Somatostatin receptor 2 expression in the human endometrium through the menstrual cycle

OBJECTIVE: Somatostatin mediates its many inhibitory functions through five G-protein-coupled receptors (sstr1-5); however, it is not known whether somatostatin or its receptors are present in the endometrium. DESIGN: We have used immunohistochemistry on formalin-fixed paraffin-embedded sections of normal human endometrium from the menstrual (n = 6), proliferative (n = 15) and secretory (n = 10) stages of the endometrial cycle to determine the pattern of expression of somatostatin receptor (sstr) subtype 2. In addition, we have used quantitative polymerase chain reaction (PCR) to determine the level of expression of the sstr2 mRNA in 17 samples of normal human endometrium. PATIENTS: Endometrial tissue had been removed from patients undergoing dilation and curettage (D&C) for menorrhagia and had been determined to be normal histologically. MEASUREMENTS: Immunostaining in the epithelium, endothelium and the stroma of the endometrial sections was characterized and was scored positive or negative. The PCR results were analysed using the software provided to standardize the expression of sstr2 against that of constitutively expressed beta-glucoronidase in the same sample. A final percentage value of the level of sstr2 expression was then determined. RESULTS: sstr2 was expressed variably throughout all the stages of the menstrual cycle in the epithelium, the endothelium and the stroma. In particular, the position of sstr2 expression varied in the epithelial cells surrounding the endometrial glands from being basal or diffuse in the proliferative and secretory phase to being lumenal in the menstrual stage. Quantitative PCR showed that 15 of 17 samples expressed sstr2 mRNA and the level of expression between individual samples varied dramatically. CONCLUSIONS: These data show that sstr2 is present in the endometrium and its location seems to vary through the menstrual cycle.     

Measurement of phospholipase A2 activity in human endometrium during the menstrual cycle

Phospholipase A2 activity was measured in human endometrium throughout the menstrual cycle using an assay based on the liberation of oleic acid from 1-palmitoyl-2-[14C]oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8-9 and an apparent Michaelis constant of 110 mumol/l. Enzyme activity was low in early proliferative-phase tissue (6.08 +/- 1.42 (S.E.M.) pmol oleic acid released/mg protein per min) but rose significantly (P less than 0.01) during the late proliferative phase (10.86 +/- 2.79 pmol/mg per min). There was a tenfold increase in activity 2-4 days after ovulation (45.6 +/- 13.6 pmol/mg per min) which thereafter declined to reach values which at menstruation were not significantly different from those of the proliferative phase (4.5 +/- 1.76 pmol/mg per min). The results indicate that phospholipase A2 activity in human endometrium is related to the stage of the menstrual cycle and suggest that arachidonic acid release may be influenced by oestradiol and progesterone.     

Colony-stimulating factor-1 and c-fms expression in human endometrial tissues and placenta during the menstrual cycle and early pregnancy.

Colony-stimulating factor-1 (CSF-1), a growth factor produced by monocytes, macrophages, fibroblasts, and endothelial cells, has been implicated in the functional regulation and growth of the murine placenta through the presence of the CSF-1 receptor, c-fms, found in this tissue. In this study we examined the tissue levels of CSF-1 by RIA and the relative expression of CSF-1 and c-fms mRNA by Northern blot analysis in human endometrial, decidual, and placental tissues during the normal menstrual cycle and early pregnancy. All endometrial, decidual, and placental tissues demonstrated extractable immunoreactive CSF-1 and expressed the 4.0-kilobase CSF-1 mRNA species. First trimester decidual tissue expressed higher levels of CSF-1 mRNA than proliferative (3.2-fold higher; P less than 0.01) or secretory (2.4-fold higher; P less than 0.01) endometrial tissues, whereas proliferative and secretory endometrial tissues expressed similar levels of CSF-1 mRNA. Tissue extractable levels of immunoreactive CSF-1 were 3.2-fold (P less than 0.05) higher in first trimester decidual tissue and 2.9-fold (P less than 0.05) higher in secretory endometrial tissue compared to levels in proliferative endometrial tissue, whereas first trimester decidua and secretory endometrial tissues had similar levels of immunoreactive CSF-1. There was expression of c-fms mRNA in all endometrial and first trimester decidual tissue samples, with little change during the menstrual cycle and early pregnancy. In placenta, there was a positive correlation of increasing CSF-1 and c-fms mRNA expression with increasing gestational age. These results suggest that there is increased local production of CSF-1 in tissues found at the maternal-fetal interface during the time of implantation and early pregnancy. This increased production of CSF-1 may play a role in decidual function and placental growth through the presence of c-fms in these tissues.     

