Molecular Human Reproduction - Advance Access

Detection of novel copy number variants in uterine leiomyomas using high-resolution SNP arrays

Bowden, W., Skorupski, J., Kovanci, E., Rajkovic, A. — 2009-06-30

Uterine leiomyomas (ULs) are benign monoclonal tumors originating from myometrial tissue in the uterus. Genetic pathways that lead to myometrial transformation into leiomyomas are largely unknown. Approximately 40% of ULs are karyotypically abnormal by G-banding, however the remaining 60% of leiomyomas do not contain cytogenetically visible genomic rearrangements. Recent technological advances such as array based comparative genomic hybridization (array CGH) and dense single nucleotide polymorphism (SNP) arrays have enabled genome-wide scanning for genomic rearrangements missed by karyotype banding analysis. In the current study, we employed a high resolution SNP microarray on 16 randomly selected ULs and normal myometrium samples to detect submicroscopic (<5Mb) chromosomal aberrations. The SNP array identified gene dosage changes in 56% of the fibroids (9/16), 25% of which (4/16) had aberrations greater than 5 Mb, while 31% of which (5/16) contained only submicroscopic copy number changes (< 5 Mb). We corroborated 3/5 submicroscopic changes using quantitative PCR, meaning that ultimately, 19% of our samples (3/16) were found to contain only submicroscopic changes. Novel submicroscopic aberrations on chromosomal segments 1q42.13, 11q13.1 and 13q12.13 and large, previously unreported deletions on 15q11.2-q23, 17p-q21.31, and 22q12.2-q12.3 were identified. Previously reported deletions on 1p, 3q, 7q, 13, and chromosome 14q were also noted. RHOU, MAP3K11 and WASF3 gene copy numbers were changed in the subset of leiomyomas with submicroscopic aberrations, and these genes have previously been implicated in tumorigenesis. Our findings support the hypothesis that a significant fraction of ULs without visible cytogenetic changes harbor submicroscopic genomic rearrangements which may in turn contribute to transformation of normal myometrial tissue into leiomyomas.

New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and postmeiotic differentiation of testicular germ cells

2009-06-27

Culture and differentiation of male germ cells has been performed for various purposes in the past. To date, none of the studies aimed at in-vitro spermatogenesis have resulted in a sufficient number of mature gametes. Numerous studies have revealed worthy pieces of information, building up a body of information on conditions that are required to maintain and mature male germ cells in-vitro. In this review, we report on previously published and unpublished experiments addressing murine germ cell differentiation in three-dimensional in-vitro culture systems. In a systematic set of experiments, we examined the influence of two different matrices (soft agar and methylcellulose) as well as the need for gonadotropin support. For the first time, we demonstrate that pre-meiotic male germ cells (revealed by the absence of meiotic marker expression (e.g. Boule)) obtained from immature mice pass through meiosis in vitro. After several weeks of culture, we obtained morphologically normal spermatozoa embedded in the matrix substance. Complete maturation relied on support from somatic testicular cells and the presence of gonadotropins but appeared independent from the matrix in a three-dimensional culture environment. Further research efforts are required to reveal the applicability of this culture technique for human germ cells and the functionality of the spermatozoa for generating offspring.

Quantification of oocyte-specific transcripts in follicle-enclosed oocytes during antral development and maturation in vitro

Sanchez, F., Adriaenssens, T., Romero, S., Smitz, J. — 2009-06-24

Oocyte cytoplasmic maturation is influenced by the quantity of synthesized RNA and proteins accumulated and stored during growth. Transcriptional repression and degradation of transcripts occur during oocyte nuclear maturation and prolonged transcriptional arrest might compromise RNA stores for early development. RNA quantification of key genes in oocytes might be valuable when setting-up in vitro cultures that lack the normal hormonal interplay found in vivo.

