Laura Diaz-Cueto

Functional Relationships between Capacitation-dependent Cell Signaling and Compartmentalized Metabolic Pathways in Murine Spermatozoa

Spermatozoa are highly polarized cells with specific metabolic pathways compartmentalized in different regions. Previously, we hypothesized that glycolysis is organized in the fibrous sheath of the flagellum to provide ATP to dynein ATPases that generate motility and to protein kinases that regulate motility. Although a recent report suggested that glucose is not essential for murine sperm capacitation, we demonstrated that glucose (but not lactate or pyruvate) was necessary and sufficient to support the protein tyrosine phosphorylation events associated with capacitation. The effect of glucose on this signaling pathway was downstream of cAMP, and appeared to arise indirectly as a consequence of metabolism as opposed to a direct signaling effect. Moreover, the phosphorylation events were not affected by uncouplers of oxidative respiration, inhibitors of electron transfer, or by a lack of substrates for oxidative respiration in the medium. Further experiments aimed at identifying potential regulators of sperm glycolysis focused on a germ cell-specific isoform of hexokinase, HK1-SC, which localizes to the fibrous sheath. HK1-SC activity and biochemical localization did not change during sperm capacitation, suggesting that glycolysis in sperm is regulated either at the level of substrate availability or by downstream enzymes. These data support the hypothesis that ATP specifically produced by a compartmentalized glycolytic pathway in the principal piece of the flagellum, as opposed to ATP generated by mitochondria in the mid-piece, is strictly required for protein tyrosine phosphorylation events that take place during sperm capacitation. The relationship between these pathways suggests that spermatozoa offer a model system for the study of integration of compartmentalized metabolic and signaling pathways.

The Influence of Growth Factors on the Development of Preimplantation Mammalian Embryos

The development of the preimplantation mammalian embryo from a fertilized egg to a blastocyst capable of implanting in the uterus is a complex process. Cell division must be carefully programmed. The embryonic genome must be activated at the appropriate stage of development, and the pattern of gene expression must be carefully coordinated for the initiation of the correct program of differentiation. Cell fates must be chosen to establish specific cell types such as the inner cell mass and the trophectoderm, which give rise to the embryo proper and the placenta, respectively. This review summarizes recent findings concerning the influence of growth factors on the development of preimplantation mammalian embryos. Maternal factors secreted into the lumen of the female reproductive tract as well as substances synthesized by the developing embryo itself help to regulate this process. Studies of embryos in culture and investigations using homologous recombination to create embryos and animals null for specific genes have enabled the identification of several growth factors that appear essential for preimplantation mammalian embryo development. Some of the factors are required maternal factors; others are embryo-derived autocrine and paracrine factors. Studies using molecular biology are beginning to identify differences in the patterns of genes expressed by naturally derived embryos and those developing in culture. The knowledge gained from studies on growth factors, media, embryonic development, and gene expression should help improve culture conditions for embryos and will provide for safer outcomes from assisted reproductive procedures in human and animal clinics.

Effects of Progranulin on Blastocyst Hatching and Subsequent Adhesion and Outgrowth in the Mouse

Abstract Using cDNA microarray methodology, we have shown previously that transcripts of progranulin gene (Grn, also known as acrogranin), a recently identified autocrine growth factor, were upregulated in mouse blastocysts adhered to the filter membrane in an in vitro-culture system. In the present study, we investigated the expression and effects of progranulin on blastocyst hatching, adhesion, and embryo outgrowth during the peri-implantation period in the mouse. During this period, substantial amounts of Grn mRNA were present in both inner cell mass (ICM) and trophectoderm. Progranulin was localized exclusively to the surface of the trophectoderm in early and pre- and postadhesion blastocysts as well as in trophoblast cells and ICM of outgrowth embryos, being secreted as a single, 88-kDa form into the surrounding medium. NIH3T3 cells that had been transfected with a progranulin expression construct secreted the 88-kDa form of the protein, from which a 68-kDa form could be generated by deglycosylation. In vitro treatment of blastocysts with recombinant progranulin promoted blastocyst hatching, adhesion, and outgrowth, whereas rabbit anti-mouse progranulin immunoglobulin G reduced the incidence of blastocyst hatching, adhesion, and outgrowth. Studies of bromodeoxyuridine incorporation and immunodissection of the ICM revealed that progranulin was effective on the trophectoderm but not on the ICM. These results indicate that progranulin is an important factor for the processes of blastocyst hatching, adhesion, and outgrowth, and they suggest that the effects of progranulin on blastocyst adhesion and outgrowth may have been triggered by the previous action of progranulin to induce hatching of the blastocysts.

