Expression of progranulin (Acrogranin/PCDGF/Granulin-Epithelin Precursor) in benign and malignant ovarian tumors and activation of MAPK signaling in ovarian cancer cell line.

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.

Generation of flagella by cultured mouse spermatids.

During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained by enzymatic treatments and round spermatids (greater than 90% pure) were purified by unit gravity sedimentation. As determined by Nomarski or phase-contrast microscopy, no cells had flagella immediately after isolation; flagella were first clearly detected after 6 1/2 h of culture in Eagle's minimal essential medium containing 10% fetal bovine serum and 6 mM lactate. After 24 h, approximately 20% of round spermatids had formed flagella. Multinucleated round spermatids often formed multiple flagella, the number never exceeding the number of nuclei per symplast. Round spermatids were the only spermatogenic cells capable of tail formation. Flagella elongation was blocked by 1 microM demecolcine, an inhibitor of tubulin polymerization. Indirect immunofluorescence localized tubulin in the flagella. As seen by scanning electron microscopy, flagella developed as early as 2 h after culture and continued to elongate over the next 20 h, reaching lengths of at least 19 micron. Transmission electron microscopy demonstrated that flagella formed in culture resembled flagella from Golgi-phase round spermatids in situ; the flagella consisted of "9+2" axonemes lacking other accessory structures such as outer dense fibers and the fibrous sheath. As determined by acridine orange staining of the developing acrosomes, all spermatids that formed flagella in culture were Golgi-phase spermatids. By these criteria, the structures are indeed true flagella, corresponding in appearance to what others have described for early mammalian spermatid flagella in situ. We believe this is the first substantiated report of limited in vitro differentiation by isolated mammalian spermatids.

Targeting of a germ cell-specific type 1 hexokinase lacking a porin-binding domain to the mitochondria as well as to the head and fibrous sheath of murine spermatozoa.

Multiple isoforms of type 1 hexokinase (HK1) are transcribed during spermatogenesis in the mouse, including at least three that are presumably germ cell specific: HK1-sa, HK1-sb, and HK1-sc. Each of these predicted proteins contains a common, germ cell-specific sequence that replaces the porin-binding domain found in somatic HK1. Although HK1 protein is present in mature sperm and is tyrosine phosphorylated, it is not known whether the various potential isoforms are differentially translated and localized within the developing germ cells and mature sperm. Using antipeptide antisera against unique regions of HK1-sa and HK1-sb, it was demonstrated that these isoforms were not found in pachytene spermatocytes, round spermatids, condensing spermatids, or sperm, suggesting that HK1-sa and HK1-sb are not translated during spermatogenesis. Immunoreactivity was detected in protein from round spermatids, condensing spermatids, and mature sperm using an antipeptide antiserum against the common, germ cell-specific region, suggesting that HK1-sc was the only germ cell-specific isoform present in these cells. Two-dimensional SDS-PAGE suggested that all of the sperm HK1-sc was tyrosine phosphorylated, and that the somatic HK1 isoform was not present. Immunoelectron microscopy revealed that HK1-sc was associated with the mitochondria and with the fibrous sheath of the flagellum and was found in discrete clusters in the region of the membranes of the sperm head. The unusual distribution of HK1-sc in sperm suggests novel functions, such as extramitochondrial energy production, and also demonstrates that a hexokinase without a classical porin-binding domain can localize to mitochondria.

A-kinase anchor proteins in endocrine systems and reproduction.

Over the past few years, significant progress has been made in characterizing the expression and localization of proteins that act as scaffolds for cAMP-dependent protein kinase (PK-A). These A-kinase anchor proteins (AKAPs) tether PK-A to intracellular organelles and structures, sequestering the kinase near its physiological substrates. The compartmentalization of distinct pockets of PK-A activity serves to provide spatial regulation of this signaling pathway. In addition, other signaling proteins bind to AKAPs, as do some newly described proteins of unknown function, suggesting that proteins of various pathways are anchored through AKAPs.

Exon/intron organization of the gene encoding the mouse epithelin/granulin precursor (acrogranin).

