Postmenopausal Human Ovaries Research Articles

FOXO3 Expression in the Human Ovary [6OP

INTRODUCTION: To assess FOXO3 gene expression levels and members of the KL-PI3K-AKT-FOXO3 pathway in pre- and postmenopausal human ovaries. To evaluate in a temporal manner protein localization of FOXO3 in pre- and postmenopausal human ovaries by immunohistochemistry (IHC). METHODS: Total RNA was isolated from human ovary collected from pre- and postmenopausal patients undergoing oophorectomy and used for real-time qPCR for members of the KL-PTEN-PI3K-FOXO3 pathway, as well as other genes. IHC was performed on archived paraffin embedded ovaries to visualize the presence and localization of FOXO3 in human ovaries ranging from fetal to menopausal. RESULTS: FOXO3 is expressed in premenopausal human ovary and to a significantly reduced level in postmenopausal ovary. Nine additional genes in the KL-PTEN-PI3K-FOXO3 pathway were also expressed in premenopausal ovaries but like FOXO3 displayed significantly decreased levels in the postmenopausal cohort. IHC analysis revealed strong nuclear FOXO3 staining in primordial follicles from pre-pubertal ovaries through menarche/post-pubertal. A dominant population of primordial follicles were identified in older post-pubertal ovaries and in the reproductive/premenopausal groups that displayed light to negative nuclear staining for FOXO3, with a second much less abundant population of primordial follicles retaining strong positive nuclear FOXO3 staining. Postmenopausal ovaries were devoid of follicles but had strong stromal FOXO3 staining. CONCLUSION: As predicted from mouse genetic models, FOXO3 mRNA and protein is present in premenopausal human ovary as are nine members of the KL- PI3K-AKT-FOXO3 pathway. FOXO3 localization in the human ovary undergoes an age-dependent transition from strongly positive nuclei in young ovaries to predominantly light to negative staining in reproductive/premenopausal. Given FOXO3's importance to genome stability, primordial follicles with high nuclear FOXO3 levels may define high quality oocytes lacking detrimental levels of DNA damage, so that the ratio of FOXO3 positive to negative oocytes may explain the consequences of decreasing ovarian reserve.

Follicle Depletion Provides a Permissive Environment for Ovarian Carcinogenesis.

We modeled the etiology of postmenopausal biology on ovarian cancer risk using germ cell-deficient white-spotting variant (Wv) mice, incorporating oncogenic mutations. Ovarian cancer incidence is highest in peri- and postmenopausal women, and epidemiological studies have established the impact of reproductive factors on ovarian cancer risk. Menopause as a result of ovarian follicle depletion is thought to contribute to higher cancer risk. As a consequence of follicle depletion, female Wv mice develop ovarian tubular adenomas, a benign epithelial tumor corresponding to surface epithelial invaginations and papillomatosis frequently found in postmenopausal human ovaries. Lineage tracing using MISR2-Cre indicated that the tubular adenomas that developed in Wv mice were largely derived from the MISR2 lineage, which marked only a fraction of ovarian surface and oviduct epithelial cells in wild-type tissues. Deletion of p27, either heterozygous or homozygous, was able to convert the benign tubular adenomas into more proliferative tumors. Restricted deletion of p53 in Wv/Wv mice by either intrabursal injection of adenoviral Cre or inclusion of the MISR2-Cre transgene also resulted in augmented tumor growth. This finding suggests that follicle depletion provides a permissive ovarian environment for oncogenic transformation of epithelial cells, presenting a mechanism for the increased ovarian cancer risk in postmenopausal women.

Neo-oogenesis: Has its existence been proven?

Neo-oogenesis: Has its existence been proven?

Aged mouse ovaries possess rare premeiotic germ cells that can generate oocytes following transplantation into a young host environment