This study quantitates gene expression levels in relation to follicle culture time and time of oocyte maturation in a mouse model. RNA levels of Gdf-9, Bmp-15, Mater, Zar-1, Npm-2 and Fgf-8 were measured in germinal vesicle (GV) oocytes along fixed times during in vitro follicle development. For all genes the highest mRNA levels were detected in oocytes in the preantral follicle stage. Antrum formation was associated with a progressive shutdown in transcription in extended in-vitro culture leading to lower mRNA values than those of control in vivo preovulatory oocytes. In contrast to in vitro matured oocytes, the in vivo oocytes from 22 and 29 day-old prepubertal animals obtained after pregnant mare's serum globulin and hCG priming did not downregulate transcripts upon maturation stimulus except for Mater.

These findings show that oocyte gene expression patterns under in vitro conditions can, at certain times, mimic what is reported to occur under in vivo conditions. Moreover, they also show that meiotically competent oocytes kept in a prolonged transcriptionally inactive stage express altered levels of key transcripts compared to in vivo in both immature and mature oocytes.

The role of centrosomes in mammalian fertilization and its significance for ICSI

Schatten, H., Sun, Q.-Y. — 2009-06-23

Centrosome integrity is critically important for successful fertilization and embryo development. In humans, the sperm contributes the dominant centrosomal material containing centrioles and centrosomal components onto which oocyte centrosomal proteins assemble after sperm incorporation to form the sperm aster that is essential for uniting sperm and oocyte pronuclei. Increasingly, dysfunctional sperm centrosomes have been identified as a factor for sperm-derived infertility and heterologous Intracytoplasmic Sperm Injection (ICSI) has been used to assess centrosome and sperm aster formation and clearly established a relationship between infertility and sperm centrosomal dysfunction. ICSI has been used successfully to provide novel treatment to overcome male factor infertility and it may open up new possibilities to correct specific sperm-related centrosome dysfunctions at molecular levels. New data indicate that it is now possible to replace dysfunctional centrosomes with functional donor sperm centrosomes which may provide new treatment for couples in which infertility is a result of centrosome-related sperm dysfunctions.

MATER PROTEIN AS SUBSTRATE OF PKC{epsilon} IN HUMAN CUMULUS CELLS

2009-06-20

High activity of the phosphoinositide (PI) 3-kinase/Akt pathway in cumulus cells plays an important role in FSH regulation of cell function and Protein Kinase C epsilon (PKC) collaborates with these signalling pathways to regulate cell proliferation. Relevant roles in follicular development are played by MATER (Maternal Antigen That Embryos Require) that is a cumulus cell- and oocyte-specific protein dependent on the maternal genome. We recently demonstrated that human MATER localizes at specific domains of oocytes and, for the first time, also in cumulus cells. MATER contains a carboxy-terminal leucine-rich repeat domain involved in protein-protein interactions regulating different cellular functions. Here we investigated the functional role of MATER. Thus, we performed coimmunoprecipitation experiments using HEK293T cells expressing human MATER; a similar approach was then followed in human cumulus/follicular cells. In Mater⁺HEK293T cells, we observed that this protein acts as a phosphorylation substrate of PKC. Western blot experiments indicate that, unlike oocytes, human cumulus cells express PKC. Immunoprecipitation and confocal analysis suggest for the first time that MATER protein interacts with this protein kinase, in cumulus cells under physiological conditions. Since PKC is known to collaborate with antiapoptotic signalling pathways this suggests a novel mechanism for the function of MATER in follicular maturation.

Inhibin alpha gene and susceptibility to premature ovarian failure: a data synthesis

