Normal Sexual Development and Fertility in testatin Knockout Mice

The testatin gene was previously isolated in a screen focused on finding novel signaling molecules involved in sex determination and differentiation. testatin is specifically upregulated in pre-Sertoli cells in early fetal development, immediately after the onset of Sry expression, and was therefore considered a strong candidate for involvement in early testis development. testatin expression is maintained in the adult Sertoli cell, and it can also be found in a small population of germ cells. Testatin shows homology to family 2 cystatins, a group of broadly expressed small secretory proteins that are inhibitors of cysteine proteases in vitro but whose in vivo functions are unclear. testatin belongs to a novel subfamily among the cystatins, comprising genes that all show expression patterns that are strikingly restricted to reproductive tissue. To investigate a possible role of testatin in testis development and male reproduction, we have generated a mouse with targeted disruption of the testatin gene. We found no abnormalities in the testatin knockout mice with regard to fetal and adult testis morphology, cellular ultrastructure, body and testis weight, number of offspring, spermatogenesis, or hormonal parameters (testosterone, luteinizing hormone, and follicle-stimulating hormone)

Fetal but not adult Leydig cells are susceptible to adenoma formation in response to persistently high hCG level; a study on hCG overexpressing transgenic mice

We have previously demonstrated that male transgenic (TG) mice overexpressing human chorionic gonadotropin (hCG+) develop reproductive organ defects, but no tumors, in adult age. In this study, the effects of persistently elevated hCG were followed in TG males between day 5 postpartum and adulthood. Leydig cell (LC) adenomas were found in prepubertal mice, most prominently at the age of 10 days, but not in adult age. Serum testosterone concentrations were significantly increased in TG males at all ages studied. The phenotype of the prepubertal hCG+ males resembled that found in boys upon expression of constitutively activating luteinizing hormone (LH) receptor mutations. The temporal expression patterns of the fetal LC marker gene, thrombospondin 2, and those of adult LCs, hydroxysteroid dehydrogenase-6, delta5-3-beta and prostaglandin D synthase, were similar in wild-type and hCG+ males. Hence, the postnatal adenomas resemble functionally fetal LCs, and only these cells are susceptible to hCG-induced tumorigenesis. Our findings demonstrate a novel intriguing difference between the fetal and adult LC populations and provide further insight into the potential tumorigenic effects of gonadotropins.Fil: Ahtianen, Petteri. University of Turku; FinlandiaFil: Rulli, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Shariatmadari, Ramin. University of Turku; FinlandiaFil: Pelliniemi, Lauri J.. University of Turku; FinlandiaFil: Toppari, Jorma. University of Turku; FinlandiaFil: Poutanen, Matti. University of Turku; FinlandiaFil: Huhtaniemi, Ilpo T.. University of Turku; Finlandia. Imperial College London; Reino Unid

Trimeric Form of Intracellular ATP Synthase Subunit β of Aggregatibacter actinomycetemcomitans Binds Human Interleukin-1β.

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation

Effect of IL-1β on the formation of <i>A. actinomycetemcomitans</i> biofilm.

<p>The tested strains D7S (serotype a) and SA1151 (serotype c) were rough-colony forming clinical isolates, which produced fimbriae. Pre-grown (approximately 18 h) biofilms were grown with/without IL-1β (10 ng/ml) in RPMI 1640 medium, and the formation of biofilm was estimated with crystal violet staining after 6 h incubation. The box-plot represents data from four independent experiments.</p

Binding of IL-1β on <i>A. actinomycetemcomitans</i> cells.

<p>The binding on fixed <i>A. actinomycetemcomitans</i> cells was studied using flow cytometry. The cells were first treated with biotinylated IL-1β after which they were stained with avidin-FITC. Panel A presents the percentage of positively stained cells as detected with flow cytometer. Panel B shows the mean fluorescence intensity in positively stained cells. Results are shown as means + SD of three independent experiments. Statistically significant (Two sample paired T-test) differences between the test strain and Δ<i>flp1-flp2</i> mutant (flp1-flp2-) are marked with * and **, indicating p≤0.05 and p≤0.01, respectively. Solid line indicates the level obtained with the Δ<i>flp1-flp2</i> mutant. Biotinylated soybean trypsin inhibitor was used as a negative control protein.</p

IL-1β binding capacity of recombinant ATP synthase subunit β of <i>A. actinomycetemcomitans</i>.

<p>Recombinant ATP synthase subunit β (88 µM) was incubated with or without IL-1β (0.29 µM) for 1 h, after which the samples were run in native-PAGE and immunoblotted with anti-IL-1β (Panel A), or silver stained (Panel B). Three prominent forms could be observed from recombinant ATP synthase subunit β (Panel B). IL-1β bound to the trimeric form of recombinant ATP synthase subunit β. IL-1β was not detectable from immunoblotted native-PAGE without pre-incubation with ATP synthase subunit β (Panel A). However, IL-1β was released from the trimeric form of ATP synthase subunit β under denaturing conditions of SDS-PAGE (Panel C). The sizes of ATP synthase subunit β and IL-1β were 51 kDa and 17 kDa, respectively. The binding of the biotinylated control protein soy bean trypsin inhibitor (STI) to ATP synthase subunit β was was estimated similarly (Panel D and E) by using streptavidin-HRP in detection, except the SDS-page was not run. The control protein bound only to the ATP synthase subunit β monomer.</p