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). Crossbreeding of the two TG mouse lines resulted in double TG mice (Inhalpha/TK-Inhalpha/Tag), which also developed gonadal tumors. The single (Inhalpha/Tag) and double TG (Inhalpha/TK-Inhalpha/Tag) mice, both bearing gonadal tumors, were treated at the age of 5.5-6.5 months with ganciclovir (GCV, 150 mg/kg body weight twice daily i.p.) for 14 days, or with aciclovir (ACV, 300-400 mg/kg body weight per day perorally) for 2 months. During GCV treatment, the total gonadal volume including the tumor, decreased in double TG mice by an average of 40% (P<0.05), while in single TG mice, there was a concomitant increase of 60% in gonadal size (P<0.05). GCV was also found to increase apoptosis in gonads of the double TG mice. Peroral treatment with ACV was less effective, it did not reduce significantly the gonadal volume. We also analyzed the in vitro efficacy of ACV and GCV treatments in transiently HSV-TK-transfected KK-1 murine granulosa tumor cells, originating from a single-positive Inhalpha/Tag mouse. GCV proved to be more effective and more specific than ACV in action. These results prove the principle that targeted expression of the HSV-TK gene in gonadal somatic cell tumors is potentially useful for tumor ablation by antiherpes treatment. The findings provide a lead for further development of somatic gene therapy for gonadal tumors.

Long-term testosterone treatment prevents gonadal and adrenal tumorigenesis of mice transgenic for the mouse inhibin-alpha subunit promoter/simian virus 40 T-antigen fusion gene.

We have developed a transgenic (TG) mouse model for tumorigenesis of gonadal somatic cells using a 6 kb fragment of the mouse inhibin-alpha subunit promoter (Inh-alpha) fused with the simian virus 40 T-antigen (Tag) coding sequence. Gonadal tumors, of Leydig or granulosa cell origin, develop in the TG mice with 100% penetrance by the age of 5-8 months. Conspicuously, if the mice are gonadectomized, they develop adrenal tumors. Gonadal and adrenal tumorigenesis in these mice seem to be gonadotropin dependent. On the other hand, testosterone stimulates the proliferation of a cell line (C alpha 1) established from one of the adrenal tumors. The purpose of the present study was therefore to investigate further whether testosterone affects the growth of these gonadal and adrenal tumors in vivo. Two experimental models were used: (1) Tag TG/hypogonadotropic (hpg) double mutant mice and (2) castrated Tag TG mice. Both were treated between 1-2 and 7-8 months of age with Silastic rods (length 2 cm) containing testosterone. None of the control or testosterone-treated Tag/hpg mice developed gonadal or adrenal tumors. The castrated Tag TG mice displayed, upon microscopical examination, early stages of adrenal tumors, whereas those receiving testosterone did not show such changes. Testosterone increased the weights of gonads in the Tag/hpg mice, and those of uteri and seminal vesicles in both groups. In contrast, the adrenal weights were significantly reduced in both groups by testosterone treatment. Gonadal histology of the testosterone-treated mice showed hyperplasia of testicular Leydig cells and ovarian stroma. Spermatogenesis was induced by testosterone in the Tag/hpg mice. Adrenal histology of the testosterone-treated animals demonstrated the disappearance of the X-zone. Serum levels of FSH in testosterone-treated Tag/hpg mice were significantly increased, while those of serum LH were decreased. In conclusion, the present result indicate that the suppression of gonadotropins by testosterone implants in castrated Inh-alpha/Tag TG mice prevents the tumorigenesis of their adrenals. In intact Tag/hpg mice, testosterone implants were not able to induce gonadal or adrenal tumorigenesis. Although testosterone treatment was able to induce interstitial cell hyperplasia in gonads of the Inh-alpha/Tag mice, direct gonadotropin action is responsible for gonadal and adrenal tumorigenesis.

Characterisation of three novel cationic lipids as liposomal complexes with DNA.