Zintzaras, E. — 2009-06-19

Candidate-gene association studies that examined the association between polymorphisms of the inhibin alpha INHA gene (G769A, C16T and A124G) and premature ovarian failure (POF) have reported contradictory results. Thus, a meta-analysis of these studies was carried out. The random effects odds ratio (OR) with the corresponding 95% confidence interval and the heterogeneity among studies were estimated. Existence of potential bias and consistency of effect sizes across ethnicities were explored. Cumulative meta-analysis was also performed. The studies provided 1030/1660, 936/1398 and 938/1446 cases/controls for G769A, C16T and A124G polymorphisms, respectively. The meta-analysis showed significant heterogeneity among the studies (P_Q=0.01, I²=74%) and lack of evidence that carriers of the G769A variant confer risk of POF: OR=1.38 (0.48-3.94). Asian Indians (only two studies) produced significant association [OR=8.10 (1.27-51.6)]. Regarding C16T and A124G polymorphisms, 16T and 124G alleles were not associated with POF: OR=0.94 (0.76-1.16) and OR=0.98 (0.86-1.11), respectively. The cumulative meta-analysis for G769A and C16T polymorphisms showed a trend towards to non significance for both polymorphisms. Cumulative meta-analysis indicated that more evidence is needed to draw safer conclusions regarding the effect sizes. There was no differential magnitude of effect in large versus small studies. In conclusion, there is no evidence of association between the studied polymorphisms and POF.

Differential actions of estrogen and SERMs in regulation of the actin cytoskeleton of endometrial cells

2009-06-18

Estrogen and selective estrogen receptor modulators (SERMs) differentially impact endometrial cell function, however, the biological basis of these differences is not established. Deregulated cell adhesion to the extracellular matrix, cell movement and invasion are related to endometrial disorders, such as endometriosis or endometrial cancer. Remodeling of the actin cytoskeleton is required to achieve cell adhesion and movement. Estrogen receptor (ER) regulates actin and cell membrane remodeling through extra-nuclear signaling cascades. In this paper we show that administration of 17β-estradiol (E2) and tamoxifen (TAM) to immortalized Ishikawa endometrial cells or to human endometrial stromal cells (ESC) results in remodeling of actin fibres and cell membrane. This is linked to rapid phosphorylation on Thr⁵⁵⁸ of the actin-binding protein moesin and enhanced migration and invasion of normal and Ishikawa cells. Raloxifene (RAL) does not result in moesin activation or actin remodeling. When endometrial cells are exposed to E2 in the presence of TAM or RAL, both SERMs interfere with the recruitment of moesin, with the remodeling of the cytoskeleton, and with cell movement and migration induced by E2. The differential actions of E2, TAM and RAL are linked to a distinct modulation of the extra-nuclear signaling of ER to G proteins and to the Rho-associated kinase(ROCK). These findings increase our understanding of the actions of estrogen and SERMs in endometrial cells and highlight potential molecular targets to interfere with the estrogen-related altered cell adhesion encountered in endometrial disorders.

Changes in myometrial contractions in response to altered dimethylarginine dimethylaminohydrolase during gestation of the rat

Ito, E., Obayashi, S., Nagai, A., Imamura, M., Azuma, H. — 2009-06-15

There has been little information demonstrating the roles of dimethylarginine dimethylaminohydrolase (DDAH), which is the hydrolyzing enzyme of endogenous nitric oxide synthase (NOS) inhibitors and, in turn, modulates the intracellular concentrations of NOS inhibitors, in the myometrium during the course of pregnancy. Therefore, the present experiments were designed to investigate whether or not DDAH activity, protein and mRNA expression levels are altered during gestation of the rat and, if altered, those changes reflect on the levels of endogenous NOS inhibitors and endothelin-1 (ET-1), and NO-dependent cyclic GMP generation in the myometrium. The up-regulated changes in DDAH activity, DDAH-2 protein and DDAH-2 mRNA expression at mid-gestation were accompanied by the reduced monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) as NOS inhibitors, and endothelin-1 (ET-1) levels, and by the enhanced NO-dependent cyclic GMP production. At term gestation, on the other hand, down regulated changes in DDAH activity, DDAH-2 protein and DDAH-2 mRNA expression were accompanied by the increased NOS inhibitors and ET-1 levels, and decreased NO-dependent cyclic GMP generation. These results suggest that alterations in DDAH/NOS inhibitors/NO-dependent cyclic GMP/ET-1 pathway are possibly involved in maintaining myometrial quiescence during gestation and controlling delivery at term.

