Proliferation Of Ovarian Surface Epithelium Research Articles

Microscopic Evaluation of Ovarian Surface Epithelium Following Treatment with Conjugated Estrogens in a Mouse Model.

Background:This study was designed to evaluate the effect of different concentrations of conjugated equine estrogens (CEE) on the ovarian epithelium of female CD1 mice. Methods:Twenty-four female mice at 7 months with irregular estrus cycles were randomly divided into four groups of 6 mice each. Group one was considered as a control group and received a daily dose of 0.5ml of propylene glycol, for three weeks, while those in the treatment groups received a daily dose of 14μg/kg, 28μg/kg and 56μg/kg conjugated equine estrogens, respectively. Results:The results from this study showed a strong correlation between elevated concentrations of CEE and histological changes in ovarian surface epithelium (OSE). They also showed that administration of high-dose estrogen created the conditions for excessive proliferation of OSE which may progress into the development of cysts in the ovaries. Conclusion:This study concluded that high concentrations of CEE may increase the chances of developing epithelial ovarian cancer.

Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries

Significance This study improves our understanding of the role of Müllerian inhibiting substance (MIS/AMH) in antagonizing primordial follicle activation. By applying scRNA-seq methods to neonatal mouse ovaries, we revealed transcriptional signatures associated with MIS treatment in ovarian cell types present during the first wave of folliculogenesis and follicle development. We showed that MIS inhibits pregranulosa cell differentiation and the proliferation of ovarian surface epithelium and stromal cells. MIS also uncoupled granulosa and germ cell maturation, leading to abnormal development of activated follicles. These findings identify markers and pathways related to primordial follicle quiescence that could be targeted in contraception, preservation of ovarian reserve during aging or chemotherapy, or synchronization of preantral follicle growth for IVF.

Abstract 3571: Oxidative stress and the ovarian surface epithelium: TGF-β1 stimulation of IOSE cell lines reveals novel upregulation of NADPH oxidase 4, a membrane bound hydrogen peroxide producing enzyme

Abstract Normal ovarian surface epithelium (OSE) has been widely studied as the origin of ovarian cancer. Repetitive ovulation has been implicated in cancer initiation based on damage and inflammation incurred to the OSE upon continuous cycles of tear and repair. Transforming growth factor-beta (TGF-β) plays a role in this normal ovulatory cycle, having been shown to inhibit human OSE proliferation and induce apoptosis, which may prevent the over-proliferation of cells. NADPH oxidase 4 (NOX4), a membrane-associated hydrogen peroxide-producing enzyme, has previously been shown to be sensitive to TGF-β1, with increased expression stimulated in smooth muscle cells and fibroblasts. Here we describe for the first time TGF-β1 induced upregulation of NOX4 in immortalized OSE (IOSE) cells (IOSE121, IOSE144, IOSE364, IOSE386 and IOSE397). This time-dependent increased expression (>5 fold at 2 ng/ml TGF-β1, 24 h) was simulated by direct overexpression of NOX4 in IOSE397 cells and has demonstrated that increased NOX4, in the absence of further growth factor related effects, affords induction of p18 and p21. In addition, using immunohistochemical evaluation of human ovarian cancers in tissue microarrays (n=17 patients), we observed significantly increased expression of NOX4, suggesting that NOX4 upregulation may contribute to the malignant transformation of ovarian cells. Citation Format: Jennifer L. Meitzler, Becky A. Diebold, Smitha Antony, Yongzhong Wu, Agnes Juhasz, Iris Dahan, Guojian Jiang, Jiamo Lu, Stephen Wang, Mariam M. Konaté, Krishnendu Roy, James H. Doroshow. Oxidative stress and the ovarian surface epithelium: TGF-β1 stimulation of IOSE cell lines reveals novel upregulation of NADPH oxidase 4, a membrane bound hydrogen peroxide producing enzyme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3571.

Regulation of HGF and c-MET Interaction in Normal Ovary and Ovarian Cancer.

