High levels of luteinizing hormone analog stimulate gonadal and adrenal tumorigenesis in mice transgenic for the mouse inhibin-alpha-subunit promoter/Simian virus 40 T-antigen fusion gene.

Transgenic (TG) mice expressing the Simian virus 40 T-antigen under the control of the murine inhibin-alpha promoter (Inhalpha/Tag) develop granulosa and Leydig cell tumors at the age of 5-6 months, with 100% penetrance. When these mice are gonadectomized, they develop adrenocortical tumors. Suppression of gonadotropin secretion inhibits the tumorigenesis in the gonads of intact animals and in the adrenals after gonadectomy.

Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae.

Introduction of an active xylose utilization pathway into Saccharomyces cerevisiae, which does not naturally ferment pentose sugars, is likely to have a major impact on the overall cellular metabolism as the carbon introduced to the cells will now flow through the pentose phosphate pathway. The metabolic responses in the recombinant xylose-fermenting S. cerevisiae were studied at the proteome level by comparative two-dimensional gel electrophoresis of cellular proteins within a pH range of 3-10.

Adenosine triphosphate induces Ca2+ signal in epithelial cells of the mouse caput epididymis through activation of P2X and P2Y purinergic receptors.

In the present study, we developed a novel method to analyze the calcium (Ca2+) signal in living slices of mouse caput epididymides by applying calcium imaging on Fura-2-loaded vibratome slices. The data revealed that in epithelial cells of mouse caput epididymides, ATP induces a rapid Ca2+ signal that is sustained after 60 sec. Preincubating the sections in Ca2+-free medium in the presence of EGTA did not affect the amplitude of the ATP-induced Ca2+ signal, indicating the presence of P2Y type purinergic receptors and phospholipase C activity.

A fluorescent Tie1 reporter allows monitoring of vascular development and endothelial cell isolation from transgenic mouse embryos.

Tie1 is an endothelial receptor tyrosine kinase essential for development and maintenance of the vascular system. Here we report generation of transgenic mice expressing enhanced green fluorescent protein (EGFP) or a chimeric protein consisting of a Zeosin resistance marker and EGFP under the control of mouse Tie1 promoter. Intravital monitoring of fluorescence showed that the EGFP reporter recapitulates the Tie1 expression pattern in the developing vasculature, and flow cytometry using EGFP allowed the isolation of essentially pure Tie1-expressing endothelial cells from transgenic mouse embryos.

Epididymal dysfunction initiated by the expression of simian virus 40 T-antigen leads to angulated sperm flagella and infertility in transgenic mice.

We have generated two transgenic mouse lines (GPX5-Tag1 and GPX5-Tag2) by expressing the Simian virus 40 large and small T-antigens under a 5-kb promoter of the murine glutathione peroxidase 5 (GPX5) gene. In GPX5-Tag1 mice, with a high level of T-antigen expression, severe dysplasia was found in the epididymis and seminal vesicles. These mice also developed adrenal and prostate tumors, and spermatogenesis was disrupted.

Developmental, estrogen induced infravesical obstruction is reversible in adult male rodents.

Purpose: We treat neonatally estrogenized rats and aromatase over expressing AROM+ male mice with infravesical obstruction using the specific aromatase inhibitors finrozole and letrozole, and analyzed whether developmentally induced alterations in urodynamics and rhabdosphincter are reversible in adulthood.

Materials And Methods: Adult estrogenized rats and AROM+ mice were treated with aromatase inhibitors for 6 weeks. Maximal and mean bladder pressure, the urinary flow rates and electromyography activity were recorded from the proximal rhabdosphincter.

Transgenic mice harboring murine luteinizing hormone receptor promoter/beta-galactosidase fusion genes: different structural and hormonal requirements of expression in the testis, ovary, and adrenal gland.

In vivo regulation of the LH receptor (LHR) promoter was studied using transgenic (TG) mice harboring fusion genes containing three different lengths of the LHR promoter (7.4 kb, 2.1 kb, and 173 bp), fused with coding sequence of the Escherichia coli beta-galactosidase (beta-GAL) reporter gene.

