E2-induced degradation of uterine insulin receptor substrate-2: requirement for an IGF-I-stimulated, proteasome-dependent pathway.

The insulin receptor substrates are docking proteins that bind various receptor tyrosine kinases and signaling proteins. Previous studies have shown that E2 or progesterone can regulate the relative abundance of insulin receptor substrate-1 and -2 in cells and tissues. For instance, uterine insulin receptor substrate-2 was decreased markedly at 24 h after E2 treatment of mice. In the present study we used various in vivo experimental approaches to examine the mechanism by which E2 influences uterine insulin receptor substrate-2 expression. Uterine insulin receptor substrate-2 mRNA levels were diminished after E2 treatment, but this diminution did not account for the total reduction in insulin receptor substrate-2 protein, suggesting that the E2-induced decrease in insulin receptor substrate-2 is not regulated solely at the mRNA level. Cotreatment with progesterone prevented the E2-stimulated reduction in insulin receptor substrate-2 protein at 24 h after hormone exposure. In addition, MG-132 and epoxomicin, inhibitors of proteasomal protease activity, inhibited the E2-induced decrease in uterine insulin receptor substrate-2 protein levels, and this correlated to an increase in uterine protein ubiquitination. Insulin receptor substrate-2 protein was diminished in uteri of E2-treated insulin receptor substrate-1-null mutant mice, but not in E2-treated IGF-I-null mutant mice. Furthermore, E2-induced diminution of uterine insulin receptor substrate-2 protein was only partially inhibited in the presence of wortmannin, a PI3K inhibitor. Collectively, these data suggest that the E2-induced decrease in uterine insulin receptor substrate-2 requires IGF-I signaling, is not dependent solely on insulin receptor substrate-1 and PI3K, and is blocked by progesterone as well as by pharmacological inhibition of proteasomal protease activity. We speculate that the IGF-I-activated IGF-I receptor, in response to E2, directly or indirectly modifies insulin receptor substrate-2, probably through phosphorylation, leading to ubiquitination and subsequent degradation of this docking protein by the proteasome. This degradation could be a regulatory step to inhibit insulin receptor substrate-2-dependent signaling in the uterus.

Advances in uterine leiomyoma research: conference overview, summary, and future research recommendations.

Uterine leiomyomas (fibroids, myomas) are the most common tumors occurring in the genital tract of women over 30 years of age. These benign uterine smooth-muscle tumors are estimated to be clinically significant in at least 25% of the American female population during their reproductive years. Furthermore, when thorough pathologic examination of hysterectomy specimens has been performed in patients with or without clinical history of myomatous uteri, the incidence of fibroids is 77%, suggesting that these tumors are far more prevalent than estimated by clinical cases. In spite of their high prevalence, little is known concerning the etiology or the molecular basis of their development and growth. It is well known that leiomyoma growth is regulated by ovarian steroid hormones, yet the exact molecular pathway(s) involved in tumor growth and the role of genetic susceptibility/predisposition and the environment are unclear. This article is an overview of some of the topics addressed at the conference on Women's Health and the Environment: The Next Century--Advances in Uterine Leiomyoma Research. A summary of research needs and recommendations for future research directions based on conference discussions are also presented.

Evidence of juxtacrine signaling for transforming growth factor alpha in human endometrium.

To determine the mechanism of signaling for transforming growth factor alpha (TGFalpha) in human endometrium, uterine luminal fluid proteins were retrieved by lavage followed by collection of the adjacent endometrium at hysterectomy. In the endometrium we observed the presence of the full-length transmembrane TGFalpha protein and the phosphorylation of its only known receptor, the epidermal growth factor receptor (EGFR), by immunoprecipitation-Western blot; TGFalpha mRNA via reverse transcription-polymerase chain reaction; and immunolocalization of TGFalpha to the surface endometrium adjacent to the uterine lumen. Despite this demonstration of TGFalpha in functional endometrium, we could not detect measurable amounts of TGFalpha in any of the 16 endometrial washings by either immunoprecipitation-Western blot or by ELISA. Recovery rate for intraluminal fluid spiked with TGFalpha control peptide was 93.4-97%. The inability to detect TGFalpha in intraluminal fluid despite its high concentration in cells directly adjacent to the uterine lumen, along with the absence of any cleaved TGFalpha species identified in the endometrium, suggests that TGFalpha signals its receptor as a transmembrane ligand. Since the EGFR is present in the endometrium and on the surface of embryos, these data are consistent with a juxtacrine mode of signaling for TGFalpha between endometrial cells, and between the luminal surface epithelium and preimplantation embryos.