Mouse genomic clones encoding the epithelin/granulin gene and its 5'- and 3'-flanking regions have been isolated and sequenced. This gene was found to be a single-copy gene, and contained 13 exons interrupted by 12 introns. Eight out of the 12 introns are classified as phase 0, and are located within the central part of each of the tandem repeats in the amino acid sequence of the epithelin/granulin precursor. The first intron is unique because of the interruption of the 5'-untranslated region and its fairly large size (approximately 2.4 kbp). Consensus sequences for several of the potential regulatory elements are present in the 5'-flanking sequence, including a common CCAAT sequence.

Fucosylation events during mammalian spermatogenesis.

It will be necessary to conduct further studies to establish more precisely the localization of FT on mouse male germ cells. Antibodies to FTs are not yet available, so an immunocytochemical approach is not currently feasible. Additional cell fractionation protocols can be designed to compare plasma membrane fractions with enriched fractions of Golgi apparatus and to compare directly the activities of multiple glycosyltransferase enzymes and Golgi-specific markers in these preparations. Schachter et al. and Nyquist and colleagues have already provided experimental techniques for the isolation of Golgi fractions of good purity from rodent pachytene spermatocytes and spermatids. Ample opportunity exists, then, for a detailed analysis of the number, specificity, and localization of FT enzymes during mammalian spermatogenesis. All available data imply that these enzymes will prove to be vital components in the differentiation of cells within the seminiferous epithelium.

A serum proteomics approach to the diagnosis of ectopic pregnancy.

An ectopic pregnancy (EP) occurs when implantation of the embryo occurs outside of the uterus. If left untreated, the developing fetus will continue to grow, leading to life-threatening consequences for the mother. A major difficulty with the diagnosis of ectopic pregnancy is that methods of detection are limited, and some, such as ultrasound, are not very reliable in the earliest days of gestation. Currently, no effective serum test exists to distinguish an ectopic pregnancy from a normal intrauterine pregnancy. The incidence of ectopic pregnancy is increasing and has doubled in the last 20 years. It is now the second most common cause of maternal death in the first trimester of pregnancy. To address this issue, we initiated a project to identify serum markers of ectopic pregnancy. The subjects for these studies presented at the Hospital of the University of Pennsylvania. We obtained over 140 serum samples from women with suspected ectopic pregnancy: women presenting with pain and/or bleeding in the first trimester of pregnancy. The approximate racial breakdown of the subjects is as follows: African American, 36%; Caucasian, 3%; Asian, 2%; Hispanic, 1%; unknown, 58%. Serum samples from 139 women (62 with ectopic pregnancy and 77 with a normal intrauterine pregnancy) were applied to WCX2 (weak ion exchange) protein chip surfaces and analyzed for serum markers using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Several proteins in the 7500-18,000 Da mass range were identified that may discriminate an ectopic pregnancy from an intrauterine pregnancy. The most promising markers were analyzed using classification and regression tree analysis (CART) with and without clinical variables (serum hCG value, length of amenorrhea). Two different algorithms were developed that classify the patients on the basis of sensitivity (number of EPs who screen positive/# of EPs) or specificity (# of healthy patients who screen negative/# of healthy). Our current approach is to refine these two "rule sets" to segregate patients into three groups: those who need immediate intervention for a probable ectopic pregnancy, those who appear to have a normal pregnancy, and those who need further monitoring for diagnosis.

Molecular evaluation of two major human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82, in stump tail sperm.

OBJECTIVE: To determine whether mutations in the pro-hAKAP82 gene and the resulting pro-hAKAP82 and hAKAP82 proteins were associated with the infertility seen in a patient with stump tail sperm. DESIGN: Case report. SETTING: Academic research and teaching environment, tertiary care hospital. PATIENT(S): A single, infertile Caucasian male diagnosed with essentially 100% stump tail sperm. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Electrophoresis, silver staining, and immunoblotting of patient and control sperm proteins; RII (type II regulatory subunit of protein kinase-A) overlay assay of patient and control sperm proteins, partial DNA sequence analysis of patient's pro-hAKAP82 gene; indirect immunofluorescence and immunogold electron microscopy of patient and control sperm. RESULTS(S): No significant abnormalities in the size or amount of pro-hAKAP82 and hAKAP82 or in the ability of these proteins to bind the regulatory subunit of protein kinase-A were identified in the patient's sperm. Partial sequence analysis of the patient's pro-hAKAP82 gene was identical to the published normal sequence. Indirect immunofluorescence and immunoelectron microscopy of sperm localized pro-hAKAP82/hAKAP82 to the sperm flagellum and demonstrated that the proteins were present in a disorganized, amorphous region, which apparently represented the fibrous sheath. CONCLUSION(S): These results suggest that, although pro-hAKAP82 and hAKAP82 localize to the correct structural component of the flagellum and are not directly responsible for the stump tail phenotype, they are unable to assemble normally into the fibrous sheath. Although this study did not identify abnormalities in the pro-hAKAP82 gene or its resulting proteins in a patient with stump tail sperm, several regions of the gene and protein remain to be examined.

