Tubulin Complexity in Cancer and Metastasis.

Tubulin plays a fundamental role in cellular function and as the subject for microtubule-active agents in the treatment of ovarian cancer. Microtubule-binding proteins (e.g., tau, MAP1/2/4, EB1, CLIP, TOG, survivin, stathmin) and posttranslational modifications (e.g., tyrosination, deglutamylation, acetylation, glycation, phosphorylation, polyamination) further diversify tubulin functionality and may permit additional opportunities to understand microtubule behavior in disease and to develop microtubule-modifying approaches to combat ovarian cancer. Tubulin-based structures that project from suspended ovarian cancer cells known as microtentacles may contribute to metastatic potential of ovarian cancer cells and could represent an exciting novel therapeutic target.

Microtubule-Targeting Agents: Disruption of the Cellular Cytoskeleton as a Backbone of Ovarian Cancer Therapy.

Microtubules are dynamic polymers composed of α- and ß-tubulin heterodimers. Microtubules are universally conserved among eukaryotes and participate in nearly every cellular process, including intracellular trafficking, replication, polarity, cytoskeletal shape, and motility. Due to their fundamental role in mitosis, they represent a classic target of anti-cancer therapy. Microtubule-stabilizing agents currently constitute a component of the most effective regimens for ovarian cancer therapy in both primary and recurrent settings. Unfortunately, the development of resistance continues to present a therapeutic challenge. An understanding of the underlying mechanisms of resistance to microtubule-active agents may facilitate the development of novel and improved approaches to this disease.

Haploidy in somatic cells is induced by mature oocytes in mice.

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

Insulin-like growth factor 2 is produced by antral follicles and promotes preantral follicle development in macaques†.

Insulin-like growth factors (IGFs) are known for their involvement in endocrine and paracrine regulation of ovarian function. Although IGF2 is the predominant circulating and intraovarian form of IGFs in primate species, the stage-specific follicular expression, action, and regulation of IGF2 are not well defined. Therefore, experiments were conducted to investigate the follicular IGF production in response to steroid hormone regulation and the direct IGF actions on follicular development and function in vitro. Preantral follicles were isolated from rhesus macaque ovaries and cultured to the antral stage in media supplemented with follicle-stimulating hormone and insulin. Follicles were randomly assigned to treatment groups: (a) control, (b) trilostane (a steroid synthesis inhibitor), (c) trilostane + estradiol, (d) trilostane + progesterone, and (e) trilostane + dihydrotestosterone. Media was analyzed for IGF concentrations, which were correlated to follicle growth. Follicles produced IGF2, but not IGF1, at the antral stage. Steroid depletion decreased, whereas steroid replacement increased, IGF2 production by antral follicles. Media IGF2 levels correlated positively with antral follicle diameters. Macaque preantral follicles and granulosa cells were subsequently cultured without (control) and with recombinant human IGF2 supplementation. Follicle survival, growth, and paracrine factor production, as well as granulosa cell proliferation and gonadotropin receptor gene expression, were assessed. IGF2 addition increased follicle survival rates, diameters and inhibin B production, as well as granulosa cell proliferation. These data demonstrate that IGF2 produced by antral follicles, in response to steroid hormone regulation, could act as a paracrine factor that positively impacts preantral follicle development and function in primates.

Association between vitamin D and ovarian cancer development in BRCA1 mutation carriers.

OBJECTIVE: Women with inherited mutations in BRCA1 gene have a high (40-70%) genetic risk of developing ovarian cancer. Epidemiological studies suggest an inverse correlation between serum vitamin D (VD) levels and the risk of ovarian cancer, but there is a lack of data from BRCA1 mutation (BRCA1 mut) carriers. Therefore, we investigated VD levels and actions in cancer free women with BRCA1 mutations. MATERIALS AND METHODS: Blood, ovary and fallopian tube samples were collected from healthy pre-menopausal women with BRCA1 mut and without BRCA1 mutations (BRCA wt). Serum calcifediol (major circulating form of VD) concentrations were measured by electrochemiluminescence immunoassay. Immunohistochemistry was performed on paraffin-embedded ovarian and fallopian tube sections to determine vitamin D receptor (VDR) expression. Ovarian surface epithelial cells (OSEs) from BRCA1 mut carriers were cultured with or without calcitriol supplementation for 72 hrs. VDR protein levels, cell proliferation and cell viability were analyzed. RESULTS: BRCA1 mut women had lower serum calcifediol levels compared to BRCA wt women (p = 0.003). VDR protein expression was evident in ovarian and the fallopian tube epithelium of BRCA wt patients, but was reduced in BRCA1 mut women. Calcitriol (biologically active VD) supplementation elevated VDR expression in cultured BRCA1 mut OSEs (p = 0.005) and decreased cell proliferation rates in a dose-dependent manner without inducing apoptosis. CONCLUSIONS: VD biosynthesis and signaling via VDR in the ovarian and fallopian tube epithelium are impaired in BRCA1 mut women. VD treatment may limit BRCA1 mut epithelial cell proliferation without affecting cell viability, providing a rationale for exploring the potential for VD in ovarian cancer prevention in BRCA1 mut carriers.