Cationic lipids (CLs) are being increasingly exploited as transfection vectors for the delivery of DNA into eukaryotic cells. To obtain further insight to the complex formation and interactions between cationic liposomes and DNA, we characterised three novel cationic lipids, viz. bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide, N-hexadecyl-N-¿10-[O-(4-acetoxy)-phenylundecanoate]ethyl¿- dimethylammonium bromide, and bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide. These lipids bear the same charged headgroup yet have different hydrophobic parts. Accordingly, we may anticipate their electrostatic interactions with DNA to be similar while differing in both thermal phase behaviour and physicochemical properties of their complexes with DNA. In keeping with the above all three lipids formed complexes with DNA as evidenced by light scattering, fluorescence spectroscopy and Langmuir film balance. Differential scanning calorimetry revealed very different phase behaviours for the binary mixtures of the three CLs with dimyristoylphosphatidylcholine and also provided evidence for DNA-induced lipid phase separation. These data were confirmed by compression isotherms and fluorescence microscopy of monolayers residing on an aqueous buffer, recorded both in the presence and absence of DNA. Importantly, binding to cationic liposomes appears to prevent thermal denaturation of DNA upon heating of the complexes. Likewise, renaturation of heat-treated DNA complexed with the cationic liposomes appears to be abolished as well.

Oestrogen-dependent expression of LH/hCG receptors in pig Fallopian tube and their role in relaxation of the oviduct.

The current studies investigated the concentration and distribution of LH receptors in the oviduct of ovariectomized gilts at various times after administration of oestradiol benzoate (10 micrograms kg-1 body weight) to determine whether LH participates in the regulation of oviductal contractions. Polyclonal antibodies to the LH receptor were used in immunocytochemical and western blot analyses of oviductal tissues. The mechanical activity of the isthmus and ampullar segments of oviduct, collected from 16 cyclic gilts, was recorded for 30 min after LH or hCG treatment. In the oviduct, there was little competition for receptor occupancy between hCG and pig FSH, bovine thyroid-stimulating hormone (TSH), pig growth hormone (GH) and pig prolactin (1.2, 0.1, 0.01 and < 0.001%, respectively) but pig LH could completely inhibit the binding of [125I]hCG. Oestradiol benzoate increased (P < 0.01) the number of LH binding sites in oviduct 24, 48 and 72 h (0.60 +/- 0.08, 1.62 +/- 0.15, 2.48 +/- 0.35 fmol mg-1 protein; n = 4 per treatment, respectively) after injection compared with the control gilts treated with corn oil (0.20 +/- 0.04 fmol mg-1 protein; n = 4). The affinity of oviductal LH/hCG binding sites (Ka) varied from 4.0 to 8.5 x 10(10) l mol-1 and was similar to that of luteal cell binding sites (6.1 x 10(10) l mol-1). Oestradiol benzoate also resulted in more intense LH receptor immunostaining of the tubal mucosal epithelium, smooth muscle cells and blood vessels as compared with controls. Western blotting has revealed that the pig oviduct, similar to the corpus luteum, contains 75, 48 and 45 kDa immunoreactive LH receptor proteins. Treatment with LH in vitro (100 ng ml-1) affected the contractility of oviduct. During the peri-ovulatory stage of the oestrous cycle, the amplitude, frequency and area under curve(s) of the isthmus decreased (P < 0.05), as did the frequency and area under curve (P < 0.05 and P < 0.01, respectively) of the ampulla (n = 4). The frequency and area under curve of the oviductal contractions were also significantly reduced during the early follicular phase of the oestrous cycle (P < 0.05). There was no effect of LH (or hCG) on the frequency and area under curve of the oviductal contractions during luteal stages of the oestrous cycle (n = 8). These data indicate that (1) the pig oviduct possesses immunoreactive and functional LH receptor, (2) oestradiol promotes the synthesis of LH receptor in the epithelium and smooth muscles, and (3) LH causes the relaxation of oviduct, especially during the peri-ovulatory stage of the oestrous cycle. In summary, the results of the present study indicate that LH can control oviductal contractions directly and may be partially responsible for the relaxation of isthmus during fertilization in pigs.

Persistence of biological activity of biotinylated human chorionic gonadotropin and its use for visualization of rat luteinizing hormone receptors in tissue sections.