Of all the major organ systems in the body, the ovaries of females are the first to exhibit impaired function with advancing age. Until recently, traditional thinking was that female mammals are provided with a non-renewable pool of oocyte-containing follicles at birth that are depleted during postnatal life to exhaustion, driving ovarian failure. However, a growing body of evidence, including the isolation of germline stem cells (GSC) from adult mouse ovaries that produce developmentally-competent oocytes, has challenged this belief. In addition, rare germline stem-like cells capable of generating oocytes in vitro that undergo parthenogenesis to form blastocyst-like structures have recently been identified in postmenopausal human ovaries. Here we show that the germline-specific meiosis-commitment genes,Stimulated by retinoic acid gene 8 (Stra8) and Deleted in azoospermia-like (Dazl), are highly expressed in aged mouse ovaries. However, histological and marker analyses fail to demonstrate the presence of oocytes, supporting that Stra8 and Dazl are expressed in premeiotic germ cells that do not undergo further differentiation. Through the use of aged germline-specific GFP-expressing transgenic mice, we further show that these germ cells can generate GFP-positive oocytes that co-express the primordial oocyte marker NOBOX and form follicles when grafted into young adult wild-type female hosts. Thus, aged mouse ovaries possess a rare population of premeiotic germ cells that retain the capacity to form oocytes if exposed to a young host environment.

Immune physiology and oogenesis in fetal and adult humans, ovarian infertility, and totipotency of adult ovarian stem cells

It is still widely believed that while oocytes in invertebrates and lower vertebrates are periodically renewed throughout life, oocytes in humans and higher vertebrates are formed only during the fetal/perinatal period. However, this dogma is questioned, and clashes with Darwinian evolutionary theory. Studies of oogenesis and follicular renewal from ovarian stem cells (OSCs) in adult human ovaries, and of the role of third-party bone marrow-derived cells (monocyte-derived tissue macrophages and T lymphocytes) could help provide a better understanding of the causes of ovarian infertility, its prevention, and potential treatment. We have reported differentiation of distinct cell types from OSC and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. OSCs are also capable of producing neural/neuronal cells in vitro after sequential stimulation with sex steroid combinations. Hence, OSC represent a unique type of totipotent adult stem cells, which could be utilized for autologous treatment of premature ovarian failure and also for autologous stem cell therapy of neurodegenerative diseases without use of allogeneic embryonic stem cells or somatic cell nuclear transfer. The in vivo application of sex steroid combinations may augment the proliferation of existing neural stem cells and their differentiation into mature neuronal cells (systemic regenerative therapy). Such treatment may also stimulate the transdifferentiation of autologous neural stem cell precursors into neural stem cells useful for topical or systemic regenerative treatment.

Potential new strategies for the treatment of ovarian infertility and degenerative diseases with autologous ovarian stem cells

The 50-year-old and currently prevailing view that all oocytes in adult human ovaries persist from the fetal period of life is controversial as it clashes with Darwinian evolutionary theory. Studies of oogenesis and follicular renewal in adult human ovaries, and of the role of hormonal signals and third-party cells (tissue macrophages and T cells), could all be helpful in providing better understanding of the causes of ovarian infertility, its prevention and potential therapy. In addition, the authors recently reported differentiation of distinct cell types and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. It is possible that fertilisation of such eggs will open up new opportunities for providing genetically related children to infertile women for whom conventional in vitro fertilisation has failed. As ovarian stem cells appear to represent a new type of totipotent adult stem cell, they could also be utilised for autologous stem cell therapy of degenerative diseases, without any involvement of allogeneic embryonic stem cells and somatic cell nuclear transfer.

Cycle and age-related changes in corticotropin-releasing hormone levels in human endometrium and ovaries

Corticotropin-releasing hormone (CRH) is synthesized in most female reproductive tissues such as the ovaries and the uterus. In the non-pregnant uterus ,it is mainly produced by epithelial cells of the endometrium. Recent in vitro experimental findings show that endometrial CRH is under the positive control of progesterone ,participating in the decidualization process of endometrial stroma and the progression of blastocyst implantation. CRH is also produced in the thecal compartment of the human ovary ,controlling ovarian steroid hormone biosynthesis. In the present study we compared the concentration of immunoreactive CRH (ir-CRH) in biopsies from proliferative and secretory human endometria ,and from pre- and postmenopausal human ovaries. We found that the concentration of ir-CRH was significantly higher in the secretory (92 ± 8 pg/mg protein; n = 10) than the proliferative (75 ± 9 pg/mg protein; n = 12; p < 0.05) endometria. This observation supports the experimental in vitro findings associating endometrial CRH in intrauterine phenomena of the secretory phase of the menstrual cycle (decidualization and implantation). Additionally ,we have shown that the concentration of ir-CRH was significantly higher in the premenopausal (125 ± 12 pg/mg protein; n = 14) than the postmenopausal (100 ± 12 pg/mg protein; n = 12; p < 0.05) ovaries, suggesting that ovarian CRH is related to normal ovarian function during the reproductive lifespan.