The dilemma of early preventive oophorectomy in familial small cell carcinoma of the ovary of hypercalcemic type.

•New pathogenic familial SMARCA4 variant, c.3081+1G>T.•Prophylactic surgery in healthy carrier of germline SMARCA4 mutation.•Long term hormone therapy in a 13-year-old girl.

Vitamin D3 Regulates Follicular Development and Intrafollicular Vitamin D Biosynthesis and Signaling in the Primate Ovary.

There is an increasing recognition that vitamin D plays important roles in female reproduction. Recent studies demonstrated that 1α,25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, improved ovarian follicle survival and growth in vitro. Therefore, we investigated the direct effects of VD3 at the specific preantral and antral stages of follicular development, and tested the hypothesis that vitamin D receptor (VDR) and enzymes critical for vitamin D biosynthesis are expressed in the primate ovary. Fourteen adult rhesus macaques provided ovarian tissue. Secondary and antral follicles were isolated for PCR analysis on VDR, vitamin D3 25-hydroxylase, and 25-hydroxyvitamin D3-1α-hydroxylase. VDR protein localization was determined by immunohistochemistry on ovarian sections. Isolated secondary follicles were cultured under conditions of control and VD3 supplementation during the preantral or antral stage. Follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, as well as oocyte maturation were evaluated. In vivo- and in vitro-developed follicles were also assessed for genes that are critical for vitamin D biosynthesis and signaling, gonadotropin signaling, steroid and paracrine factor production, and oocyte quality. The mRNA encoding VDR, 25-hydroxylase, and 1α-hydroxylase was detectable in in vivo- and in vitro-developed preantral and antral follicles. The 25-hydroxylase was elevated in cultured follicles relative to in vivo-developed follicles, which further increased following VD3 exposure. VD3 treatment increased 1α-hydroxylase in in vitro-developed antral follicles. The absence of VD3 during culture decreased VDR expression in in vitro-developed antral follicles, which was restored to levels comparable to those of in vivo-developed antral follicles by VD3 supplementation. Positive immunostaining for VDR was detected in the nucleus and cytoplasm of granulosa cells and oocytes. While only survival was improved in preantral follicles treated with VD3, VD3 supplementation promoted both survival and growth of antral follicles with increased estradiol and AMH production, as well as oocyte maturation. Thus, Vitamin D biosynthesis and signaling systems are expressed in primate ovarian follicles. Our findings support a role for VD3 in regulating follicular development in a stage-dependent manner, as well as the intrafollicular vitamin D biosynthesis and signaling, directly in the ovary.

Anti-Müllerian hormone is a survival factor and promotes the growth of rhesus macaque preantral follicles during matrix-free culture.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector + recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.

Subcutaneous ovarian tissue transplantation in nonhuman primates: duration of endocrine function and normalcy of subsequent offspring as demonstrated by reproductive competence, oocyte production, and telomere length.