Preparation and short-term culture of enriched populations of guinea pig spermatocytes and spermatids.

In order to study acrosome biogenesis in vitro, the authors developed techniques for the isolation and culture of enriched populations of guinea pig spermatogenic cells using a modification of the technique of Romrell et al (1976). The modifications include changes in the medium, enzyme concentrations, cell loads for gradient separations, and sedimentation times. Three major cell populations were pooled: Pachytene spermatocytes (PS: 2.0 x 10(7), 80-85% pure), round spermatids, (RS: 1.2 x 10(8), 80-85% pure), and condensing spermatids (CS: 2.0 x 10(8), 50-60% pure, contaminated with residual bodies). The ultrastructural properties of the isolated cells appeared similar to those of cells in situ. PS were 3-4 times more active than RS and 10 times more active than CS in synthesizing proteins. These experiments demonstrate that highly enriched populations of guinea pig spermatogenic cells can be isolate, and that these cells can be cultured in the presence of radioactive precursors for studies of protein synthesis during spermatogenesis.

Molecular genetic analysis of two human sperm fibrous sheath proteins, AKAP4 and AKAP3, in men with dysplasia of the fibrous sheath.

Dysplasia of the fibrous sheath (DFS) is characterized by male infertility, asthenozoospermia, and morphologically abnormal flagella that possess a severely malformed fibrous sheath. In many cases, DFS is familial, suggesting a genetic component. Human AKAP4 and AKAP3 are structural proteins of the fibrous sheath that also function to anchor protein kinase A to this structure via the regulatory subunit of the kinase. We hypothesized that defects in either AKAP4 or AKAP3 might cause DFS. No quantitative or qualitative differences between patients with DFS and normal controls were detected when sperm proteins were analyzed by either silver staining or immunoblot analysis using antibodies raised against AKAP4 and AKAP3. Additionally, AKAP4 and AKAP3 from DFS sperm retained the ability to bind the regulatory subunit of protein kinase A. Localization at the light and electron microscopic levels showed that AKAP3 and AKAP4 localized correctly to the FS of the amorphous flagellum in DFS sperm. Partial sequence analysis of the AKAP4 and AKAP3 genes in patients with DFS did not identify any significant alterations in potential AKAP4/AKAP3 binding regions, suggesting that the two proteins interact normally in DFS sperm. Our results did not find evidence to support the hypothesis that mutations in either gene are responsible for DFS in humans.

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.

Expression of progranulin and the epithelin/granulin precursor acrogranin correlates with neoplastic state in renal epithelium.

BACKGROUND: Current traditional pathological parameters, including staging and grading, are not sufficient in predicting outcome in patients with renal cell carcinoma (RCC). Acrogranin is an epithelial growth factor and has been demonstrated to play a role in teratocarcinogenesis and tumorigenesis. The aim of this study was to examine levels of acrogranin in renal cancer. MATERIALS AND METHODS: Western blot analysis was performed on renal tissue protein lysates. In addition, immunohistochemical (IHC) analysis of acrogranin expression was conducted on tissue sections of various histological types and grades of RCC. RESULTS: Western analysis showed that acrogranin levels were low in benign renal tissue and increased in malignant renal tissue. In addition, IHC revealed that high-grade RCC exhibited higher levels of expression than low-grade RCC and normal tissue. CONCLUSION: These data suggest that acrogranin may be a functional important growth factor in RCC and may be a potential molecular marker for high-grade RCC.

Biochemical studies of the envelope transformations in Xenopus laevis eggs.