Cycle and age-related changes in corticotropin-releasing hormone levels in human endometrium and ovaries

Corticotropin-releasing hormone (CRH) is synthesized in most female reproductive tissues such as the ovaries and the uterus. In the non-pregnant uterus ,it is mainly produced by epithelial cells of theendometrium. Recent in vitro experimental findings show that endometrial CRH is under the positive control of progesterone ,participating in the decidualization process of endometrial stroma andthe progression of blastocyst implantation. CRH is also produced in the thecal compartment of the human ovary ,controlling ovarian steroid hormone biosynthesis. In the present study we compared the concentrationof immunoreactive CRH (ir-CRH) in biopsies from proliferative and secretory human endometria ,and from pre- and postmenopausal human ovaries. We found that the concentration of ir-CRH was significantlyhigher in the secretory (92 ± 8 pg/mg protein; n = 10) than the proliferative (75 ± 9 pg/mg protein; n = 12; p < 0.05) endometria. This observation supports the experimentalin vitro findings associating endometrial CRH in intrauterine phenomena of the secretory phase of the menstrual cycle (decidualization and implantation). Additionally ,we have shown that the concentrationof ir-CRH was significantly higher in the premenopausal (125 ± 12 pg/mg protein; n = 14) than the postmenopausal (100 ± 12 pg/mg protein; n = 12; p < 0.05) ovaries,suggesting that ovarian CRH is related to normal ovarian function during the reproductive lifespan.

Expression of P450c17 messenger ribonucleic acid in postmenopausal human ovary tissues

Objective: To investigate the expression of the P450c17 gene in postmenopausal human ovaries compared with normal cycling ovaries. Design: Prospective nonrandomized clinical research study. Setting: Servei de Medicina Reproductiva and Centre d’Investigacions en Bioquı́mica i Biologia Molecular, Hospitals Vall d’Hebron, Barcelona, Spain. Patient(s): Six premenopausal women and four postmenopausal women undergoing bilateral oophorectomy for nonovarian gynecologic disease. Intervention(s): Extraction of 10 mL of peripheral venous blood for hormone measurements. Extraction of RNA from surgically removed ovaries for Northern blot, ribonuclease protection, and reverse transcriptase polymerase chain reaction Southern blot assays. Main Outcome Measure(s): Definition of the reproductive cycle state of each patient and determination of the level of P450c17 gene expression in all samples with the use of the semiquantitative reverse transcriptase polymerase chain reaction Southern blot assay. Result(s): P450c17 messenger RNA levels in postmenopausal ovaries varied considerably between samples. Although the levels were similar to those detected in the early follicular phase, one of the samples had levels as high as those observed in the late follicular phase. Conclusion(s): Although the degree varied from one sample to another, all the postmenopausal ovaries studied expressed the P450c17 gene at the messenger RNA level. In a sample from a patient with endometrial adenocarcinoma, the level was as high as the levels observed in the late follicular phase.

Ovarian transforming growth factor-beta (TGF-beta) receptors: in-vitro effects of follicle stimulating hormone, epidermal growth factor and TGF-beta on receptor expression in human preantral follicles.

The expression patterns of transforming growth factor (TGF)-beta receptor type I (TbetaRI) and -II (TbetaRII) in pre- and post-menopausal human ovaries, and the in-vitro effects of follicle stimulating hormone (FSH), epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1) on receptor expression in preantral follicles were evaluated using immunohistochemistry and immunoblotting. Both types of receptor were present in granulosa, theca and interstitial cells; however, more mural than antral granulosa cells were TbetaRII positive. Overall, more cells expressed TbetaRI than TbetaRII. TbetaRI and TbetaRII expressions were detected in the membrane as well as in the soluble fractions. However, marked increases in TbetaRII and TbetaRI expression in the soluble fraction and corresponding decreases in the membrane fraction were noted in the post-menopausal ovary. Preantral follicles in class 1 and 2 responded in vitro to FSH and EGF with increased growth and a corresponding increase in TbetaRII expression in granulosa cells; however, TGF-beta did not influence follicular growth or receptor expression. There was no apparent change in follicular TbetaRI immunoreactivity regardless of test factors or follicular growth status. These results suggest that TbetaRI and TbetaRII are expressed differentially in human ovarian cells, particularly in the granulosa cells. The shift in TbetaR-I and -II from transmembrane to soluble fraction after menopause indicates that the endocrine milieu provided by the granulosa cells positively influences receptor induction in the membrane and, hence, the biological action of TGF-beta. FSH- and EGF-induced preantral follicular development is associated with a selective induction of TbetaRII and may indicate a mechanism whereby gonadotrophins and growth factor(s) regulate preantral folliculogenesis.