PURPOSE: The main purposes of the study were to investigate the endocrine function of ovarian tissue transplanted to heterotopic subcutaneous sites and the reproductive competence and telomere length of a nonhuman primate originating from transplanted tissue. METHODS: Ovarian cortex pieces were transplanted into the original rhesus macaques in the arm subcutaneously, in the abdomen next to muscles, or in the kidney. Serum estradiol (E2) and progesterone (P4) concentrations were measured weekly for up to 8 years following tissue transplantation. A monkey derived from an oocyte in transplanted ovarian tissue entered time-mated breeding and underwent controlled ovarian stimulation. Pregnancy and offspring were evaluated. Telomere lengths and oocytes obtained following controlled ovarian stimulation were assessed. RESULTS: Monkeys with transplants in the arm and abdomen had cyclic E2 of 100 pg/ml, while an animal with arm transplants had E2 of 50 pg/ml. One monkey with transplants in the abdomen and kidney had ovulatory cycles for 3 years. A monkey derived from an oocyte in transplanted tissue conceived and had a normal gestation until intrapartum fetal demise. She conceived again and delivered a healthy offspring at term. Controlled ovarian stimulations of this monkey yielded mature oocytes comparable to controls. Her telomere length was long relative to controls. CONCLUSIONS: Heterotopic ovarian tissue transplants yielded long-term endocrine function in macaques. A monkey derived from an oocyte in transplanted tissue was reproductively competent. Her telomere length did not show epigenetically induced premature cellular aging. Ovarian tissue transplantation to heterotopic sites for fertility preservation should move forward cautiously, yet optimistically.

Hypoxia-related microRNA-210 is a diagnostic marker for discriminating osteoblastoma and osteosarcoma.

Osteoblastoma is a benign bone tumor that can often be difficult to distinguish from malignant osteosarcoma. Because misdiagnosis can result in unfavorable clinical outcomes, we have investigated microRNAs as potential diagnostic biomarkers for distinguishing between these two tumor types. Next generation RNA sequencing was used as an expression screen to evaluate >2,000 microRNAs present in tissue derived from rare formalin fixed paraffin embedded (FFPE) archival tumor specimens. MicroRNAs displaying the greatest ability to discriminate between these two tumors were validated on an independent tumor set, using qPCR assays. Initial screening by RNA-seq identified four microRNA biomarker candidates. Expression of three miRNAs (miR-451a, miR-144-3p, miR-486-5p) was higher in osteoblastoma, while the miR-210 was elevated in osteosarcoma. Validation of these microRNAs on an independent data set of 22 tumor specimens by qPCR revealed that miR-210 is the most discriminating marker. This microRNA displays low levels of expression across all of the osteoblastoma specimens and robust expression in the majority of the osteosarcoma specimens. Application of these biomarkers to a clinical test case showed that these microRNA biomarkers permit re-classification of a misdiagnosed FFPE tumor sample from osteoblastoma to osteosarcoma. Our findings establish that the hypoxia-related miR-210 is a discriminatory marker that distinguishes between osteoblastoma and osteosarcoma. This discovery provides a complementary molecular approach to support pathological classification of two diagnostically challenging musculoskeletal tumors. Because miR-210 is linked to the cellular hypoxia response, its detection may be linked to well-established pro-angiogenic and metastatic roles of hypoxia in osteosarcomas and other tumor cell types. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1137-1146, 2017.

Anti-Müllerian hormone is produced heterogeneously in primate preantral follicles and is a potential biomarker for follicle growth and oocyte maturation in vitro.

PURPOSE: The main goals of this study were to investigate the expression of anti-Müllerian hormone (AMH) and its receptor (AMHR2) during follicular development in primates, and to evaluate the potential of AMH as a biomarker for follicle growth and oocyte maturation in vitro. METHODS: The mRNA and protein expression of AMH and AMHR2 were determined using isolated follicles and ovarian sections from rhesus macaques (n = 4) by real-time PCR and immunohistochemistry, respectively. Isolated secondary follicles were cultured individually. Follicle growth and media AMH concentrations were assessed by ELISA. The mRNA expression profiles, obtained from RNA sequencing, of in vitro- and in vivo-developed antral follicles were compared. Secondary follicles from additional animals (n = 35) were cultured. Follicle growth, oocyte maturation, and media AMH concentrations were evaluated for forecasting follicular development in vitro by AMH levels. RESULTS: AMH immunostaining was heterogeneous in the population of preantral follicles that were also stained for AMHR2. The mRNA expression profiles were comparable between in vivo- and in vitro-developed follicles. AMH levels produced by growing follicles were higher than those of nongrowing follicles in culture. With a cutoff value of 1.40 ng/ml, 85 % of nongrowing follicles could be identified while eliminating only 5 % of growing follicles. Growing follicles that generated metaphase II-stage oocytes secreted greater amounts of AMH than did those yielding immature germinal vesicle-stage oocytes. CONCLUSIONS: AMH, co-expressed with AMHR2, was produced heterogeneously by preantral follicles in macaques with levels correlated positively with follicle growth and oocyte maturation. AMH may serve as a biomarker for primate follicular development in vitro.