The envelopes that enclose the eggs of the amphibian Xenopus laevis were isolated and examined for biochemical correlates of the ultrastructural and sperm penetrability differences among the coelomic egg envelope (CE), the vitelline envelope (VE), and the fertilization envelope (FE). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the 43,000 molecular weight glycoproteins of CEs were found to be converted to components with molecular weights of 41,000 in VEs; also, a protein with a molecular weight of 57,000 was added to the envelope during the CE-to-VE conversion. The molecular weights of two components decreased during the VE-to-FE conversion, from 69,000 and 64,000 in the VE to 66,000 and 61,000 in the FE. Components from the cortical granules and the innermost jelly coat were also added to the newly-formed FE. As detected by iodination with lactoperoxidase or IODOGEN, both the CE-to-VE and the VE-to-FE conversions caused conformational changes in envelope glycoproteins. Peptide mapping demonstrated that the 43,000 molecular weight components of CE were precursors to the 41,000 molecular weight components of VE and the 69,000 and 64,000 molecular weight components of VE were precursors to the 66,000 and 61,000 molecular weight components of FE. The CE-to-VE conversion presumably occurs in the first portion of the oviduct. Experiments probing the VE-to-FE conversion demonstrated the need for an intact jelly coat for the molecular weight changes to occur. Sperm were not required for the envelope alteration; the SDS-PAGE pattern of envelopes from jellied eggs activated with the Ca++-ionophore A23187 were indistinguishable from the FE. These studies show that there are molecular correlates of the morphological and biological differences among the envelopes. The CE-to-VE and the VE-to-FE conversions follow a similar pattern: in both cases, material is added to the envelope and there are changes in the molecular weights of some of the components.

Thiol changes during epididymal maturation: a link to flagellar angulation in mouse spermatozoa?

Caput epididymal wild-type spermatozoa and cauda epididymal spermatozoa from mice null for the adenylyl cyclase Adcy10 gene are immotile unless stimulated by a membrane-permeant cyclic AMP analogue. Both types of spermatozoa exhibit flagellar angulation where the head folds back under these conditions. As sperm proteins undergo oxidation of sulfhydryl groups and the flagellum becomes more stable to external forces during epididymal transit, we hypothesized that ADCY10 is involved in a mechanism regulating flagellar stabilization. Although no differences were observed in global sulfhydryl status between caput and cauda epididymal spermatozoa from wild-type or Adcy10-null mice, two-dimensional fluorescence difference gel electrophoresis was performed to identify specific mouse sperm proteins containing sulfhydryl groups that became oxidized during epididymal maturation. A-kinase anchor protein 4, fatty acid-binding protein 9 (FABP9), glutathione S-transferase mu 5 and voltage-dependent anion channel 2 exhibited changes in thiol status between caput and cauda epididymal spermatozoa. The level and thiol status of each of these proteins were quantified in wild-type and Adcy10-null cauda epididymal spermatozoa. No differences in the abundance of any protein were observed; however, FABP9 in Adcy10-null cauda epididymal spermatozoa contained fewer disulfide bonds than wild-type sperm cells. In caput epididymal spermatozoa, FABP9 was detected in the cytoplasmic droplet, principal piece, midpiece, and non-acrosomal area of the head. However, in cauda epididymal spermatozoa, this protein localized to the perforatorium, post-acrosomal region and principal piece. Together, these results suggest that thiol changes during epididymal maturation have a role in the stabilization of the sperm flagellum.

The vitelline envelope to fertilization envelope conversion in eggs of Xenopus laevis.

Fertilization of the Xenopus laevis egg causes the conversion of the vitelline envelope to the fertilization envelope, a change reflected in the loss of sperm penetrability of the egg and the appearance of an electron-dense layer on the outer aspect of the fertilization envelope. As seen by one-dimensional gel electrophoresis, two components with molecular weights of 69,000 and 64,000 in the vitelline envelope were converted to 66,000 and 61,000 in the fertilization envelope. By two-dimensional gel electrophoresis, the components in the 69,000 and 64,000 molecular weight regions of the vitelline envelope were seen to shift to more basic isoelectric points upon conversion to the fertilization envelope. Peptide mapping by limited proteolysis suggested that the 69,000 and 64,000 molecular weight components shared the same polypeptide chains but the smaller glycoprotein lacked a carbohydrate side chain found on the larger species. Similar sites on each glycoprotein were affected when the vitelline envelope was converted to the fertilization envelope. No N-terminal amino acids could be identified on the envelope components, indicating that these glycoproteins have blocked N-termini. Ionophore A23187-activation of jellied eggs (but not dejellied eggs) caused the molecular weight changes in the absence of sperm. Thus, factors from the jelly and the cortical granules but not from sperm apparently are involved in the processing of the 69,000 and 64,000 molecular weight components.