Follicular development during the luteal phase of the human menstrual cycle

The aims of the present studies were to determine the number, size range, health, and steroidogenic activities of antral follicles in normal human ovaries during the luteal phase of the menstrual cycle. Steroidogenic activity was assessed from the levels of androstenedione, testosterone, and estradiol in follicular fluid and the levels of extant and FSH-stimulable aromatase activity and FSH-stimulable progestin synthesis in the granulosa cells. Data for luteal phase ovaries were compared to those obtained for ovaries from the late follicular phase. On average, 94% (range, 70-100%) of the luteal phase follicles (greater than or equal to 1 mm diameter) were atretic as assessed by oocyte viability and granulosa cell number. The largest healthy follicles during the mid- to late luteal phase were 4-4.5 mm in diameter; these contained high levels of aromatizable androgen (500-2000 ng/ml), low levels of estradiol (less than 10 ng/ml), and granulosa cells with an extant level of aromatase activity 200 times lower than that in a preovulatory follicle. Based on these biochemical criteria, healthy (luteal phase) follicles were not distinguishable from atretic follicles. Granulosa cells from the luteal phase follicles were responsive to FSH with respect to progesterone and estradiol biosynthetic activity; the aromatase system in the cells from the mid- to late luteal phase follicles was significantly more responsive to FSH than that in cells from late follicular or early luteal phase follicles (P less than 0.05). These data suggest that the number of healthy luteal phase follicles (greater than or equal to 1 mm diameter) available for subsequent preovulatory development is limited.     

The role of the interferon regulatory factors,IRF-1 and IRF-2, in LPS-induced cyclooxygenase-2 (COX-2) expression in vivo and in vitro

Cyclooxygenase (COX) exists as two isoforms: COX-1, which is constitutively expressed in most cell types; and COX-2, which is inducible by lipopolysaccharide (LPS) and cytokines in a variety of cell types. Although previous studies have implicated two DNA binding proteins, interferon regulatory factor (IRF)-1 and IRF-2, in the regulation of LPS- and IFN-gamma-induced COX-2, their effects in vivo and in vitro are not well-defined. Using real-time PCR, COX-2 gene expression in the livers and lungs of mice challenged in vivo and in macrophages stimulated with LPS in vitro was investigated in wild-type and in IRF-1 and IRF-2 knockout mice. In response to 35 mg/kg LPS, IRF-1-, but not IRF-2-deficient mice, exhibited much poorer induction of COX-2 gene expression in both the livers and lungs. In vitro, COX-2 mRNA levels were also poorly induced in IRF-1-deficient macrophages, while IRF-2- deficient macrophages exhibited higher levels than in normal macrophages. IRF-1 and IRF-2 were confirmed to activate and repress expression of the COX-2 promoter, respectively, in a transient transfection system and the role of specific DNA binding sites confirmed by site-specific mutagenesis. Collectively, these data provide evidence for an important role for IRF-1 in vivo and in vitro and for IRF-2 in vitro in the regulation of COX-2 expression by LPS.     

Regulation of fatty acid synthetase ribonucleic acid in the human endometrium during the menstrual cycle

Fatty acid synthetase (FAS) is induced by progesterone in MCF7 and T47D breast cancer cell lines. We studied a possible in vivo regulation of expression of this gene by looking for FAS RNA in human endometrial biopsies at various periods of the menstrual cycle, using a cloned cDNA FAS probe. By Northern blot analysis, we detected the 8-kilobase FAS RNA throughout the cycle in 7 uterine samples. RNA in situ hybridization analysis of frozen sections from 22 endometrial biopsies showed that FAS RNA was present during follicular and luteal phases of the menstrual cycle in stromal and epithelial cells. RNA levels were quantified by counting autoradiographic silver grains using a computer-aided image analyzer. FAS RNA levels were significantly higher in epithelial cells than in fibroblasts (P less than 2 x 10(-5]. Furthermore, in both cell types, mean FAS RNA concentrations were higher in biopsies removed during the luteal phase than the follicular phase of the menstrual cycle (P = 2 x 10(-3) and 9 x 10(-5), respectively). A 2- to 3-fold increase in FAS RNA levels between days 8-14 and days 22-24 was detected in 2 normal patients who had previously undergone 2 successive biopsies. This increase was not observed in 2 patients with low plasma estradiol and progesterone concentrations, indicating a probable dysovulation. We conclude that FAS normally increases in both stromal and epithelial endometrial cells during the luteal phase. This increase is probably due to progesterone, which implies that FAS is induced in normal endometrium, as demonstrated in breast cancer.     