Estrogen receptor {beta} gene mutations in Indian infertile men

2009-06-09

Recent studies suggest that estrogens play an important role in male fertility. Estrogen signaling is mediated by Estrogen Receptors (ER and ERβ). Association of ERβ with male infertility has not been analyzed to date except for genotyping of known polymorphisms in two different studies, which yielded controversial interpretation. Hence, we performed sequencing of all the exons and untranslated regions (UTRs) of ERβ gene in 300 infertile and 255 fertile control Indian men. We identified 8 novel mutations and 4 known single nucleotide polymorphisms (SNPs). Of the 8 novel mutations, 4 were nonsynonymous, of which 1 was detected only in infertile men, whereas the other 3 mutations were detected only in fertile men. Using different bioinformatic tools, we predicted that nonsynonymous mutations were benign and they neither altered the structure nor the function of the protein. Among synonymous novel mutations, 1 was detected in both fertile and infertile men, 2 were exclusive to infertile men and 1 was exclusive to fertile men. None of the known SNPs or novel mutations showed statistically significant difference between infertile and fertile men. Moreover, infertile men having ERβ mutations had normal reproductive tract and serum hormone levels. Our results suggest that the SNPs and mutations in ERβ gene are not a common cause of spermatogenesis failure in Indian men, although mutations specifically found in infertile men can affect transcription, translation or have synergic effect with other variants in causing infertility.

The genotype of the NK cell receptor, KIR2DL4, influences INF{gamma} secretion by decidual natural killer cells

2009-06-09

Natural killer (NK) cells are the predominant leukocyte in first trimester decidua and play a role in vascular remodelling through interferon gamma (IFN) secretion. Membrane expression of the killer immunoglobulin-like receptor (KIR) KIR2DL4 on peripheral blood NK (pNK) cells is controlled by the 9A/10A transmembrane genetic polymorphism. On peripheral NK cells (pNK), KIR2DL4 can only be detected on the membrane of cells from individuals with at least one copy of the 10A allele and ligation of KIR2DL4 results in IFN secretion. In this study, we assessed KIR2DL4 expression and IFN secretion as a result of KIR2DL4 ligation, by decidual NK cells (dNK). The 9A/10A transmembrane polymorphism was shown to control KIR2DL4 expression by dNK, as previously shown for pNK cells. Freshly isolated dNK cells from subjects with at least one 10A allele expressed KIR2DL4 whilst those from 9A homozygous subjects did not. Although freshly isolated dNK did not secrete IFN in response to KIR2DL4 ligation regardless of KIR2DL4 genotype, activation by in vitro culture with IL-2 enabled dNK cells from individuals with at least one 10A allele, but not those without a 10A allele, to secrete IFN in response to KIR2DL4 ligation. This study confirms that expression of KIR2DL4 by dNK is dependent on the 9A/10A polymorphism and that this polymorphism influences IFN secretion by dNK cells.

Induction of Endometrial Epithelial Cell Invasion and c-fms expression by Transforming Growth Factor Beta

2009-06-08

Transforming growth factor beta 1 (TGF-β1) levels are increased in the peritoneal fluid of endometriosis patients, and endometrial cells express TGF-β signaling components; however, little is known regarding the role of TGF-β in endometriosis. Our objective was to examine the effects of TGF-β1 on 1) the expression of macrophage colony-stimulating factor receptor encoded by the c-fms gene, 2) trans-mesothelial invasiveness of endometrial cells, 3) cellular proliferation and 4) attachment to peritoneal mesothelial cells (PMCs). Effects of TGF-β1 on c-fms mRNA expression were determined by real-time RT-PCR and c-fms cell surface expression by flow cytometry. Effects of TGF-β1 on the invasiveness of the immortalized endometrial epithelial cell (EEC) line EM42 and primary EECs were examined using a three-dimensional in vitro system modeling the peritoneum. Cellular proliferation and attachment to PMCs were also examined using established techniques. TGF-β1 had little or no effect on cellular proliferation and endometrial cell attachment to PMCs. TGF-β1 significantly induced the expression of c-fms mRNA and c-fms cell surface expression. TGF-β1 enhanced trans-mesothelial invasion by EM42 cells and EECs. Antagonists of TGF-β1 signaling significantly inhibited both the induction of c-fms expression and cellular invasiveness, suggesting that additional studies are warranted to assess the therapeutic potential of TGF-β antagonists in endometriosis.