Binding of hepatocyte growth factor (HGF) to the c-MET receptor has mitogenic, motogenic, and morphogenic effects on cells. The versatile biological effects of HGF and c-MET interactions make them important contributors to the development of malignant tumors. We and others have demonstrated a therapeutic value in targeting the interaction of c-MET and HGF in epithelial ovarian cancer (EOC). However, both HGF and c-MET are expressed in the normal ovary as well. Therefore, it is important to understand the differences in mechanisms that control HGF signaling activation and its functional role in the normal ovary and EOC. In the normal ovary, HGF signaling may be under hormonal regulation. During ovulation, HGF-converting proteases are secreted and the subsequent activation of HGF signaling enhances the proliferation of ovarian surface epithelium in order to replenish the area damaged due to expulsion of the ovum. In contrast, EOC cells that exhibit epithelial characteristics constitutively express both c-MET and HGF-converting proteases such as urokinase-type plasminogen activator. In EOC, mechanisms to control the activation of HGF signaling are absent since HGF is provided locally from the tissue microenvironment as well as remotely throughout the body. Potential incessant HGF signaling in EOC may lead to an increase in proliferation, invasion through the stroma, and migration to other tissues of cancer cells. Therefore, targeting the interaction of c-MET and HGF would be beneficial in treating EOC.

In vitro regulation of sheep ovarian surface epithelium (OSE) proliferation by local ovarian factors

The ovarian surface epithelium (OSE) forms a lining around the entire ovary and actively participates in the ovulatory cycle. To investigate how specific growth factors and hormones affect OSE proliferation, the present study used sheep as a model to examine the effects of follicular and luteal products on the proliferation of sheep OSE cells in culture, and to analyse the influences of large antral follicles and corpora lutea (CL) on the expression of gonadotrophin receptors (FSHR and LHR) in the OSE. In the present study, follicular fluids from medium and large follicles, and extracts of corpora lutea stimulated the growth of OSE cells. The results of the present study showed that factors in the follicular fluid can induce OSE proliferative activity, and this stimulation effect could not be attributed to steroids in the follicular fluid since oestrogen and progesterone treatments failed to stimulate OSE cells. The expression of LH and FSH receptors over large follicles (5mm or larger) was two and four times higher than those over stroma and CL, respectively. In conclusion, OSE proliferation in cycling sheep is associated with underlying developing follicles and CL, mediated by, at least in part, the up-regulation of gonadotrophin receptors, and facilitated by the action of mitogenic glycopeptides and growth factors, but not steroids.

Conditional knockout ofbrca1/2andp53in mouse ovarian surface epithelium: Do they play a role in ovarian carcinogenesis?

Alterations of genes are known to be critical for the induction of tumorigenesis, but the mechanism of ovarian carcinogenesis is little understood and remains to be elucidated. In this study, we investigated the roles of brca1, brca2 and p53 genes in the development of ovarian cancer using conditional knockout mice generated by a Cre-loxP recombinant system. Following the application of recombinant adenovirus expressing Cre in vitro, the proliferation of ovarian surface epithelium (OSE) was increased. For instance, a significant increase in cell growth was observed in OSE cells in vitro by conditional knockout isolated from the mice bearing concurrent floxed copies of brca1 and brca2/p53. However, the proliferative effect of the ovarian cells was not observed in concurrent brca1/brca2 or p53 knockout mice in vivo, indicating that we could not observe the direct evidence of the involvement of brca1, brca2, and p53 in ovarian carcinogenesis. Since morphological changes including tumor formation were not observed in mice bearing floxed copies of concurrent brca1/brca2 or p53, the inactivation of brca1/2 or p53 is not sufficient for the induction of tumor formation. Taken together, these results suggest that the deficiency of these genes may not be involved directly in the mechanism of ovarian carcinogenesis.