Biotinylation of antibodies is an established method for producing systems for detection of antigens. We currently aim to develop liposomal targeting vectors for gene transfer into transgenic gonadal tumor cells expressing the luteinizing hormone (LH) receptor (R). We have biotinylated (B) human chorionic gonadotrophin (hCG) to obtain a selective targeting molecule to be attached to biotinylated liposomes via an avidin-streptavidin bridge. The biotinylation was performed by combining biotin isothiocyanate (BITC) and hCG in alkaline reaction buffer in a 100:1 (BITC:hCG) molar ratio. B-hCG maintained its ability to bind specifically to rat testicular membranes and was also bound to streptavidin-coated polypropylene wells. cAMP production was induced in BLT-1 Leydig tumor cells in vitro after stimulation with B-hCG, as a sign of persistent bioactivity. Frozen sections of rat testicular and ovarian tissues and skeletal muscle were labeled by incubating for 2 hr at 37 degrees C with 10 ng/microliter B-hCG. The binding was subsequently visualized by the avidin-biotin-peroxidase system, followed by silver enhancement of Ni-DAB staining. In rat testicular and ovarian sections, labeling was observed in structures known to strongly express the LH-R, i.e., Leydig cells, corpora lutea, and blood vessels. The labeling was blocked by preincubation with a 100-fold excess of the native hormone, and by injecting the rats sc with a high dose of hCG (1000 IU/kg) 48 hr before sacrifice. Skeletal muscle, used as negative control, was not labeled. These data demonstrate that the bioactivity of hCG is relatively well preserved after biotinylation. The biotinylated gonadotropin offers a new nonradioactive alternative for visualization of bioactive LH receptors in tissue sections.

Direct luteinizing hormone action triggers adrenocortical tumorigenesis in castrated mice transgenic for the murine inhibin alpha-subunit promoter/simian virus 40 T-antigen fusion gene.

Transgenic (TG) mice, expressing the Simian Virus 40 T-antigen (Tag) under a 6-kb fragment of the murine inhibin alpha-subunit promoter (inh alpha p), develop gonadal tumors of granulosa/theca or Leydig cell origin. We showed previously that adrenocortical tumors develop if the TG mice are gonadectomized but never develop in intact animals. However, if functional gonadectomy was induced by GnRH antagonist treatment or by cross-breeding the TG mice into the hypogonadotropic hpg genetic background, neither gonadal nor adrenal tumors appeared. Since the most obvious difference between the gonadectomized and GnRH-antagonist-treated or Tag/hpg double mutant mice is the elevated gonadotropin secretion in the first group, we examined whether the adrenal tumorigenesis would be gonadotropin-dependent. Surprisingly, both the adrenal tumors and a cell line (C alpha 1) derived from one of them expressed highly functional LH receptors (LHR), as assessed by Northern hybridization, immunocytochemistry, ligand binding, and human CG (hCG)-stimulated cAMP and steroid production. No FSH receptor expression was found in the adrenal tumors by RT-PCR. hCG treatment of the C alpha 1 cells stimulated their proliferation, as measured by [3H]thymidine incorporation. This effect was related to hCG-stimulated steroidogenesis since progesterone, testosterone, and estradiol, at physiological concentrations, also stimulated the C alpha 1 cell proliferation. Different adrenocortical cells expressed initially LHR and Tag, whereas both were highly expressed in the tumor cells. In conclusion, the high level of functional LHR in the adrenal tumors indicates that this receptor can function as tumor promoter when ectopically expressed and stimulated by the ligand hormone.

Transgenic mouse models for gonadal tumorigenesis.

The versatile transgenic (TG) techniques allow the production of in vivo animal models for a variety of diseases, including malignant tumors, through tissue-specific expression of oncogenes. We have created a TG mouse model for gonadal somatic cell tumors by expressing the powerful viral oncogene, Simian virus 40 T-antigen (Tag) under regulation of the murine inhibin alpha-subunit promoter (inh alpha). Ovarian granulosa and theca cell tumors were formed in the female, and those of testicular Leydig cells, in the male TG mice at the age of 5-6 months, with 100% penetrance. The tumors produced high levels of inhibin peptides, especially the alpha-subunit, and were steroidogenically active, mainly producing progesterone. The gonadal tumorigenesis was gonadotropin-dependent, since TG mice rendered gonadotropin-deficient by crossbreeding them into the hypogonadotropic hpg genetic background, or by treating them with a gonadotropin-releasing hormone (GnRH) antagonist, did not develop tumors. In order to study the possibility of using the tumor mouse model for testing gene therapy, we created another TG mouse model expressing under the same inhibin-alpha promoter the Herpes Simplex virus (HSV) thymidine kinase (TK) transgene. The inh alpha/HSV-TK mice were crossbred with the inh alpha/Tag mice and the double mutant mice also developed gonadal tumors. When they were treated with antiherpes drugs (acyclovir or gancyclovir), further growth of the tumors was blocked. These preliminary findings prove the principle that tumor ablation in our TG mouse model can be achieved by transduction of the HSV-TK gene into the tumor cells. Besides studies of formation, regulation and therapy of the tumors in vivo, immortalized cell lines derived from them provide models for studies of gonadal somatic cell functions in vitro.