The Pluripotency Transcription Factor Kruppel-like Factor 4 Is Strongly Expressed in Intratubular Germ Cell Neoplasia Unclassified (IGCNU) and Seminoma

2009-06-08

Germ cell tumors of the testis are the most frequent tumors in men between 20 and 40 years. Their most common subtype is the seminoma, which arises like the embryonal carcinoma from an intratubular germ cell neoplasia unclassified (IGCNU), i.e. fetal germ cells that escaped from the control of the developing testicular stem cell niche, eventually leading to a fully developed seminoma (or embryonal carcinoma). The molecular causes for the development of an IGCNU are still unknown. However, IGCNU cells share the expression of several factors with primordial germ cells and gonocytes and, interestingly, also with pluripotent embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. One factor playing important roles in both, iPS and ES cells is the transcription factor Krüppel-like factor 4 (KLF4). This study examined

KLF4 expression data from totalling 179 human testicular samples including normal controls and seminoma, deposited in Gene Expression Omnibus repository for microarray data at the National Centre for Biotechnology Information, were analysed. Immunohistochemistry was used to detect KLF4 protein expression in IGCNU (n=6), seminoma (n=14) and fetal human testes (n=14).

Microarray data from three independent sources suggest higher mRNA expression in seminoma than in normal testis. Normal spermatogonia, which are the stem cells of spermatogenesis, controlled by their stem cell niche, do not express KLF4. In contrast, IGCNU and seminoma cells strongly express KLF4.

In conclusion, this finding suggests that KLF4 may be an important factor for the maintenance of the developmental and the tumorigenic potential of IGCNU as well as for the malignancy of seminoma.

Dienogest, a synthetic progestin, inhibits the proliferation of immortalized human endometrial epithelial cells with suppression of cyclin D1 gene expression

2009-06-05

Dienogest is a specific progesterone receptor agonist with potent oral endometrial activity and is used in the treatment of endometriosis. In this study, we examined the direct effects of dienogest on the proliferation of human endometrial epithelial cells using an immortalized cell line. 5-Bromo-2’-deoxyuridine incorporation into the cells was inhibited by dienogest and by progesterone (P₄) in dose-dependent fashion at concentrations of 10^–8 mol/l or higher. To identify the target genes of dienogest and P₄, we screened the expression of 84 genes related to cell cycle regulation by real-time polymerase chain reaction after 6 h of treatment at a concentration of 10^–7 mol/l. Results showed that only cyclin D1 expression was significantly down-regulated, while expression of the other genes did not significantly change after dienogest or P₄ treatment compared with the control. In a time-course study during the first 24 h after drug treatment, dienogest and P₄ each produced a lasting decrease in the expression of cyclin D1 mRNA, followed by a decrease in cyclin E1 mRNA but not an increase in the expression of cell cycle inhibitor genes (p21, p27, and p53). These findings suggest that dienogest directly inhibits the proliferation of human endometrial epithelial cells with suppression of cyclin D1 gene expression.

Abnormal Methylation at the KvDMR1 Imprinting Control Region in Clinically Normal Children Conceived by Assisted Reproductive Technologies

Gomes, M.V., Huber, J., Ferriani, R.A., Amaral Neto, A.M., Ramos, E.S. — 2009-06-03