Reproductive disturbances, pituitary lactotrope adenomas, and mammary gland tumors in transgenic female mice producing high levels of human chorionic gonadotropin.

To assess the consequences of prolonged exposure to elevated levels of LH/human chorionic gonadotropin (hCG) in the female, we developed a transgenic (TG) mouse model (hCGbeta+) that overexpresses the hCGbeta-subunit cDNA. Because of the promoter used, ubiquitin C, the transgene is expressed in multiple tissues, including the pituitary gland, in which coupling with the endogenous common alpha-subunit results in synthesis of high levels of bioactive hCG. The TG females presented with precocious puberty, infertility, enhanced ovarian steroidogenesis, and abnormal uterine structure.

Mammary gland development in transgenic male mice expressing human P450 aromatase.

We recently generated a transgenic mouse strain that expresses the human aromatase gene under the ubiquitin C promoter (AROM+). We have previously shown that in these mice the serum estradiol concentration is highly elevated, whereas the testosterone concentration is decreased. In the present study we examined mammary gland development in AROM+ male mice at different ages and found that the mammary glands of AROM+ males undergo ductal and alveolar development morphologically resembling that of terminally differentiated female mammary glands, expressing mRNA for a milk protein gene (beta-casein).

Sperm volume regulation: maturational changes in fertile and infertile transgenic mice and association with kinematics and tail angulation.

Laser light scatter analyzed by flow cytometry was used to monitor the volume of viable maturing murine spermatozoa. Upon release, dispersion, and dilution, epididymal sperm from fertile heterozygous c-ros knockout mice were smallest in the cauda region and largest in the corpus region. Cauda sperm from both infertile homozygous c-ros knockout and GPX5-Tag2 transgenic mice were abnormally large.

Infravesical obstruction in aromatase over expressing transgenic male mice with increased ratio of serum estrogen-to-androgen concentration.

Purpose: The potential role of estrogen in the development of infravesical obstruction is still unresolved. Aromatase over expressing transgenic mice provide a novel instrument for investigating the consequences of prolonged systemic or local increases in endogenous estrogen concentrations. Two aromatase over expressing transgenic mouse strains with different prostatic phenotypes (reduced and normal size, respectively) were compared in urodynamic studies with each other and with the wild-type strain.

Transgenic and knockout mouse models for the study of luteinizing hormone and luteinizing hormone receptor function.

The main functions of luteinizing hormone (LH) are concerned with regulation of gonadal function, and these functions are today well delineated through previous physiological studies. However, novel information of less well-known aspects of actions of this hormone is currently emerging from studies on genetically modified mouse models, with either enhanced or suppressed LH/LH receptor (LHR) function. The novel functions of LH include its role, in specific situations, as promoter of formation and growth of gonadal and extragonadal tumors.

Murine relaxin-like factor promoter: functional characterization and regulation by transcription factors steroidogenic factor 1 and DAX-1.

The gene for mouse relaxin-like factor (RLF), a member of the insulin/IGF/relaxin family of hormones, appears to be predominantly expressed in testicular Leydig cells. Mice deficient in RLF have revealed a role for this peptide in testicular descent, but the regulatory mechanisms of its function are still insufficiently characterized. In the present study we showed that the RLF promoter was active in both mLTC-1 Leydig cells and luteinized KK-1 granulosa tumor cells.

Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites.

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim.

Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis.

Current analytical techniques in protein identification by mass spectrometry are based on the generation of peptide mass maps or sequence tags that are idiotypic for the protein sequence. This work reports on the development of the use of mass spectrometric methods for protein identification in research on metabolic pathways of a genetically modified strain of the baker's yeast Saccharomyces cerevisiae. This study describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag analysis in identification of yeast proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).

Gonadal tumors of mice double transgenic for inhibin-alpha promoter-driven simian virus 40 T-antigen and herpes simplex virus thymidine kinase are sensitive to ganciclovir treatment.

We have previously produced transgenic (TG) mice expressing the mouse inhibin alpha-subunit promoter/Simian virus 40 T-antigen (Inhalpha/Tag) fusion gene. The mice develop gonadal somatic cell tumors at the age of 5-7 months; the ovarian tumors originate from granulosa cells, and those of the testes from Leydig cells. In the present study another TG mouse line was produced, expressing under the same inh-alpha promoter the herpes simplex virus thymidine kinase gene (Inhalpha/TK).