The coelomic envelope to vitelline envelope conversion in eggs of Xenopus laevis.

An amphibian egg recovered from the body cavity is enclosed by a coelomic egg envelope. Upon transport down the oviduct, the envelope is converted to the vitelline envelope. The coelomic and vitelline envelopes are distinct in terms of sperm penetrability, ultrastructural morphology, and radioiodination profiles. In this study, the macromolecular compositions of these two envelopes were determined. Isolated envelopes were compared by one- and two-dimensional gel electrophoresis, peptide mapping, and radiolabeling. A protein with a molecular weight of 57,000 (57K) was present in the vitelline envelope but was absent in the coelomic envelope. A glycoprotein with a molecular weight of 43K in the coelomic envelope was converted to a component with a molecular weight of 41K in the vitelline envelope. The 43K-molecular weight component of the coelomic envelopes could be radioiodinated by lactoperoxidase but no labeling of the 41K-molecular weight component occurred in the vitelline envelope. Peptide mapping using limited proteolysis established that the 43K-molecular weight component of the coelomic envelope was a precursor to the 41K-molecular weight component of the vitelline envelope. These molecular alterations may underlie the ultrastructural and physiological changes occurring in these envelopes.

Stage-specific synthesis and fucosylation of plasma membrane proteins by mouse pachytene spermatocytes and round spermatids in culture.

Little is known about the ability of mammalian spermatogenic cells to synthesize plasma membrane components in the presence or absence of Sertoli cells. In this study, purified populations (greater than 90%) of pachytene spermatocytes or round spermatids were isolated by unit gravity sedimentation and cultured for 20-24 h in the presence of [35S]methionine or [3H]fucose. Cell viabilities remained over 90% during the course of these experiments. Plasma membranes were purified from these cells and analyzed by two-dimensional gel electrophoresis. Qualitatively, the same plasma membrane proteins were synthesized by both cell types with the exception of the major Concanavalin A-binding glycoprotein, p151; the synthesis of p151 is greatly diminished or inhibited after meiosis. [3H]Fucose was incorporated into at least 6 common glycoproteins of both cells. Eight components fucosylated with molecular weights from 35,000 to 120,000 were specific to pachytene spermatocyte membranes. One fast-migrating fucosylated component may represent an uncharacterized lipid whose synthesis is terminated after meiosis. Round spermatids specifically fucosylated two components with molecular weights of 45,000 and 80,000. These results demonstrate the viability of germ cells of the male mouse in short-term culture and show that they are capable of synthesizing and fucosylating plasma membrane components in the absence of Sertoli cells.

Proacrosin/acrosin during guinea pig spermatogenesis.

Enriched populations of guinea pig spermatogenic cells were isolated by sedimentation velocity at unit gravity. Each cell population was analyzed for the presence of members of the proacrosin/acrosin family by enzymography, immunoblotting, and immunofluorescence. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gels containing 0.1% gelatin, protease activities with molecular weights of 55,000 (major) and 50,000 (minor) were detected in round spermatid extracts. Condensing spermatid extracts contained protease activities with molecular weights between 55,000 and 50,000. These major protease activities had molecular weights similar to antigens detected by immunoblotting with a monospecific rabbit antiserum directed against purified boar acrosin. Extracts of guinea pig sperm and the soluble acrosomal components released following the acrosome reaction induced with ionophore A23187 contained three major protease activities (Mr 32,000, 34,000, 47,000) but only the 47,000 Mr protease cross-reacted with the antibody. The spermatid and sperm protease activities were inhibited and activated by classical effectors of acrosin activity from other species. Immunofluorescence demonstrated that proacrosin/acrosin was present as early as the Golgi phase of spermiogenesis. In addition, immunoreactivity was confined to the acrosomes in a manner characteristic of each spermatid stage. These results demonstrate that proacrosin/acrosin can be detected in the earliest spermiogenic stages by electrophoretic and immunological techniques and suggest that changes in the molecular weights of proacrosin/acrosin occur as spermatids mature.