Genomic imprinting alterations have been shown to be associated to assisted reproductive technologies (ART) in animals. At present, data obtained in humans are inconclusive, however, some epidemiological studies have demonstrated an increased incidence of imprinting disorders in children conceived by ART. In the present study, we focused on the effect of ART (IVF and ICSI) on the epigenetic reprogramming of the maternally methylated imprinting control region KvDMR1 in clinically normal children. Qualitative and quantitative methylation at KvDMR1 were assessed by the methylation-specific PCR approach (MS-PCR) and by the methylation-sensitive enzymatic digestion associated to real-time PCR method (MSED-qPCR), respectively. DNA was obtained from peripheral blood of 12/18 and umbilical cord blood and placenta of 6/18 children conceived by IVF or ICSI. The methylation patterns observed in this group were compared with the patterns observed in 30 clinically normal naturally conceived children (negative controls) and in three naturally conceived BWS patients (positive controls). Hypomethylation at KvDMR1 was observed in 3/18 clinically normal children conceived by ART (2 conceived by IVF and 1 by ICSI). A discordant methylation pattern was observed in the three corresponding dizygotic twins. Our findings corroborate the hypothesis of vulnerability of maternal imprinting to ART. Furthermore, the discordant methylation at KvDMR1 observed between dizygotic twins could be consequent to one of the following possibilities: (i) a differential vulnerability of maternal imprints among different embryos; or (ii) epimutations that occurred during gametogenesis resulting in the production of oocytes without the correct primary imprint at KvDMR1.

GRP78 as a marker of preeclampsia: an exploratory study

2009-05-29

Although the exact mechanisms that lead to shallow invasion or defective trophoblastic differentiation in preeclampsia are still unknown, it is widely admitted that the etiology of preeclampsia is a defect in trophoblast invasion of the uterine spiral arteries. We have previously observed that the status of a chaperone protein, glucose regulated protein 78 (GRP78) is associated with the invasive properties of cytotrophoblastic cells, we therefore hypothesized that circulating GRP78 could serve as a diagnostic tool in preeclampsia. In a prospective case-control study, we quantified GRP78 autoantibodies, complexes of GRP78 with autoantibodies, and GRP78 (C-term fragment, N-term fragment, and full-length GRP78) by ELISA. Plasma from women diagnosed with preeclampsia (n=16), from women during the first trimester of pregnancy who subsequently developed preeclampsia (n=10) and from healthy pregnant women (controls, n=58 at term, n=26 at first trimester) were analyzed and compared. We observed no significant difference between preeclamptic and healthy pregnant women for autoantibodies-GRP78 complexes or total GRP78 at both first trimester and at delivery. In contrast, the ratio of C-terminal GRP78 over full length GRP78 was significantly different in plasma of preeclamptic patients as compared to controls both during first trimester (p<0.004) and at term (p<0.0001). Our findings suggest that circulating C-terminal GRP78 reflect the invasive properties of cells, and could be used as a predictive marker for preeclampsia early in pregnancy.

PDE8A Genetic Variation, Polycystic Ovary Syndrome and Androgen Levels in Women

2009-05-29

Polycystic ovary syndrome (PCOS) is characterized by excessive theca cell androgen secretion, dependent upon LH, which acts through the intermediacy of adenosine 3’, 5’-cyclic monophosphate (cAMP). cAMP signaling pathways are controlled through regulation of its synthesis by adenylyl cyclases, and cAMP degradation by phosphodiesterases (PDEs). PDE8A, a high-affinity cAMP-specific PDE is expressed in ovary and testis cells. Leydig cells from mice with a targeted mutation in the PDE8A gene are sensitized to the action of LH in terms of testosterone production. These observations led us to evaluate the human PDE8A gene as a PCOS candidate gene and the hypothesis that reduced PDE8A activity or expression would contribute to excessive ovarian androgen production. We identified a rare variant (R136Q; NM_002605.2 c.407 G>A) and studied another known single nucleotide polymorphism (SNP) (rs62019510, N401S) in the PDE8A coding sequence causing non-synonymous amino acid substitutions, and a new SNP in the promoter region (NT_010274.16:g.490155G>A). Although PDE8A kinetics were consistent with reduced activity in theca cell lysates, study of the expressed variants did not confirm reduced activity in cell-free assays. Cellular localization of the enzyme was also not different among the coding sequence variants. The PDE8A promoter SNP and a previously described promoter SNP did not affect promoter activity in vitro assays. The more common coding sequence SNP (N401S), and the promoter SNPs were not associated with PCOS in our transmission/disequilibrium test (TDT)-based analysis, nor where they associated with total testosterone or hydroepiandrosterone sulphate (DHEAS) levels. These findings exclude a significant role for PDE8A as a PCOS candidate gene and a major determinant of androgen levels in women.