Prolactin gene expression and prolactin protein in premenopausal and postmenopausal human ovaries

Objective: To investigate intraovarian prolactin and prolactin-receptor gene expression and to assess local prolactin synthesis with emphasis on possible differences between premenopausal and postmenopausal status. Design: The RNA extracted from human premenopausal and postmenopausal tissues was subjected to reverse transcription and polymerase chain reaction by using prolactin-specific intron- and exon-spanning primers. Prolactin-receptor expression was investigated accordingly. The amplified complementary DNA fragments were analyzed by gel electrophoresis and restriction enzyme mapping. Local prolactin hormone synthesis was verified by a time-resolved immunofluorometric assay based on our monoclonal antibodies. Result(s): Prolactin and prolactin-receptor gene expression was observed in all analyzed human ovaries ( n = 18). Several other human tissue specimens, such as lung and kidney, served as negative control tissues. Significantly elevated concentrations of prolactin were detected in cytosolic extracts of premenopausal ( nf = 6; mean ± SD; 20.6 ± 3.3 ng/g tissue wet weight) versus postmenopausal ( n = 6; 3.6 ± 3.0 ng/g tissue wet weight) ovaries. Conclusion(s): The human ovary not only serves as a target for endocrine prolactin action but also as a site of local prolactin hormone production. In agreement with previous reports on extrapituitary sources of prolactin, we consider prolactin as a hormone as well as an autocrine or paracrine growth or regulatory factor. Significantly increased concentrations of prolactin in premenopausal ovarian tissue verifies its role in human reproduction.

Proteinhormone als lokal wirksame Regulatoren in humanen Ovarien*

Das klassische Konzept der endokrin regulierten Ovarfunktion wurde in letzter Zeit durch zahlreiche Berichte über lokal wirksame Faktoren deutlich erweitert. Da für die humanen Proteinhormone Prolaktin (PRL) und Wachstumshormon (GH), deren endokrine Effekte auf die Ovarialfunktion gut beschrieben sind, bereits in anderen Geweben ein lokaler Wirkmechanismus beschrieben ist, war die Abklärung einer möglichen intraovariellen auto/parakinen Wirkung dieser Hormone Ziel dieser Arbeit. Durch Reverse-Transkriptase-Poly-merase-Ketten-Reaktion (RT-PCR) und nachfolgender Restriktionsendonukleasenanalyse der PCR-Produkte konnte in dieser Untersuchung eine eutrope Genexpression für Proteinhormone außerhalb der klassischen Produktionsorgane, Hypophyse und Plazenta, gezeigt werden. Mittels genspezifischer Oligonukleotidprimer wurde im Ovar die mRNA einerseits für verschiedene Genprodukte des GH/plazentaren Laktogen(PL)-Genclusters, andererseits für PRL nachgewiesen. Hierbei zeigte sich, daß in humanen Ovarien diese Gene nicht nach dem Muster der Hypophyse oder der Plazenta transkribiert werden, sondern, daß für dieses Organ ein eigenes, unterschiedliches Expressionsmuster des GH/PL-Genclusters besteht. So konnten neben PL-A/B zusätzlich noch GH-N-Transkripte detektiert werden. Da diese Proteinhormone in viel geringerem Ausmaß als in den klassischen Produktionsorganen gebildet werden, postulieren wir eine lokale Wirkung im Ovar über autokrine/parakrine Mechanismen, wobei die wesentlichsten Effekte über eine Regulation und Modulation der Gonadotropinwirkung bzw. deren Rezeptoren vermittelt sein dürften.