Effect of repeated gonadotropin stimulation on ovarian reserves and proliferation of ovarian surface epithelium in mice

This study aimed to evaluate the effect of repeated ovarian stimulation (OS) on the ovarian follicular population and morphology in female mice and its influence on the embryo’s developmental ability, and the profile of the ovarian surface epithelium (OSE). A total of 75 mice were enrolled in this experiment and randomly assigned into three groups: repeated ovarian stimulated group [n = 25; receiving 5 IU pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotropin (hCG) at 6 day intervals for 5 cycles]; single ovarian stimulated group (n = 25; receiving 5 IU PMSG and hCG for 1 cycle), and control group (n = 25; without additional treatment). The follicle number at various stages and the morphologies were recorded respectively in the three groups. The harvested oocytes or embryos, cleavage rate, good quality embryo rate, and blastocyst production rate were counted and calculated, and the proliferations of ovarian surface epithelium were evaluated respectively. In the three groups, the single ovarian stimulation treatment significantly increased the mean number of ovarian oocytes or embryos (39.25±10.77 one-cell embryos/female); on the other hand, repeated gonadotropin stimulation obtained the lowest mean number (5.15± 2.81 eggs/female, P<0.01). Repeated ovarian stimulation also tended to decrease normal follicles of primary follicles (66.67%) and secondary follicles (72.86%), and got the lowest cleavage rate (67.47%), lowest good quality embryo rate (2.41%), and lowest blastocyst production rate (0). The OSE cells adjacent to the antral follicles and corpus luteum (CL) in the repeated ovarian stimulated group (81.8%) had a significantly higher proliferation rate than the other groups. The proliferation rate of the OSE in the single ovarian stimulated group (56.4%) was significantly higher than that in the control group (37.5%) (P<0.01). In conclusion, single ovarian stimulation may produce more oocytes/embryos. However, repeated gonadotropin stimulation may have a negative effect on the ovarian follicular quality, the number of mature retrieved oocytes, and the embryo quality, even increasing the chance of ovarian cancer.

Regulation of the proliferative activity of ovarian surface epithelial cells by follicular fluid

Despite critical roles of the ovarian surface epithelium (OSE) in ovulation and post-ovulatory wound repair, little is known about the physiological mechanism regulating OSE proliferation. A role of follicles and corpora lutea in locally regulating the proliferative activity of OSE has been suggested. In this study, the effects of follicular and luteal products on proliferation of cultured OSE cells were tested using cells obtained from seasonally anoestrous ewes. Follicular fluid but not luteal extracts induced OSE cell proliferation (2.5-fold relative to untreated controls; P < 0.0001). The response of OSE cells was not affected by follicle size or previous charcoal-extraction of follicular fluid ( P > 0.1). Treatment with IGF-1 (2.2-fold; P < 0.01), EGF (1.9-fold; P < 0.01) and, to a lesser extent, FSH ( P < 0.05) also induced OSE cell proliferation. In contrast, oestradiol or progesterone did not induce cell proliferation or enhance the effects of FSH on proliferation ( P > 0.1). It was concluded that follicular fluid can directly stimulate ovine OSE cell proliferation and that this effect is attributable to non-steroidal mitogens.

Induction of Proliferation in the Primate Ovarian Surface Epithelium In Vivo

Epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy in women, has resisted direct testing of possible causes in part because, among other mammals, only primates are affected. Proliferation has been proposed as a mode of transformation of the ovarian surface epithelium (OSE). This study examined OSE proliferation in the rhesus macaque, a monkey whose reproductive physiology resembles that of humans. In particular, the question was addressed of whether primate OSE can undergo proliferative repair in vivo in a manner that might relate to the cause of EOC. Macaque ovaries were collected at 3 stages of the menstrual cycle. Very late luteal-phase ovaries were gently brushed during laparoscopy to remove part of the OSE, and groups of ovaries numbering 3 or less were collected after 1 to 4 days. Ovaries also were sampled from 10 women ranging in age from 33 to 74 years. Ovarian tissue sections were evaluated with OSE markers (keratin, β-catenin, E- and N-cadherin); markers of proliferation (proliferating cell nuclear antigen [PCNA], phosphorylated histone H3 [phospho-H3], and phosphorylated retinoblastoma [pRb]); or labels of collagen and basement membrane. As is evident in vitro, both monkey and human OSE were positive for keratin, β-catenin, and N-cadherin in vivo. Brushing of the ovaries partially removed the OSE without causing tissue damage or adhesions, and it increased the frequency of PCNA, phospho-H3, and pRb in the remaining OSE. As many as 50% of cells in brushed regions were marked, compared to fewer than 0.1% of cells in nonbrushed areas. Brushing did not induce the proliferation of underlying stromal cells. Further studies confirmed that the residual OSE was taking part in a true mitotic response (beyond merely transitioning from G1 to S phase). Proliferation of the OSE correlated more closely with brushing than with specific cell morphology. The investigators conclude that, because the OSE is able to undergo proliferative repair, its normal regulation might contribute to the development of EOC. It remains unclear whether the OSE is a necessary part of ovarian function, or whether abnormalities in the OSE contribute to ovarian defects such as polycystic ovary syndrome or infertility.