Expression of Progranulin (Acrogranin/PCDGF/Granulin-Epithelin Precursor) in Benign and Malignant Ovarian Tumors and Activation of MAPK Signaling in Ovarian Cancer Cell Line

ABSTRACT It has been recently demonstrated that progranulin is overexpressed in ovarian cancer and that this protein is involved in the stimulation of cell proliferation, malignancy, and chemoresistance in ovarian cancer. The goal of the present study was to establish the differences in progranulin expression among normal, benign, and malignant ovarian tissues and to identify the signal transduction pathways activated by progranulin in an ovarian cancer cell line. Compared with benign tumors and normal ovarian tissue, progranulin mRNA and protein were overexpressed in malignant tumors. Survival analysis by the Kaplan–Meier method showed a correlation between high mRNA expression levels with poor survival outcome. Progranulin activated the MAPK-signaling pathway in NIH-OVCAR-3 cells. Progranulin expression may be potentially involved in the pathogenesis and malignant progression of ovarian cancer, and thus may represent a therapeutic target for this particular malignancy.

Vaginal matrix metalloproteinase levels in pregnant women with bacterial vaginosis.

Objective: To compare matrix metalloproteinase (MMP)-8 and MMP-9 levels in the vaginal secretions of pregnant women with or without asymptomatic bacterial vaginosis (BV). Methods: In this study, vaginal levels and molecular forms of MMP-8 and MMP-9 were studied in 36 pregnant women between 28 and 34 weeks of gestation with asymptomatic BV and 41 pregnant women, matched for gestational age, without BV. Results: Vaginal MMP-8 concentrations were significantly higher (P = .023) in BV-positive women. There was no significant difference in MMP-9 levels between healthy pregnant controls and BV-positive pregnant women. The presence of MMP-8 was confirmed by a 38-kd band on Western blots. Conclusions: Our findings show that BV is associated with increased levels of MMP-8 in vaginal fluid. Increased production of collagen-degrading enzymes such as MMP-8 is a possible causeof spontaneous spreterm delivery in pregnant women with asymptomatic BV.

PKC signaling is involved in the regulation of progranulin (acrogranin/PC-cell-derived growth factor/granulin-epithelin precursor) protein expression in human ovarian cancer cell lines.

Objective Overexpression of progranulin (also named acrogranin, PC-cell-derived growth factor, or granulin-epithelin precursor) is associated with ovarian cancer, specifically with cell proliferation, malignancy, chemoresistance, and shortened overall survival. The objective of the current study is to identify the signaling pathways involved in the regulation of progranulin expression in ovarian cancer cell lines. Methods We studied the relation of protein kinase C (PKC), phosphatidylinositol 3-kinase, protein kinase A, P38, extracellular signal-regulated kinase, and Akt pathways on the modulation of progranulin expression levels in NIH-OVCAR-3 and SK-OV-3 ovarian cancer cell lines. The different pathways were examined using pharmacological inhibitors (calphostin C, LY294002, H89, SB203580, PD98059, and Akt Inhibitor), and mRNA and protein progranulin expression were analyzed by reverse transcriptase polymerase chain reaction and Western blot techniques, respectively. Results Inhibition of PKC signal transduction pathway by calphostin C decreased in a dose-dependent manner protein but not mRNA levels of progranulin in both ovarian cancer cell lines. LY294002 but not wortmannin, which are phosphatidylinositol 3-kinase inhibitors, also diminished the expression of progranulin in both cell lines. In addition, LY294002 treatment produced a significant reduction in cell viability. Inhibition of protein kinase A, P38, extracellular signal-regulated kinase, and Akt did not affect progranulin protein expression. Conclusions These results suggest that the PKC signaling is involved in the regulation of progranulin protein expression in 2 different ovarian cancer cell lines. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the cellular proliferation and invasion in ovarian cancer produced by progranulin.