Effect of progestin on the ovarian epithelium of macaques: cancer prevention through apoptosis?

OBJECTIVE: The apoptosis pathway is a vital mechanism in vivo that functions to eradicate genetically damaged cells prone to malignancy. The purpose of this study was to determine whether oral contraceptives, which confer significant protection against subsequent epithelial ovarian cancer, induce apoptosis in the ovarian epithelium. METHODS: Female cynomolgus macaques (N = 75) were randomized to receive a diet for 35 months containing either no hormones, the oral contraceptive Triphasil (Wyeth-Ayerst Laboratories, Philadelphia, PA), the estrogenic component of Triphasil (ethinyl estradiol) alone, or the progestin component of Triphasil (levonorgestrel) alone, each administered in a cyclic fashion. At study termination, the animals underwent ovariectomy and the ovarian epithelium was examined morphologically and immunohistochemically for apoptosis. The percentage of ovarian epithelial cells undergoing apoptosis was measured in each animal and compared between the treatment groups. RESULTS: The median percentage of ovarian epithelial cells undergoing apoptosis by treatment was control (3.8%), ethinyl estradiol (1.8%), Triphasil (14.5%), and levonorgestrel (24.9%). Compared with control and ethinyl estradiol-treated monkeys, a statistically significant increase in the proportion of apoptotic cells was noted in the ovarian epithelium of monkeys treated with the oral contraceptive Triphasil (P < or = .01) or levonorgestrel (P < .001), with a maximal effect (six-fold) seen in the group treated with levonorgestrel alone. CONCLUSION: Oral contraceptive progestin induces apoptosis in the ovarian epithelium. Given the importance of the apoptosis pathway for cancer prevention, an effective chemopreventive strategy may be possible using progestins or other agents that selectively induce apoptosis in the ovarian epithelium to prevent the development of ovarian cancer.

Effects of dietary soybean estrogens on the reproductive tract in female rats.

The estrogenic actions of dietary phytoestrogens have raised concerns regarding the potential DES-like developmental effects on the female genital tract, but the growing evidence of cardioprotective benefits of dietary soybean estrogens provides the impetus to assess the effects of these compounds in adult female models of the menopause. We conducted an experiment in ovariectomized rats to determine the independent effects of dietary soybean estrogens (SBE) and the interactions of these agents with the commonly used pharmaceutical estrogen preparation (conjugated equine estrogens, CEE) in the vagina and uterus. We looked at the effects of SBE and CEE, alone and in combination, on uterine weight, body weight, vaginal cytology, uterine luminal epithelial height, and immunohistochemical staining for proliferating cell nuclear antigen (PCNA), lactoferrin (Ltf), and apoptosis. Ovariectomized rats were fed diets containing casein or soybean protein (SBE, low dose = 11.6 mg isoflavones/ 1800 cal; high dose = 117.8 mg/1800 cal), with no CEE, low dose CEE (0.313 mg/1800 cal), or high dose CEE (0.625 mg/1800 cal) added. In this study, SBE did not demonstrate estrogenic activity for uterine weight or vaginal cytology. We also found no estrogenic effects of these doses of SBE for PCNA, apoptosis, Ltf staining, or for LEH measurements. In addition, our results regarding the interactions of SBE and CEE do not show any evidence that the combination is additive in effect. On the contrary, the LEH response induced by low levels of CEE, was reduced by high levels of SBE. Furthermore, the Ltf response induced by CEE also was reduced by high levels of SBE. This suggests that high doses of SBE may antagonize the estrogen-agonist actions of low doses of CEE in the rat uterus. Our results in the ovariectomized rat model of menopause suggest that dietary soybean estrogens will not elicit a pattern of effects that simply recapitulates those of steroidal estrogens.

Toxicity of methoxyacetic acid in cultured human luteal cells.