Induction of proliferation in the primate ovarian surface epithelium in vivo

Epithelial ovarian cancer (EOC) is the primary ovarian malignancy affecting women. Proposed etiologies of EOC resist direct testing due to the absence of a suitable animal model, as EOC affects only primates, not other mammals. The role of proliferation in ovarian surface epithelium (OSE) transformation has been suggested but not demonstrated, nor has OSE proliferation been widely reported. We selected the rhesus macaque as a model to evaluate the unique primate OSE in vivo, and to determine whether it can undergo proliferative repair, which may relate to EOC etiology. Macaque ovaries were collected at three stages of the cycle. Very late luteal phase ovaries were gently brushed during laparoscopy to remove a portion of the OSE, and ovaries (< or =3 per group) were collected 1-4 days later. Ovary samples were also collected from 10 women aged 33-74 years. Ovarian tissue sections were probed with OSE markers (keratin, beta-catenin, E- and N-cadherin), proliferation markers [proliferating cell nuclear antigen (PCNA), phosphorylated histone H3 (phospho-H3), and phosphorylated Retinoblastoma (pRb)], or labels of collagen and basement membrane. Brushing partially removed the OSE; did not cause tissue damage/adhesions; elevated the frequency of PCNA, phospho-H3 and pRb in the residual OSE, marking as many as 10-50% of cells in brushed regions (unbrushed areas contained <0.1% positive cells), and; did not induce proliferation in underlying stromal cells. The OSE can undergo proliferative repair, and thus its normal regulation could contribute to EOC etiology.

Gonadotropin-Induced Superovulation Drives Ovarian Surface Epithelia Proliferation in CD1 Mice

The ovarian surface epithelium (OSE) is a monolayer of cells that surround the ovary and accommodate repeated tear and repair in response to ovulation. OSE cells are thought to be the progenitors of 90% of ovarian cancers. Currently, the total amount of proliferation of the OSE has not been reported in response to one ovulatory event. In this study, proliferation of the OSE was quantified in response to superovulation induced by ip injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) in immature 27-d-old CD1 mice using bromodeoxyuridine (BrdU). BrdU incorporation into the OSE cells was measured from the time of hCG injection for a total cumulative label of 12 h. BrdU incorporation was also measured from the time of PMSG injection for a total label of 60 h to correlate proliferation with specific gonadotropin stimulation. The OSE proliferation was significantly higher in superovulated animals compared with control mice at all time points. Proliferation was also analyzed in discrete anatomical sections and indicated that OSE covering antral follicles and corpora lutea proliferated more rapidly than OSE distal to follicular growth. Finally, apoptosis was assessed in response to ovulation, and virtually no cell death within the OSE was detected. These data demonstrate that the OSE, especially near antral follicles and corpora lutea, proliferates significantly in response to the gonadotropins PMSG and hCG. Therefore, ovarian surface cell division in response to ovulation could contribute to ovarian cancer by proliferation-induced DNA mutations and transformed cell progression.

Mechanisms of Hormone-Mediated Carcinogenesis of the Ovary

Experimental ovarian carcinogenesis has been investigated in inbred and hybrid strains of mice and induced by a diversity of mechanisms including X-irradiation, oocytotoxic xenobiotic chemicals, ovarian grafting to ectopic or orthotopic sites, neonatal thymectomy, mutant genes reducing germ cell populations, and aging. Disruptions in the function of graafian follicles by a variety of mechanisms results in a spectrum of ovarian proliferative lesions, including tumors. The findings in mutant and genetically engineered mice support the concept of a secondary (hormonally mediated) mechanism of ovarian tumorigenesis in mice associated with sterility. Multiple pathogenic factors that either destroy or diminish the numbers of graafian follicles in the ovary result in decreased sex hormone secretion, especially estradiol-17beta, leading to a compensatory overproduction of pituitary gonadotrophins, particularly LH, which places the mouse ovary at an increased risk for developing tumors in chronic studies. The intense proliferation of ovarian surface epithelium and stromal (interstitial) cells with the development of unique tubular adenomas in response to sterility does not appear to have a counterpart in the ovaries of adult human females.