Improved technique for detection of enhanced green fluorescent protein in transgenic mice.

One of the most exciting recent advances in cell biology is the possibility to use the green fluorescent protein and its various mutated forms as reporter proteins in studies carried out in vitro and in vivo. In the present study, several detection techniques for the enhanced green fluorescent protein (EGFP) were compared in transgenic mice, using fluorescence and confocal microscopy. In addition, different tissue preparation techniques (squash preparations, vibratome sections, frozen sections) were evaluated.

Altered structure and function of reproductive organs in transgenic male mice overexpressing human aromatase.

Aromatization of androgens is a key step in estrogen production, and it regulates the delicate balance between estrogens and androgens in the gonads and sex steroid target tissues. In the present study, we generated transgenic mice (AROM(+)) bearing the human ubiquitin C promoter/human P450 aromatase fusion gene. AROM(+) male mice are characterized by an imbalance in sex hormone metabolism, resulting in elevated serum E(2) concentrations, combined with significantly reduced testosterone and FSH levels, and elevated levels of PRL and corticosterone.

Promoter function of different lengths of the murine luteinizing hormone receptor gene 5'-flanking region in transfected gonadal cells and in transgenic mice.

The purpose of these studies was to explore the sex- and tissue-specific expression of the LH receptor (LHR) gene. Fusion genes containing three different lengths of the 5'-flanking region of the mouse LHR gene (7.4 kb, 2.

A novel transgenic model to characterize the specific effects of follicle-stimulating hormone on gonadal physiology in the absence of luteinizing hormone actions.

Gonadal function is wholly reliant on the two pituitary-derived gonadotropins, FSH and LH. Identifying the specific effects of FSH has been difficult because of the intimate relationship between LH and FSH action and inherent limitations of classic research paradigms. We describe a novel transgenic model to characterize the definitive actions of FSH alone, distinct from LH effects, created by combining transgenic FSH expression with the gonadotropin-deficient background of the hypogonadal (hpg) mouse.

Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis.

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice.

Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice.

To study further the role of gonadotropins in reproductive functions, we generated mice with LH receptor (LHR) knockout (LuRKO) by inactivating, through homologous recombination, exon 11 on the LHR gene. LuRKO males and females were born phenotypically normal, with testes, ovaries, and genital structures indistinguishable from their wild-type (WT) littermates. Postnatally, testicular growth and descent, and external genital and accessory sex organ maturation, were blocked in LuRKO males, and their spermatogenesis was arrested at the round spermatid stage.

Activin-A, but not inhibin, regulates 17beta-hydroxysteroid dehydrogenase type 1 activity and expression in cultured rat granulosa cells.

17beta-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes the reduction of estrone (E(1)) to biologically more active estradiol (E(2)). In the present study, the effect of activin, inhibin, and follistatin on 17HSD activity and 17HSD type 1 expression in cultured, unluteinized rat granulosa cells was examined. Furthermore, the effects of these hormones on 17HSD type 1 expression were compared with the expression of P450 aromatase (P450arom).

Analysis of residual solvents in pharmaceuticals with purge-and-membrane mass spectrometry.

A method using purge-and-membrane mass spectrometry (PAM-MS) was developed for the analysis of residual solvents in pharmaceutical products. The method combines dynamic headspace and membrane inlet mass spectrometry. The limits of detection for the compounds studied, benzene, toluene, chloroform, 2-pentene and 2-methyl- and 3-methylpentane, were 0.

A common polymorphism in the human relaxin-like factor (RLF) gene: no relationship with cryptorchidism.

The incidence of impaired testicular descent (cryptorchidism) is high; 1-2% of boys at the age of 3 mo are diagnosed for this condition in western countries. Recent data on mice with targeted disruption of the Insl3/relaxin-like factor (RLF) gene proposed that this factor plays a role in testicular descent in fetal life. Male RLF-/- mice exhibit bilateral cryptorchidism due to developmental abnormalities of the gubernaculum, associated with abnormal spermatogenesis and infertility.