Expression, localization, and signaling of PGE(2) and EP2/EP4 receptors in human nonpregnant endometrium across the menstrual cycle

This study was designed to elucidate the sites of synthesis and action of PGE(2) in the nonpregnant human uterus across the menstrual cycle. The sites of expression of PGE synthase and synthesis of PGE(2) were investigated by immunohistochemistry using full thickness uterine biopsies. Expression of PGE synthase and synthesis of PGE(2) were localized to glandular epithelial and endothelial cells in both basalis and functionalis regions of the human endometrium. By contrast, stromal staining was predominantly localized in the functionalis layer. Some cyclical variation in expression of PGE synthase and PGE(2) synthesis was observed, with reduced expression/synthesis detected in the stromal compartment of the functionalis during the late secretory phase of the menstrual cycle. Subsequently, we assessed the site of action of PGE(2) by investigating the expression of two PGE(2) receptor isoforms, namely EP2 and EP4. Cyclical variation in endometrial EP2 and EP4 receptor mRNA expression was quantified by TaqMan quantitative RT-PCR using RNA isolated from endometrial tissue collected across the menstrual cycle. No differences in EP2 receptor mRNA expression were detected; however, EP4 receptor mRNA expression was significantly higher in late proliferative stage (P < 0.05) than in early, mid, and late secretory stage endometrium. Expression patterns of EP2 and EP4 receptors were localized by nonradioactive in situ hybridization using fluorescein isothiocyanate end- labeled oligonucleotide probes. Expression of both receptors was observed in endometrial glandular epithelial and vascular cells, with no notable spatial or temporal variation. Finally, signaling of EP2/EP4 receptors was assessed by investigating cAMP generation in vitro after stimulation with PGE(2). Endometrial cAMP generation in response to PGE(2) was significantly greater in proliferative tissue compared with early and midsecretory stage tissue (3.77 +/- 0.85 vs. 1.96 +/- 0.28 and 1.38 +/- 0.23, respectively; P < 0.05). In conclusion, this study demonstrates glandular and vascular coexpression of PGE synthase, PGE(2), EP2, and EP4 receptors and suggests an autocrine/paracrine role for PGE(2) in epithelial/endothelial cell function in the human endometrium.     

Expression and localization of endothelial monocyte-activating polypeptide II in the human endometrium across the menstrual cycle: regulation of expression by prostaglandin E(2)

A role for prostaglandins (PGs) in the regulation of endometrial functions, such as menstruation, has been established, although the mechanisms by which this is achieved are not fully elucidated. In the present study, cDNA array analysis has identified endothelial monocyte-activating polypeptide II (EMAP II) as a PGE(2)-regulated gene in endometrial epithelial cells. Incubation of endometrial epithelial cells with 100 nM PGE(2) for 4 and 24 h resulted in a 2.3- and 16-fold decrease in EMAP II expression, respectively. In endometrial tissue collected across the menstrual cycle, a significant increase in EMAP II mRNA was observed during the late secretory phase, compared with the proliferative and early-midsecretory phases. The temporal pattern of EMAP II expression was confirmed further by Western blotting; EMAP II protein expression was detected as a 43-kDa band. In situ hybridization and immunohistochemistry localized EMAP II mRNA and protein expression in glandular epithelial, endothelial, and stromal cells in the functionalis and basalis layers of the endometrium. Finally, the role of PGE(2) in the regulation of EMAP II expression in the endometrium was assessed. Incubation of fresh endometrial tissue (n = 5) with 3 micro g/ml indomethacin resulted in an increase in EMAP II protein expression, compared with control untreated tissue. However, cotreatment of the cells with 100 nM PGE(2) resulted in a significant decrease in EMAP II protein expression, compared with tissue incubated with indomethacin alone (P < 0.05). These data confirm temporal variation in EMAP II expression in the human endometrium across the menstrual cycle and localize expression to glandular epithelial, endothelial, and stromal cells. Moreover, EMAP II expression is negatively regulated by PGE(2).