered that much, if not all, of E2 in the systemic circulation is derived from the stomach in immature rats. In this study, it becomes apparent that E2 synthesis and secretion in the stomach begins to elevate after 20 days from aromatase protein investigated by western blotting and immunohistochemistry (Figs 1 and 2), aromatase mRNA levels (Fig. 3), and E2 levels in the portal vein (Fig. 4). These results are the first findings that focused on the E2 synthesized and secreted from parietal cells in gastric mucosa during the postnatal development, and it is considered that these findings are associated with previous reports on the serum E2 level (Germain et al. 1978, Greenstein 1992). It is well known that the stomach functions not only as a gastrointestinal system but also as an endocrine system, and gastrin (Thompson 1969), somatostatin (Lucey 1986), and ghrelin (Kojima et al. 1999) can be enumerated as the major gastric peptide hormones. These peptide hormones are observed in the stomach in the fetal period by immunohistochemistry (Braaten et al. 1976, Hayashida et al. 2002), and the speculated onset mechanism of gastric E2 synthesis is different from that of other gastric endocrine systems. Parietal cells, which also synthesize and secrete gastric acid, are changed ultrastructurally and histochemically after weaning around 20 days (Ekelund et al. 1985). Before 20 days, the synthesis and secretion of E2 is very low, and after 20 days, it increases by some regulatory mechanism of E2 synthesis and secretion in the stomach.
As the gastric E2 is synthesized from progesterone by metabolic enzymes, 17α-hydroxylase, 17β-HSD(3), and aromatase (Ueyama et al. 2004), the change in serum E2 in the portal vein is significantly correlated with the expression of the enzyme mRNAs (Fig. 4). A large amount of gastric E2 binding to liver ERs is normal; however, the level of arterial E2 is dependent on the pathophysiological conditions of the liver (Kobayashi et al. 2013). The present results show that gastric E2 was correlated with the expression of liver Esr1 and the liver weight (Fig. 5) and that the serum E2 levels of systemic circulation, which passes through the liver, was reduced to keep the constant level of arterial E2 levels. However, the E2 level in the portal vein at 15 days tends to be slightly higher than that at 20 days when some of the epithelial cells in the stomach start to synthesize E2. Döhler & Wuttke (1975) reported that serum E2 levels were high for the first 2 days in newborn male and female rats and were decreased to one fourth and then increased again to two thirds of those in newborns between 9 and 19 days. Furthermore, they described that the question arises why serum E2 levels are high in newborn rats and increase again after a transient drop in both sexes during the second and third weeks of life. It is considered that the serum E2 in newborn rats may be maternal and that the high levels of serum E2 decrease gradually by onset of E2 synthesis in the stomach. As a result of the combined effects of elevated Esr1 expression and of liver weight, several hundred-fold increases in the total ERα per liver were observed during this period (Fig. 5). Thus, the serum E2 levels in the artery decreased with the increase in liver ERα contents in marked contrast to those in the portal vein.
It is well known that serum E2 in perinatal rats is maternal and binds to plasma estrogen binding proteins (Michel et al. 1974). One of them, α-fetoprotein, is well known as the major fetal serum protein and has a very high affinity for estrogens (Lai et al. 1976, Mizejewski 2004). Although α-fetoprotein is synthesized in fetal liver and yolk sacs abundantly, it reduces exponentially postnatally and regresses by 20 days (Lai et al. 1976, Nayak & Mital 1977, Greenstein 1992). On the other hand, the aromatase protein and mRNAs levels reached a plateau per unit at 30 days, it is indicated that E2 levels in 40 days tend to keep increasing. In Fig. 2A, B, C, D, E, F, G, H, and I, light micrographs of gastric mucosa show a thickening height of the gastric mucosal epithelium. It is feasible to surmise that the elevation is induced by the growth of gastric mucosa and/or the stomach gradually increasing blood flow.
This is the first study to report the changes of gastric E2 levels in the portal vein during the postnatal development; however, the mechanism of regulation of gastric E2 is unknown and needs further studies to clarify the overview of gastric E2.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Life Science Laboratory Research Grant (grant numbers K10G05, K11G05); the Yamagata Health Support (grant number H21012); and the Yuki Plan of Yamagata University (grant number DAY0010).
Acknowledgements
The authors acknowledged Mr Nathan Strenge (Yamagata High School, Tokai University) for his suggestions and critically reading the manuscript.
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