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2.

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Dynamics of Immune Cell Types Within the Macaque Corpus Luteum During the Menstrual Cycle: Role of Progesterone.

The goal of the current study was to characterize the immune cell types within the primate corpus luteum (CL). Luteal tissue was collected from rhesus females at discrete intervals during the luteal phase of the natural menstrual cycle. Dispersed cells were incubated with fluorescently labeled antibodies specific for the immune cell surface proteins CD11b (neutrophils and monocytes/macrophages), CD14 (monocytes/macrophages), CD16 (natural killer [NK] cells), CD20 (B-lymphocytes), and CD3epsilon (T-lymphocytes) for analysis by flow cytometry. Numbers of CD11b-positive (CD11b(+)) and CD14(+) cells increased significantly 3 to 4 days after serum progesterone (P4) concentrations declined below 0.3 ng/ml. CD16(+) cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases and were 3-fold increased 3 to 4 days after serum P4 decreased to baseline levels. CD3epsilon(+) cells tended to increase 3 to 4 days after P4 decline. To determine whether immune cells were upregulated by the loss of luteotropic (LH) support or through loss of LH-dependent steroid milieu, monkeys were assigned to 4 groups: control (no treatment), the GnRH antagonist Antide, Antide plus synthetic progestin (R5020), or Antide plus the estrogen receptor agonists diarylpropionitrile (DPN)/propyl-pyrazole-triol (PPT) during the mid-late luteal phase. Antide treatment increased the numbers of CD11b(+) and CD14(+) cells, whereas progestin, but not estrogen, replacement suppressed the numbers of CD11b(+), CD14(+), and CD16(+) cells. Neither Antide nor steroid replacement altered numbers of CD3epsilon(+) cells. These data suggest that increased numbers of innate immune cells in primate CL after P4 synthesis declines play a role in onset of structural regression of primate CL.

SWI/SNF-Mediated Lineage Determination in Mesenchymal Stem Cells Confers Resistance to Osteoporosis.

Redirecting the adipogenic potential of bone marrow-derived mesenchymal stem cells to other lineages, particularly osteoblasts, is a key goal in regenerative medicine. Controlling lineage selection through chromatin remodeling complexes such as SWI/SNF, which act coordinately to establish new patterns of gene expression, would be a desirable intervention point, but the requirement for the complex in essentially every lineage pathway has generally precluded selectivity. However, a novel approach now appears possible by targeting the subset of SWI/SNF powered by the alternative ATPase, mammalian brahma (BRM). BRM is not required for development, which has hindered understanding of its contributions, but knockdown genetics here, designed to explore the hypothesis that BRM-SWI/SNF has different regulatory roles in different mesenchymal stem cell lineages, shows that depleting BRM from mesenchymal stem cells has a dramatic effect on the balance of lineage selection between osteoblasts and adipocytes. BRM depletion enhances the proportion of cells expressing markers of osteoblast precursors at the expense of cells able to differentiate along the adipocyte lineage. This effect is evident in primary bone marrow stromal cells as well as in established cell culture models. The altered precursor balance has major physiological significance, which becomes apparent as protection against age-related osteoporosis and as reduced bone marrow adiposity in adult BRM-null mice.

Quantification of dynamic changes to blood volume and vascular flow in the primate corpus luteum during the menstrual cycle.

BACKGROUND: The objective of the current study was to determine changes to vascular parameters of nonhuman primate dominant ovarian structures by dynamic contrast-enhanced ultrasound (DCE-US). MATERIALS AND METHODS: Dynamic contrast-enhanced ultrasound with intravenous microbubble infusion was performed on the rhesus macaque ovary bearing the pre-ovulatory follicle and corpus luteum (CL) sequentially during the natural luteal phase (n = 8) and GnRH antagonist (antide)-induced luteal regression (n = 6). RESULTS: Changes in luteal blood volume (BV) and vascular flow (VF) were observed between stages of the luteal phase Luteal BV was highest in early stage CL, before decreasing 2.5-fold in late stage CL (P < 0.06); in contrast, luteal VF peaked at mid luteal stage (P < 0.01). Two females identified with luteal insufficiency trended toward lower peak BV, compared to typical CLs. Another female was identified with a luteal cyst on the contralateral ovary, and a CL that regressed before P levels declined. After 72 hours of antide exposure, BV was reduced 2.3-fold (P = 0.03). CONCLUSIONS: DCE-US provides a sensitive, non-invasive measurement of the dynamics of blood volume and flow in dominant ovarian structures.