Selective Growth Hormone/Placental Lactogen Gene Transcription and Hormone Production in Pre- and Postmenopausal Human Ovaries

Selective Growth Hormone/Placental Lactogen Gene Transcription and Hormone Production in Pre- and Postmenopausal Human Ovaries

Selective Growth Hormone/Placental Lactogen Gene Transcription and Hormone Production in Pre- and Postmenopausal Human Ovaries1

In addition to effects of pituitary-derived gonadotropins, human GH modulates and regulates intraovarian reproductive processes in a dose-dependent manner via the endocrine GHRH/GH/insulin-like-growth-factor I (IGF-I) axis. Based on increasing evidence that ovarian regulation involves a complex system of putative para/autocrine factors, we investigated the possibility of gene-selective intraovarian GH/placental lactogen (PL) hormone production, with emphasis on differences between pre- and postmenopause. Analysis of both premenopausal (n = 8) and postmenopausal (n = 10) ovarian-derived messenger ribonucleic acid by reverse transcription-PCR, which amplifies all major gene products of the five-member GH/PL gene cluster GH-N, GH-V, PL-A/B, and PL-L, revealed specific transcripts in all specimens. Their share in gene selective expression by analytical restriction enzyme digestion was determined. The expression pattern of GH/PL messenger ribonucleic acid shows PL-A/B > GH-N, which sets it apart from those of pituitary and placenta. Local production of the respective protein hormones was verified by two time-resolved immunofluorometric assays for human PL-A/B and GH-N; significant amounts of these hormones were detected in cytosolic extracts of premenopausal (n = 6; 555.5 +/- 171 ng PL-A/B and 0.8 +/- 0.6 ng GH-N/g tissue wet wt) and postmenopausal (n = 6; 5.2 +/- 2.7 ng PL-A/B and 0.9 +/- 0.6 ng GH-N/g tissue wet wt) ovaries. No difference was observed between pre- and postmenopausal ovarian GH-N contents, but PL values were 2-3 orders of magnitude lower in postmenopausal tissue (P < 0.001). Serum levels of healthy premenopausal (n = 21) and postmenopausal (n = 16) women were less than 0.02 ng PL/mL. In summary, ovarian-derived GH-N and PL-A/B synthesis correlates well with the established local cascade of GHRH, GHRH receptor, GH receptor, IGF-I, and IGF-I receptor as a putative para/autocrine regulator of ovarian reproductive function.

Expression of Aromatase Cytochrome P-450 in Premenopausal and Postmenopausal Human Ovaries: an Immunocytochemical Study*

The cellular distribution of the aromatase cytochrome P-450 enzyme in human ovaries has been investigated immunocytochemically, using an aromatase-specific monoclonal antibody. Ovaries of females ranging in age from prepubertal infant girl through to postmenopausal adulthood were obtained from immediate autopsy or after surgery. The results have revealed temporal and spatial changes in expression of aromatase at different stages of development. No immunoreactive aromatase was detected in the ovary of the 2.5 month infant. In premenopausal ovaries, aromatase was absent from the stromal compartment, but in follicles, a consistent pattern in expression of aromatase was observed, related to their size and developmental stage. Aromatase was not expressed in primordial, primary, or small secondary follicles less than 250 microns diameter. In slightly larger follicles (250-700 microns diameter) aromatase was first detected in a few thecal cells (TC). In more developed secondary through to large preovulatory follicles (greater than 1 cm) TC aromatase immunostain increased in intensity and number of positive cells, and the reaction was localized to a band of theca interna (TI) cells at the TI/theca externa interface. In granulosa cells (GC), aromatase was first detected in follicles in the initial stages of antrum formation (greater than 700 microns), and staining intensified as follicle diameter and antral cavity increased, being maximal in preovulatory follicles. GC aromatase was always found in the presence of TI immunostain. These two cell populations were separated by an unstained layer of TI cells giving the follicle walls a banded appearance. Immunostain was most intense in mural GC, was weaker in antral GC cells and was absent from the cumulus GC. Immunoreactive aromatase was also detected in functional corpora lutea (CL) but was absent from involuting CL's and corpora albicans. Our findings indicate that the immunostained cells of the CL are comprised of the former GC and possibly a subpopulation of former TI cells. In perimenopausal ovaries there was no evidence of any follicular or stromal aromatase immunostain. In postmenopausal ovaries no follicles were observed, but individual cells and clusters of cells in the stromal compartment of 3/7 specimens were found to have an aromatase immunostain reaction. In all cases, the aromatase immunostain reaction was cytoplasmic. The results provide the first direct evidence of the existence of TC aromatase, and of stromal cell aromatase in postmenopausal women.

Single-stranded regions in DNA from pre- and postmenopausal human ovaries

No differences were noted between the percent (2–4%) of single-stranded regions in DNA isolated from pre- and postmenopausal human ovaries as determined by nuclease S 1 digestion.