Ethylene glycol monomethyl ether (EGME) and its proximate metabolite, 2-methoxyacetic acid (MAA), increase ovarian luteal cell progesterone production in the female rat in vivo and in cultured rat luteal cells in vitro, respectively. In order to better assess the potential hazard of EGME and MAA to women, these studies were conducted to determine whether the same concentrations of MAA increase progesterone in human luteinized granulosa cells as in rat luteal cells. Human cells were collected from healthy anonymous oocyte donors, washed, plated 25,000 viable cells per well, and treated with 10 IU hCG and 0-5 mM MAA for 6-48 hr. Progesterone in media was significantly elevated after 24 hr incubation at >/=1 mM MAA. MAA had no effect on ATP levels at 6 or 24 hr. Thus, MAA increased progesterone production in cultured human luteal cells at the same concentration as MAA increased progesterone in rat luteal cells. The implication is that EGME has the potential to alter ovarian luteal function in women. These data should be useful for determining the real health hazards and potential risks of EGME exposure.

Human endometrial transforming growth factor-alpha: a transmembrane, surface epithelial protein that transiently disappears during the midsecretory phase of the menstrual cycle.

OBJECTIVES: To characterize the forms of transforming growth factor-alpha (TGF-alpha) in normal human endometrium, to evaluate the regional and temporal changes in TGF-alpha expression, and to correlate the pattern of TGF-alpha expression with physiologic events in the endometrium. METHODS: Immunohistochemistry and Western blot analyses were performed using two TGF-alpha antisera, one raised against the active extracellular N-terminus and the other recognizing the intracellular carboxy terminus of the protein. Immunohistochemistry was performed on hysterectomy specimens from premenopausal women with normal menstrual cycles. Soluble and membrane-bound endometrial proteins were isolated from fresh tissue for Western blot analysis. RESULTS: Antibodies recognizing the intracellular and extracellular domains of TGF-alpha exhibited identical immunohistochemical staining patterns. Transforming growth factor-alpha localized primarily to endometrial epithelial cells, and the most intense staining was in the luminal surface epithelium. In the surface epithelium, TGF-alpha staining was intense in the proliferative phase, decreased during the early secretory phase, was at its nadir in the midsecretory phase, and rebounded in the late secretory phase. Western blot analysis demonstrated two transmembrane forms. The 28-kD protein contained both intracellular and extracellular antigens, and the 18-kD protein contained only the intracellular antigen. CONCLUSION: Western blot data were consistent with the hypothesis that the extracellular segment of TGF-alpha is cleaved from the transmembrane precursor in vivo, as has been demonstrated in other tissues. Immunohistochemistry demonstrated that the TGF-alpha antigens are concentrated in the luminal surface epithelium and decline and disappear in the early to midsecretory phase. These findings suggest that the most active period of membrane-bound TGF-alpha cleavage corresponds with the interval during which preimplantation embryos are in the uterine cavity.

Evaluation of the postcoital test in cycles involving exogenous gonadotropins.

OBJECTIVE: To evaluate the hypothesis that a postcoital test, optimally performed in the periovulatory period of cycles in which gonadotropin-induced superovulation was used, correlates with cycle fecundity. METHODS: Of 1135 total consecutive cycles, 367 first cycles were analyzed from the reproductive endocrinology and infertility service of a university medical center. This referral population had a mean age of 34.6 years for the female partner, a nulliparity rate of 81%, and a mean length of infertility of 4.8 years. Postcoital tests were performed 36-40 hours after hCG administration in gonadotropin-stimulated cycles. Clinical pregnancy was defined as fetal cardiac activity as seen on transvaginal ultrasound examination. RESULTS: Couples with no sperm observed per high-power field in the cervical mucus achieved a 16% fecundity rate (21 pregnancies in 129 cycles), one to ten sperm a 18% fecundity rate (28 pregnancies in 154 cycles), and more than ten sperm a 15% fecundity rate (13 pregnancies in 84 cycles). There was no significant difference between groups (n = 367, P = .85); the power to detect a statistically significant difference was .82. As validation of optimal cervical mucus, fecundity rates were compared with these postcoital test values across the entire range of peak periovulatory serum estrogen levels, and no correlation was seen (P = .61, .86, and .96 for estrogen levels of 201-500, 501-1500, and 1501-3433 pg/mL, respectively). CONCLUSION: With precise periovulatory timing and supraphysiologic estrogen levels optimizing qualitative cervical mucus characteristics in gonadotropin-induced cycles, the number of sperm observed per high-power field does not correlate with cycle fecundity.