Endocrine and local control of the primate corpus luteum.

The primate corpus luteum is a transient endocrine gland that differentiates from the ovulatory follicle midway through the ovarian (menstrual) cycle. Its formation and limited lifespan is critical for fertility, as luteal-derived progesterone is the essential steroid hormone required for embryo implantation and maintenance of intra-uterine pregnancy until the placenta develops. It is well-established that LH and the LH-like hormone, CG, are the vital luteotropic hormones during the menstrual cycle and early pregnancy, respectively. Recent advances, particularly through genome analyses and cellular studies, increased our understanding of various local factors and cellular processes associated with the development, maintenance and repression of the corpus luteum. These include paracrine or autocrine factors associated with angiogenesis (e.g., VEGF), and that mediate LH/CG actions (e.g., progesterone), or counteract luteotropic effects (i.e., local luteolysis; e.g., PGF2α). However, areas of mystery and controversy remain, particularly regarding the signals and events that initiate luteal regression in the non-fecund cycle. Novel approaches capable of gene "knockdown" or amplification", in vivo as well as in vitro, should identify novel or underappreciated gene products that are regulated by or modulate LH/CG actions to control the functional lifespan of the primate corpus luteum. Further advances in our understanding of luteal physiology will help to improve or control fertility for purposes ranging from preservation of endangered primate species to designing novel ovary-based contraceptives and treating ovarian disorders in women.

Cooperative activation of tissue-specific genes by pRB and E2F1.

The retinoblastoma tumor suppressor protein pRB is conventionally regarded as an inhibitor of the E2F family of transcription factors. Conversely, pRB is also recognized as an activator of tissue-specific gene expression along various lineages including osteoblastogenesis. During osteoblast differentiation, pRB directly targets Alpl and Bglap, which encode the major markers of osteogenesis alkaline phosphatase and osteocalcin. Surprisingly, p130 and repressor E2Fs were recently found to cooccupy and repress Alpl and Bglap in proliferating osteoblast precursors before differentiation. This raises the further question of whether these genes convert to E2F activation targets when differentiation begins, which would constitute a remarkable situation wherein pRB and E2F would be cotargeting genes for activation. Chromatin immunoprecipitation analysis in an osteoblast differentiation model shows that Alpl and Bglap are indeed targeted by an activator E2F, i.e., is E2F1. Promoter occupation of Alpl and Bglap by E2F1 occurs specifically during activation, and depletion of E2F1 severely impairs their induction. Mechanistically, promoter occupation by E2F1 and pRB is mutually dependent, and without this cooperative effect, activation steps previously shown to be dependent on pRB, including recruitment of RNA polymerase II, are impaired. Myocyte- and adipocyte-specific genes are also cotargeted by E2F1 and pRB during differentiation along their respective lineages. The finding that pRB and E2F1 cooperate to activate expression of tissue-specific genes is a paradigm distinct from the classical concept of pRB as an inhibitor of E2F1, but is consistent with the observed roles of these proteins in physiological models.

Dynamics of the transcriptome in the primate ovulatory follicle.