Suppression of gonadotropins inhibits gonadal tumorigenesis in mice transgenic for the mouse inhibin alpha-subunit promoter/simian virus 40 T-antigen fusion gene.

We have previously developed a transgenic (TG) mouse model expressing the Simian virus 40 T-antigen (Tag), driven by a 6-kb fragment of the mouse inhibin alpha-subunit promoter (inh-alpha). The mice develop metastasizing gonadal tumors, of granulosa/theca or Leydig cell origin, with 100% penetrance by the age of 5-8 months. In the present study, we examined whether the appearance and growth of the gonadal tumors are dependent on gonadotropins. Gonadotropin suppression was achieved either by treatment of 3-month-old mice for 2-3 months with a GnRH antagonist (Cetrorelix, SB-75), or by cross-breeding the TG mice to the genetic background of the gonadotropin-deficient hypogonadal mutant mouse (hpg). Gonadal tumor growth was clearly inhibited by SB-75 treatment in one of the TG mouse lines (IT6-M), as indicated by the absence of macroscopically visible tumors and by reduced gonadal weights. Despite the suppressed gonadotropin secretion and Tag expression, hyperplasia of testicular Leydig, and ovarian stromal cells persisted in some of the treated mice. In another TG mouse line (IT6-F), with more aggressive tumorigenesis, the SB-75 treatment only partially inhibited gonadal tumor growth. None of the hypogonadotropic TG mice, homozygous for the hpg mutation, developed gonadal tumors. Their gonadal histology was indistinguishable from that of the non-TG hpg mice, suggesting total inhibition of gonadal tumorigenesis in the absence of gonadotropin stimulation. Tag expression and Leydig cell hyperplasia were apparent already in the postnatal TG mice but absent in those TG mice homozygous for the hpg mutation. In conclusion, the present results indicate that the gonadal tumorigenesis in our TG mouse model starts in early age as hyperplasia in specific somatic cells. Both this, and the subsequent malignant tumor growth, are gonadotropin dependent. A sufficient level of Tag expression, a prerequisite for gonadal tumorigenesis, only occurs upon gonadotropin stimulation.

Novel cationic liposomes for DNA-transfection with high efficiency and low toxicity.

Liposomes containing the natural cationic amphiphile, sphingosine and some of its derivatives were used for transfection of DNA in vitro. Multilamellar liposomes comprised of dioleoylphosphatidylethanolamine (DOPE), different sphingosine derivatives, and diacylglycerols with varying fatty acid chains, preincubated with DNA, transfected efficiently the KK-1 murine granulosa cells. Most efficient transfection on this cell line was achieved with liposomes composed of phytosphingosine, DOPE, and dioctanoylglycerol (DC8G) (64:31:4.8, molar stoichiometry), which gave expression of the transfected gene 2-10-fold higher than the commercial reagent Lipofectin. At higher doses the new liposomes also caused markedly less cell death of KK-1 cells. On COS-7 cells these liposomes showed slightly, but significantly lower transfection, of approximately 70%, of that gained with Lipofectin. The murine Sertoli cells, MSC-1, selectively resisted transfection by the sphingosine derivative based liposomes tested, giving only 11-14% of the expression detected in Lipofectin transfected cells of the same line. In conclusion, the novel liposomes formulated offer an effective, technically easy and economical method of transfection for a variety of cultured cell lines.

The mouse inhibin alpha-subunit promoter directs SV40 T-antigen to Leydig cells in transgenic mice.