Advanced maternal age and perinatal outcome: oocyte recipiency versus natural conception.

OBJECTIVE: To compare perinatal outcome in older oocyte recipients with that in women of advanced maternal age who conceived without assisted reproductive technologies. METHODS: We performed a retrospective cohort study of 46 oocyte recipients and 49 women who conceived without assisted reproductive technologies. The obstetric courses in singleton and multiple gestations in the two groups of women were compared. RESULTS: Among singleton pregnancies, a comparable obstetric course was noted between the groups. Fifty percent of the oocyte recipients experienced multiple gestations, resulting in an increased risk for placenta previa, premature rupture of membranes, preterm labor and delivery, glucose intolerance of pregnancy, pregnancy-induced hypertension, and cesarean delivery. However, only the risks for pregnancy-induced hypertension and cesarean delivery were significantly increased in the pregnancies of oocyte recipients with multiple gestations. CONCLUSIONS: Perinatal complications in women receiving oocyte donation may be related to their higher incidence of multiple gestation.

p53lyn and p56lyn: a new signaling pathway in human endometrium and endometrial adenocarcinomas.

OBJECTIVE: To identify specific tyrosine kinases that are involved in endometrial signaling and to study their in vivo expression in normal and abnormal endometrium. We hypothesized that proteins that are differentially expressed would be more likely to be important in regulated cellular events. METHODS: Complementary DNA libraries, constructed from human secretory (n = 5) and proliferative (n = 5) endometrial specimens, were screened with a polyclonal anti-phosphotyrosine antibody. Positive clones were sequenced and screened for differential expression using immunoblotting and Northern analysis of samples from proliferative and secretory endometrium. The expression of one identified clone, lyn, a Src family member, was characterized further with Western and Northern blot analyses and immunolocalization. RESULTS: One protein identified by the above method was lyn, a member of the src family of protein tyrosine kinases, never before described in the human endometrium. Western blot analysis revealed two forms of lyn protein, p53lyn and p56lyn, that were most abundant in the late secretory phase. Immunohistochemistry demonstrated uniform protein expression by all cells in normal glandular epithelium and suggested a correlation between lyn protein expression and cell differentiation for human endometrial adenocarcinomas, with markedly-elevated levels noted in poorly differentiated adenocarcinomas compared with well-differentiated tumors (n = 3). Northern hybridization confirmed the presence of the expected 3.5-kb lyn transcript in normal and abnormal endometrium. CONCLUSIONS: Our data demonstrate that human cDNA libraries created from different phases of the menstrual cycle can be screened successfully using anti-phosphotyrosine antibodies to identify differentially expressed protein tyrosine kinases. Although p53lyn and p56lyn expression has been thought of as a predominantly lymphoid-specific tyrosine kinase, we show prominent expression of lyn protein and mRNA by normal and malignant epithelium of the human endometrium, suggesting a role in endometrial signaling and human reproduction.

Analysis of messenger ribonucleic acid and protein for the ligands and receptors of the platelet-derived growth factor signaling pathway in the placenta, extraembryonic membranes, and uterus during the latter half of murine gestation.

Previous investigation of ligand and receptor messenger RNA (mRNA) expression implicated the platelet-derived growth factor (PDGF) pathway as a participant in the maintenance of pregnancy and fetal development during the first half of murine gestation. We extended these studies using Northern and in situ RNA hybridization and immunohistochemical detection of protein to evaluate the expression kinetics and cell-specific localization of PDGF-A, PDGF-B, PDGF alpha-receptor, and PDGF beta-receptor in mouse placenta, extraembryonic membranes, and uterus during the second half of gestation (days 9.5-18.5). Northern blotting experiments reveal that mRNAs for the PDGF signaling components exhibit unique time-dependent and tissue-specific expression in the placenta and uterus, being progressively and coordinately up-regulated as gestation proceeds. Cell-specific localization of mRNA and protein by in situ hybridization and immunohistochemistry demonstrates widespread expression in multiple cell types of the placenta, gravid uterus, and extraembryonic membranes. Abundant PDGF protein and mRNA expression is exhibited in the nucleated fetal erythroid progenitor cells that originate in the extraembryonic membranes and circulate throughout the developing conceptus. Our data together with those of previous studies demonstrate that PDGF ligands and receptors are globally expressed in many cell types within fetal and maternal tissues during murine gestation and, thus, imply a potential role for PDGF in fetal development and maternal-fetal interactions.