Follicle-stimulating hormone increases cell proliferation in the ovary and the testis of the chick embryo

Previous studies have demonstrated that FSH stimulates cell proliferation in the ovary and the testis of the chick embryo. This study analyzed the presence of FSH receptor and the cell subpopulations that proliferate in response to FSH in chick embryo gonads. FSH receptor mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in the male and female gonads of the 6 to 14-day-old chick embryo. Somatic cells of the ovary expressed the FSH receptor in the 14-day-old chick embryo. Ovarian surface epithelium of the 14-day-old chick embryo increased the mitotic index 15–18 h after FSH treatment. Similarly, the mitotic index in oogonia was increased 24 h after receiving a pulse of FSH; this result was confirmed by an increase in the number of germ cells that incorporated bromodeoxyuridine (BrdU). Somatic cells of the medullary cords in the ovary displayed an increase in the mitotic index 15–21 h after the FSH injection. In the chick embryo testis, at the same stage of development, the treatment with FSH increased the mitotic index in cells of the seminiferous tubules and to a lesser extent in cells at a peritubular and interstitial location. Present results demonstrate that in the chick embryo, FSH stimulates the proliferation of ovarian surface epithelium, oogonia in the cortex, and somatic cells of the medullary cords of the chick embryo ovary. In the chick embryo testis, FSH stimulates cell proliferation in seminiferous tubules and peritubular cells.

Changes in the mouse ovarian surface epithelium with age and ovulation number

The cell biology of ovarian surface epithelium (OSE) was studied in mice of varying age and lifetime total ovulation number (OV#), to determine the relative importance of these factors in control of OSE proliferation and development of invaginations and cysts. Ovaries from Swiss Webster mice (total OV# median: range [ n mice]) were collected at 4 weeks of age (prepubertal; 0[9]), from 3-month virgins (113: 11–235 [55]), from 12-month old breeders (217: 97–386 [21]) and from 8-month virgin mice, housed in split cages alongside a male, to induce continuous oestrous cycles (629: 456–908 [16]). Scanning electron microscopy revealed cuboidal and squamous cells in OSE from all ages. Higher total OV# markedly increased the rate of OSE invagination and layering. Histology showed the incidence of cysts, which had the appearance of benign serous cystadenomata, increased with age rather than total OV#.

Expression and action of kit ligand/stem cell factor in normal human and bovine ovarian surface epithelium and ovarian cancer.

Greater than 95% of ovarian cancers originate from the epithelial cells on the surface of the ovary termed ovarian surface epithelium (OSE). A normal aspect of OSE function is repeated proliferation after ovulation, and this is postulated to be involved in part in the onset of ovarian cancer. The hypothesis tested is that locally produced growth factors have an important role in controlling OSE proliferation. The current study investigates the potential role of the growth factor kit ligand (KL)/stem cell growth factor and its receptor c-kit in normal OSE biology and ovarian cancer. Human tumors from borderline, stage I, and stage III cases of ovarian cancer were found to express KL and c-kit protein in the epithelial cell component by ICC analysis. The stromal cell component of human ovarian tumors contained little immunostaining. Bovine ovarian physiology and endocrinology are similar to the human such that cow ovaries were used as a model system to investigate normal OSE functions. KL and c-kit proteins were detected in the OSE from both normal human and bovine ovaries. Adjacent ovarian stromal tissue contained less intense but positive KL and c-kit immunostaining. To extend the ICC results, RNA was collected from normal bovine OSE and ovarian stromal cells to examine KL gene expression. KL transcripts were detected in cultured OSE and stromal cells by Northern blot analysis. KL gene expression was found to be high in freshly isolated OSE but low in freshly isolated stroma using a quantitative polymerase chain reaction procedure. Levels of KL gene expression in cultured OSE and stroma increased to high levels. Observations indicate that normal OSE expresses high levels of KL in vivo and in vitro. The actions of KL on the growth of both normal OSE cells and ovarian cancer cells was investigated. KL was found to stimulate the growth of normal OSE cells in a similar manner to epidermal growth factor. Observations demonstrate the production and action of KL by normal OSE cells and ovarian cancer cells. Coexpression of KL and c-kit by normal OSE suggests that KL can act as an autocrine factor for OSE. The local production and action of KL on OSE provides insight into normal OSE biology, and a factor that may be involved in the onset and progression of ovarian cancer.