Testicular tumorigenesis was observed in transgenic mice expressing the 6-kb mouse inhibin alpha-subunit promoter/Simian virus 40 T-antigen (SV40 Tag) fusion gene. The tumors were confined to Leydig cells using immunohistochemistry with anti-Tag antibody, specific binding of biotinylated hCG and histochemistry for 3 beta-hydroxysteroid dehydrogenase. Leydig cell hyperplasia and presence of Tag protein in the testicular interstitial tissue were already evident at 5 and 6.5 days of age, respectively. An immortalized cell line, BLT-1, was established from one testicular tumor. These cells expressed the LH receptor and P450scc mRNAs, and displayed LH-responsive cAMP and progesterone production, and low testosterone production. The cells also specifically bound 125I-labeled recombinant human LH with high affinity (36000 binding sites/cell), and the binding was regulated by 8Br-cAMP and hCG. This gonadal tumor model is valuable for further studies on endocrine functions of Leydig cells and their tumorigenesis in vivo and in vitro.

A novel role for testicular descent; temperature-dependent induction of pertussis toxin-sensitive Gi protein function in postnatal rat Leydig cells.

The role of temperature and testicular descent in postnatal appearance of inhibitory guanine nucleotide-binding regulatory protein (G(i)) function was studied in the rat testis. Dispersed testicular cells of 5-day-old rats were incubated for 24 h at 32 or 37 C, then for another 24 h at the same temperatures in the presence and absence of pertussis toxin (PT; 100 micrograms/liter), and finally for a third 24-h period with cholera toxin (CT; 500 ng/liter) with or without PT. At both temperatures, PT treatment significantly (P < 0.05) increased the CT-stimulated cAMP output, but had no effect on basal cAMP production. When testosterone (T) production, as an indicator of Leydig cell function, was measured in the same incubation, CT-stimulated T production was greater at 32 C, but PT had no effect at either temperature. A similar finding was made when hCG (10 micrograms/liter), instead of CT, was used as the stimulus of T production. Hence, a functional G(i) protein is present in seminiferous tubules of 5-day-old testes cultured for 3 days at 32 and 37 C, but not in Leydig cells. We then examined the effects of longer exposure of 5-day-old testes to the two temperatures. After culture for 7 days with 0.1 microgram/liter ovine LH, the presence of PT at 32 C significantly (P < 0.01) enhanced CT-stimulated T production during the last 24 h of culture, but the PT effect was not observed when the culture was carried out at 37 C. Hence, G(i)-mediated modulation of Leydig cell function appears to require several days of induction at the lower temperature of 32 C. As the postnatal descent also changes the ambient testicular temperature, we next studied whether this event alters the G(i) protein function of Leydig cells. Five-day-old rats were rendered bilaterally cryptorchid or sham operated, and studied after 12 days. Testis weights did not differ between the abdominal and scrotal testes. In contrast, the basal and hCG-stimulated rates of T production were significantly (P < 0.01-0.05) higher in the scrotal testes. When dispersed cells of the scrotal and abdominal testes were incubated for 24 h at 37 C in the presence of CT with or without PT, enhancement of T production by PT was only observed in cells of the scrotal testes.(ABSTRACT TRUNCATED AT 400 WORDS)

Induced ablation of gonadotropins in transgenic mice expressing herpes simplex virus thymidine kinase under the FSH beta-subunit promoter.

Suppression of gonadotropins was induced by gancyclovir or acyclovir treatment in transgenic mice carrying 2.3 kb of bovine follicle-stimulating hormone beta (FSH beta) promoter fused to Herpes simplex virus thymidine kinase (tk) coding sequence. Transgenic tk and endogenous FSH beta were immunohistochemically co-localized in the same pituitary cells. In adult castrated transgenic males, gancyclovir treatment reduced plasma FSH (30%, P < 0.001). In intact juvenile gancyclovir treated mice, the reduction of pituitary FSH, and in males also of plasma FSH, was greater (50-70%, P < 0.05-0.01). A concomitant suppression of luteinizing hormone (LH) (50%, P < 0.01) was observed in female pups. The most pronounced reduction of gonadotropins was observed in newborn transgenic pups treated in utero with acyclovir. Both males and females had significantly lower pituitary levels of FSH (75-55%), LH (80-90%) or both (P < 0.05-0.01). Less pronounced decreases (30-40%, P < 0.01) were observed in plasma FSH. No apparent defects were seen in the testes of the transgenic, acyclovir treated, newborn pups. This mouse model is applied to study the dynamics of the gonadotropes and the role of gonadotropins in the maturation of the reproductive functions.