Progesterone receptor gene polymorphisms are not associated with preterm birth in a Hispanic population

To determine whether increased frequency of mutant alleles of the progesterone receptor gene (PGR) was associated with preterm birth in a population of Hispanic women.

Effect of clindamycin treatment on vaginal inflammatory markers in pregnant women with bacterial vaginosis and a positive fetal fibronectin test.

To compare the levels of interleukin (IL)‐1β, IL‐6, and matrix metalloproteinase (MMP)‐8 in the vaginal secretions of pregnant women with a positive fetal fibronectin (fFN) test result with or without asymptomatic bacterial vaginosis (BV) before and after treatment with oral clindamycin.

Differential Effect of DDT, DDE, and DDD on COX‐2 Expression in the Human Trophoblast Derived HTR‐8/SVneo Cells

The purpose of this study was to investigate the effect of 1,1,1‐trichloro‐2,2‐bis‐(chlorophenyl)ethane (DDT), 1,1‐bis‐(chlorophenyl)‐2,2‐dichloroethene (DDE), and 1,1‐dichloro‐2,2‐bis(chlorophenyl)ethane (DDD) isomers on COX‐2 expression in a human trophoblast‐derived cell line. Cultured HTR‐8/SVneo trophoblast cells were exposed to DDT isomers and its metabolites for 24 h, and COX‐2 mRNA and protein expression were assessed by RT‐PCR, Western blotting, and ELISA. Prostaglandin E2 production was also measured by ELISA. Both COX‐2 mRNA and protein were detected under control (unexposed) conditions in the HTR‐8/SVneo cell line. COX‐2 protein expression and prostaglandin E2 production but not COX‐2 mRNA levels increased only after DDE and DDD isomers exposure. It is concluded that DDE and DDD exposure induce the expression of COX‐2 protein, leading to increased prostaglandin E2 production. Interestingly, the regulation of COX‐2 by these organochlorines pesticides appears to be at the translational level.

The follicle-stimulating hormone receptor Asn680Ser polymorphism is associated with preterm birth in Hispanic women

Abstract Objective: Recently, a study based on the analysis of accelerated evolution of related genes at birth identified the follicle-stimulating hormone receptor (FSHR) as a possible candidate for the development of preterm delivery. Additionally, FSHR expression has been described in extragonadal tissue including the placenta. Therefore, the aim of the present study was to determine the association between the N680S polymorphism of the follicle-stimulating hormone receptor and preterm birth in a population of Hispanic women. Methods: Placenta samples were obtained from 64 women who had preterm births and 54 control cases. DNA was extracted and genotyped for the N680S FSHR gene polymorphism by polymerase chain reaction-restriction fragment length polymorphism. The χ2 test and t-test were used to calculate statistical significance. Results: Statistically significant differences in genotype frequencies for the N680S polymorphism were observed between preterm and term groups (p = .04). Based on the Akaike information criterion values, the dominant model showed that the NN genotype had a significantly increased risk of preterm birth compared with the SS + NS genotype (OR 2.52, 95% CI 1.20–5.33, p = .02). Conclusions: The results herein suggest that the FSHR polymorphism N680S is significantly associated with preterm birth in the Hispanic population.