Administration of gonadotropins stimulates proliferation of normal mouse ovarian surface epithelium

Little is known concerning the proliferation of the ovarian surface epithelium or the factors which control this process. To define when and under what circumstances this epithelium proliferates, we have studied the proliferation of mouse ovarian surface epithelium (OSE) during embryogenesis, early postnatal life, various physiological circumstances in the adult and in response to gonadotropic hormones, using the bromodeoxyuridine technique. Proliferation of the OSE is greatest during embryonic development, and falls gradually after birth until sexual maturity is reached. Very little proliferation of the OSE is detectable in adult life in non-pregnant, pregnant or lactating mice. The basal proliferation of the OSE can be increased significantly by inducing follicular development with pregnant mare serum gonadotropin (PMSG) or by administration of the pure recombinant gonadotropins follicle-stimulating hormone (FSH) or luteinizing hormone (LH). These results show that administration of gonadotropins to sexually mature mice induces proliferation of ovarian surface epithelium concurrently with the process of folliculogenesis.

Mechanisms of hormone-mediated carcinogenesis of the ovary in mice

Experimental ovarian carcinogenesis has been investigated in inbred and hybrid strains of mice and induced by a diversity of mechanisms including X-irradiation, oocytotoxic xenobiotic chemicals, ovarian grafting to ectopic or orthotopic sites, neonatal thymectomy, mutant genes reducing germ cell populations, and aging. The mechanisms are briefly reviewed whereby disruptions in the function of graafian follicles results in a spectrum of ovarian proliferative lesions including tumors. The findings in mutant mice support the concept of a secondary (hormonally-mediated) mechanism of ovarian carcinogenesis in mice associated with sterility. Multiple pathogenetic factors that either destroy or diminish the numbers of graafian follicles in the ovary result in decreased sex hormone secretion (especially estradiol-17β) leading to a compensatory over-production of pituitary gonadotrophins (particularly luteinizing hormone), which places the mouse ovary at an increased risk to develop tumors. The intense proliferation of ovarian surface epithelium and stromal (interstitial) cells with the development of unique tubular adenomas in response to sterility does not appear to have a counterpart in the ovaries of women.

Periovulatory multifocal mesothelial proliferation: a possible association with malignant transformation.

This study was aimed at the identification of the site and characterization of the kinetics of tissue renewal during the repair of the ovarian surface epithelium (OSE) surrounding ovulation. Prepubescent C57BL female mice were induced hormonally either by intraperitoneal (ip) injection of pregnant mare serum gonadotropin (PMSG, 5 IU) +/- human chorionic gonadotropin (HCG, 5 IU) 46 h later, or subcutaneous (sc) diethylsilbesterol (DES) 0.1 mg or 1.0 mg in 0.5 ml corn oil, in addition to saline-injected controls. The animals were killed after various time intervals. Autoradiography was carried out on formaldehyde-fixed paraffin sections of genital organs with subsequent hematoxylin & eosin staining. The preparations were examined for nuclear [3H]thymidine incorporation (0.5 &mgr;Ci injected 1 h before killing) in the OSE, as well as the surrounding and more distal mesothelium. Surprisingly, a significant proliferative response was observed in the extra-ovarian mesothelium surrounding the uterine horns, tubes and nearby peritoneum, while the OSE proliferation rate was less than half of it. In contrast to the common notion, it seems that the expansion and repair of the OSE, associated with ovulation, is contributed mainly by its extra-ovarian mesothelial continuum. This observation may provide additional support to the multifocal theory suggested in the pathogenesis of ovarian cancer. Therefore, any search for cellular or molecular events related to malignant transformation should include the proximal and distal mesothelium in addition to the OSE.