Malignant transformation of the human endometrium is associated with overexpression of lactoferrin messenger RNA and protein.

In the mouse uterus, lactoferrin is a major estrogen-inducible uterine secretory protein, and its expression correlates directly with the period of peak epithelial cell proliferation. In this study, we examine the expression of lactoferrin mRNA and protein in human endometrium, endometrial hyperplasias, and adenocarcinomas using immunohistochemistry, Western immunoblotting, and Northern and in situ RNA hybridization techniques. Our results reveal that lactoferrin is expressed in normal cycling endometrium by a restricted number of glandular epithelial cells located deep in the zona basalis. Two thirds (8 of 12) of the endometrial adenocarcinomas examined overexpress lactoferrin. This tumor-associated increase in lactoferrin expression includes an elevation in the mRNA and protein of individual cells and an increase in the number of cells expressing the protein. In comparison, only 1 of the 10 endometrial hyperplasia specimens examined demonstrates an increase in lactoferrin. We also observe distinct cytoplasmic and nuclear immunostaining patterns under different fixation conditions in both normal and malignant epithelial cells, similar to those previously reported in the mouse reproductive tract. Serial sections of malignant specimens show a good correlation between the localization of lactoferrin mRNA and protein in individual epithelial cells by in situ RNA hybridization and immunohistochemistry. Although the degree of lactoferrin expression in the adenocarcinomas did not correlate with the tumor stage, grade, or depth of invasion in these 12 patients, there was a striking inverse correlation between the presence of progesterone receptors and lactoferrin in all 8 lactoferrin-positive adenocarcinomas. In summary, lactoferrin is expressed in a region of normal endometrium known as the zona basalis which is not shed with menstruation and is frequently overexpressed by progesterone receptor-negative cells in endometrial adenocarcinomas.

Lactoferrin expression in the mouse reproductive tract during the natural estrous cycle: correlation with circulating estradiol and progesterone.

Lactoferrin (LTF), an iron-binding glycoprotein present in most exocrine secretions and in the secondary granules of polymorphonuclear leucocytes (PMN), is regulated by estrogen in the mouse reproductive tract. We investigated the expression of LTF mRNA and protein during the natural estrous cycle to increase our understanding of how this uterine secretory protein is regulated under physiological conditions. There was a positive correlation between LTF mRNA expression in the genital tract and serum estradiol (E2) concentrations. When E2 peaked in proestrus, LTF mRNA and protein were expressed in the uterus; however, during metestrus, when both E2 and progesterone levels were high, LTF mRNA was expressed, while LTF protein was decreasing. LTF protein expression may be hindered by progesterone or some other local factor in the endometrial epithelium after ovulation. Immunohistochemistry demonstrated two distinct staining patterns for LTF in the vaginal and endometrial epithelium. In one staining pattern, the colorimetric reaction was noted over the cytoplasm, and in the other, the nuclear region stained more intensely. This suggests the possibility that in addition to its known role as a secretory protein, LTF may be transported to the nucleus, serving an autocrine role. Our results also indicated that LTF protein is a useful marker for tracking PMN. Nonproliferating epithelial cells in the vagina and endometrium may synthesize chemotactic and/or adhesion molecules for PMN.

Human granulosa-luteal and cumulus cells express transforming growth factors-beta type 1 and type 2 mRNA.

Human granulosa-luteal cells and cumulus cells obtained from women undergoing in vitro fertilization and embryo transfer (IVF-ET) were examined for the presence of TGF-beta 1 and TGF-beta 2 mRNA by reverse transcription-polymerase chain reaction (RT-PCR) analysis. RT-PCR analysis revealed that both follicle cell types express mRNA for both TGF-beta subtypes. Verification of RT-PCR products was done by restriction enzyme digestion analysis. These results suggest a role(s) for TGF-beta 1 and TGF-beta 2 in the development of human granulosa-luteal cells and the oocyte-cumulus cell complex.