Experiments were designed to evaluate changes in the transcriptome (mRNA levels) in the ovulatory, luteinizing follicle of rhesus monkeys, using a controlled ovulation model that permits analysis of the naturally selected, dominant follicle at specific intervals (0, 12, 24 and 36 h) after exposure to an ovulatory (exogenous hCG) stimulus during the menstrual cycle. Total RNA was prepared from individual follicles (n= 4-8/timepoint), with an aliquot used for microarray analysis (Affymetrix Rhesus Macaque Genome Array) and the remainder applied to quantitative real-time PCR (q-PCR) assays. The microarray data from individual samples distinctly clustered according to timepoints, and ovulated follicles displayed markedly different expression patterns from unruptured follicles at 36 h. Between timepoint comparisons revealed profound changes in mRNA expression profiles. The dynamic pattern of mRNA expression for steroidogenic enzymes (CYP17A, CYP19A, HSD3B2, HSD11B1 and HSD11B2), steroidogenic acute regulatory protein (StAR) and gonadotrophin receptors [LH/choriogonadotrophin receptor (LHCGR), FSH receptor (FSHR)] as determined by microarray analysis correlated precisely with those from blinded q-PCR assays. Patterns of mRNA expression for epidermal-growth-factor-like factors (amphiregulin, epiregulin) and processes [hyaluronan synthase 2 (HAS2), tumor necrosis factor alpha-induced protein 6 (TNFAIP6)] implicated in cumulus-oocyte maturation/expansion were also comparable between assays. Thus, several mRNAs displayed the expected expression pattern for purported theca (e.g. CYP17A), granulosa (CYP19A, FSHR), cumulus (HAS2, TNFAIP6) cell and surface epithelium (HSD11B)-related genes in the rodent/primate pre-ovulatory follicle. This database will be of great value in analyzing molecular and cellular pathways associated with periovulatory events in the primate follicle (e.g. follicle rupture, luteinization, inflammatory response and angiogenesis), and for identifying novel gene products controlling mammalian fertility.

Expression of mRNA and proteins for GnRH I and II and their receptors in primate corpus luteum during menstrual cycle.

The differential expression of mRNA and protein of GnRH I, II and their receptors (RI and RII) in the monkey corpus luteum (CL) were measured during different stages of the luteal phase of the menstrual cycle as an initial step towards considering the role and regulation of GnRH (I and II) system during luteinization and luteolysis in primates. RT-PCR confirmed the sequence identity of PCR products and real time PCR quantified specific mRNA expressions. Proteins were localized by immunohistochemistry (IHC). Changes in mRNA expression patterns of GnRH I and II (increased) and GnRH RII (decreased) were maximal at mid-late to late stages, that is, at CL regression, where as GnRH RI was low during the entire luteal phase. However, RT-PCR and IHC studies confirmed the presence of GnRH RI at both mRNA and protein levels, respectively. IHC results showed the presence of GnRH I, II and their receptors in steroidogenic cells (granulose-luteal cells and thecal-luteal cells) across the luteal phase. Hence, GnRH I and II systems may have a role on both luteinization (from early to mid stages of CL) and luteolysis (from mid-late to very-late stages of CL). These novel findings suggest that monkey luteal GnRH system may have a role in fertility regulation in paracrine and/or autocrine manner.

Cooperative control via lymphoid enhancer factor 1/T cell factor 3 and estrogen receptor-alpha for uterine gene regulation by estrogen.

Accumulating evidence indicates that estrogen regulates diverse but interdependent signaling pathways via estrogen receptor (ER)-dependent and -independent mechanisms. However, molecular relationship between these pathways for gene regulation under the direction of estrogen remains unknown. To address this possibility, our uterine analysis of Wnt/beta-catenin downstream effectors revealed that lymphoid enhancer factor 1 (Lef-1) and T cell factor 3 (Tcf-3) are up-regulated temporally by 17beta-estradiol (E2) in an ER-independent manner. Lef-1 is abundantly up-regulated early (within 2 h), whereas Tcf-3 is predominantly induced after 6 h, and both are sustained through 24 h. Interestingly, activated Lef-1/Tcf-3 molecularly interacted with ERalpha in a time-dependent manner, suggesting they possess a cross talk in the uterus by E2. Moreover, dual immunofluorescence studies confirm their colocalization in uterine epithelial cells after E2. Most importantly, using chromatin immunoprecipitation followed by PCR analyses, we provide evidence for an interesting possibility that ERalpha and Tcf-3/Lef-1 complex occupies at certain DNA regions of estrogen-responsive endogenous gene promoters in the mouse uterus. By selective perturbation of activated Lef-1/Tcf-3 or ERalpha signaling events, we provide in this study novel evidence that cooperative interactions, by these two different classes of transcription factors at the level of chromatin, direct uterine regulation of estrogen-responsive genes. Collectively, these studies support a mechanism that integration of a nonclassically induced beta-catenin/Lef-1/Tcf-3 signaling with ERalpha is necessary for estrogen-dependent endogenous gene regulation in uterine biology.