Adjunctive leuprolide therapy does not improve cycle fecundity in controlled ovarian hyperstimulation and intrauterine insemination of subfertile women.

Problems arising from controlled ovarian hyperstimulation for intrauterine insemination, such as premature luteinization and asynchronous ovarian follicular development, are identical to those encountered with controlled ovarian hyperstimulation for in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT). It has been suggested that the adjunctive use of GnRH agonists for controlled ovarian hyperstimulation improves the efficiency of GIFT and IVF cycles. We hypothesized that adjunctive use of leuprolide acetate, a GnRH agonist, would have a similarly beneficial effect on cycle quality and cycle fecundity in subfertile women treated with controlled ovarian hyperstimulation and intrauterine insemination. We randomly assigned the first cycle of controlled ovarian hyperstimulation and intrauterine insemination for each of 97 subfertile women to include either human menopausal gonadotropins (hMGs) alone or hMGs following midluteal pre-treatment with leuprolide. If a pregnancy did not occur in the first cycle, the woman was given the other treatment in the second cycle. Although the cycles that included leuprolide required a larger amount of hMGs and more days of stimulation per cycle, the mean estradiol concentrations and numbers of follicles were not different. Despite prevention of premature luteinization with leuprolide, the cycle fecundity was not different between groups (0.11 with adjunctive leuprolide treatment and 0.22 with hMGs alone). We conclude that in unselected subfertile patients, the adjunctive use of leuprolide for controlled ovarian hyperstimulation and intrauterine insemination does not improve cycle fecundity compared with treatment cycles that do not include adjunctive leuprolide therapy.

Epidermal growth factor replaces estrogen in the stimulation of female genital-tract growth and differentiation.

The in vivo studies presented here demonstrate that epidermal growth factor (EGF) is an important autocrine and/or paracrine mediator of estrogen-induced growth and differentiation in mouse uterus and vagina. An antibody specific for EGF significantly inhibited estrogen-induced uterine and vaginal growth, thereby implicating EGF involvement in estrogen action. Furthermore, EGF administered via slow-release pellets in ovariectomized mice acted as a potent uterine and vaginal mitogen as well as an inducer of vaginal keratinization. Experiments with ovariectomized, adrenalectomized, hypophysectomized mice indicated that EGF mitogenesis does not require pituitary or adrenal hormones. Treatment with EGF also mimicked estrogen in the induction of uterine lactoferrin (a major estrogen-inducible secretory protein) mRNA and protein. These data suggest that EGF has estrogen-like effects in the promotion of cell growth in the reproductive tract and that EGF may serve as an important mediator of estrogen action in vivo.

Utility of fluoroimmunoassay in detecting luteinizing hormone surges in spontaneous and human menopausal gonadotropin-stimulated menstrual cycles.

Luteinizing hormone (LH) levels measured with radioimmunoassay and time-resolved fluoroimmunoassay (FIA) in 274 serum samples correlated highly, with a linear correlation coefficient of 0.934. Through the analysis of serial samples from 43 women undergoing human menopausal gonadotropin stimulation for in vitro fertilization or gamete intrafallopian transfer and seven patients monitored in spontaneous menstrual cycles for receipt of frozen embryos, we demonstrated the utility of FIA in the detection of the LH surge. This LH assay technique, which involves no radioactive isotopes, should facilitate the monitoring of ovulation induction patients in the office/ambulatory setting.

Ovarian hyperstimulation in polycystic ovary syndrome during therapy with leuprolide acetate.

Continuous exposure to GnRH eliminates the pituitary as a source of gonadotropins and may have direct suppressive effects on the ovary. A woman with PCO syndrome received leuprolide acetate (1 mg/d SC) for 4 weeks before and simultaneously with hMG stimulation. Human chorionic gonadotropin (5,000 IU) was administered IM on the 8th day of hMG therapy. There were 10 follicles greater than 15 mm and a polycystic appearance to the ovaries with 25 follicles measuring less than 10 mm. The serum E2 concentration was 2,280 pg/mL. She developed severe ovarian hyperstimulation and required hospitalization for 12 days for fluid management. A viable intrauterine pregnancy was present. Four weeks of pretreatment with leuprolide did not prevent hyperstimulation in the presence of an intrauterine pregnancy.