Induction of female-to-male sex change in the honeycomb grouper (Epinephelus merra) by 11-ketotestosterone treatments

The honeycomb grouper, Epinephelus merra, is a protogynous hermaphrodite fish. Sex steroid hormones play key roles in sex change of this species. A significant drop in endogenous estradiol-17beta (E2) levels alone triggers female-to-male sex change, and the subsequent elevation of 11-ketotestosterone (11KT) levels correlates with the progression of spermatogenesis. To elucidate the role of an androgen in sex change, we attempted to induce female-to-male sex change by exogenous 11KT treatments. The 75-day 11KT treatment caused 100% masculinization of pre-spawning females. Ovaries of the control (vehicle-treated) fish had oocytes at various stages of oogenesis, while the gonads of the 11KT-treated fish had transformed into testes; these contained spermatogenic germ cells at various stages, including an accumulation of spermatozoa in the sperm duct. In the sex-changed fish, plasma levels of E2 were significantly low, while both testosterone (T) and 11KT were significantly increased. Our results suggest that 11KT plays an important role in sex change in the honeycomb grouper. Whether the mechanism of 11KT-induced female-to-male sex change acts through direct stimulation of spermatogenesis in the ovary or via the inhibition of estrogen synthesis remains to be clarified.     

Living in three dimensions

Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture. In vitro approximations of these nanostructured surfaces are therefore desirable for more physiologically mimetic model systems to study both normal and abnormal functions of cells, tissues, and organs. In addition, the development of 3D culture environments is imperative to achieve more accurate cell-based assays of drug sensitivity, high-throughput drug discovery assays, and in vivo and ex vivo growth of tissues for applications in regenerative medicine.     

Expression of cyclooxygenase-2 related to angiogenesis in uterine cervical cancers

Summary Angiogenesis is essential for development, growth and advancement of solid tumors. Cyclooxygenase (COX)-2 is recognized as an angiogenic factor in various tumors. This prompted us to study the clinical implications of COX-2 expression related to angiogenesis in uterine cervical cancers. There was a significant correlation between microvessel counts and COX-2 levels in uterine cervical cancers. COX-2 localized in the cancer cells, but not in the stromal cells of uterine cervical cancer tissues. COX-2 levels increased with advancement, and the prognosis of the 30 patients with high COX-2 expression in uterine cervical cancers was poor (60%), while the 24-month survival rate of the other 30 patients with low COX-2 expression was 90%. Furthermore, COX-2 levels significantly correlated with VEGF levels in uterine cervical cancers. VEGF associated with COX-2 might work on angiogenesis in advancement. Therefore, long-term administration of COX-2 inhibitors might be effective on the suppression of regrowth or recurrence after intensive treatment for advanced uterine cervical cancers.     

Changes in androgen-producing cell size and circulating 11-ketotestosterone level during female-male sex change of honeycomb grouper Epinephelus merra

11-ketotestosterone (11-KT), a potent male-specific androgen in fish, has important roles on spermatogenesis, male behavior, and nuptial coloration. The site of 11-KT synthesis and its role on male germ cell development during protogynous sex change is not clearly understood. We examined the dynamics of steroidogenic enzymes immunolocalization, viz cholesterol side-chain cleavage (P450scc), biomarker of steroids and cytochrome P45011beta-hydroxylase (P45011beta), downstream to 11-KT production, throughout the process of sex change in honeycomb grouper (Epinephelus merra). In female, P450scc immunoreactivity (-ir) was observed in the theca layer and tunica near blood vessels (BV). During the onset of sex change, P450scc reactive cells were observed in the remaining follicle layer of degenerated oocyte of the ovo-testis in early transitional (ET) and late transitional (LT). In male, P450scc-ir was localized in the interstitial Legdig cells of testis. P45011beta reactive cells were observed in the tunica near BV in female but not in theca layer. In ET and LT phases gonads, P45011beta localized in remaining follicle layer of degenerated oocyte and tunica near BV. On the other hand, in male, both interstices and tunica near BV showed strong signals against P45011beta. Moreover, in vivo and in vitro levels of 11-KT related with the changes in the nuclei diameter of P45011beta-positive cells in both tunica near BV and remaining follicle layer of degenerated oocyte to interstices during the progress of sex change. The present results suggest that 11-KT produced in the tunica near BV may provide the stimulus for female to degenerate oocytes and initiate sex change. However, 11-KT produced both in tunica near BV and remaining follicle layer of degenerated oocyte possibly plays critical role during testicular differentiation as well as gonadal restructuring at mid to late phases (ET to LT) of sex change in honeycomb grouper.     

Structural basis for ubiquitin recognition by the human ESCRT-II EAP45 GLUE domain

The ESCRT-I and ESCRT-II complexes help sort ubiquitinated proteins into vesicles that accumulate within multivesicular bodies (MVBs). Crystallographic and biochemical analyses reveal that the GLUE domain of the human ESCRT-II EAP45 (also called VPS36) subunit is a split pleckstrin-homology domain that binds ubiquitin along one edge of the beta-sandwich. The structure suggests how human ESCRT-II can couple recognition of ubiquitinated cargoes and endosomal phospholipids during MVB protein sorting.     

Variation in inflammation as a correlate of range expansion in Kenyan house sparrows

Many introduced animals harbor fewer parasites than native ones. This “enemy release” can select for individuals that bias resources away from parasite resistance traits, including immune functions, and towards traits that enhance success in new areas. One vertebrate example that supports this hypothesis involves house sparrows (Passer domesticus) and Eurasian tree sparrows (Passer montanus) introduced to St. Louis, MO, USA, over 150 years ago. Since ca. 1850, house sparrows have colonized most of North America whereas tree sparrows have expanded little from the area of introduction. The more successful house sparrows now exhibit weaker inflammatory responses than the less successful tree sparrows, which supports the possibility that diminished investments in immune defense may have been conducive to the initial colonization by the more successful species. The goal of the present study was to determine whether damped inflammation generally facilitates invasion by comparing inflammatory markers between house sparrows invading Kenya and a native congener. House sparrows arrived in Mombasa, Kenya, about 50 years ago whereas rufous sparrows (Passer ruficinctus) are native but ecologically similar. We predicted that if inflammation mediated invasion success, Kenyan house sparrows would mount weaker inflammatory responses than the native species. Complete Freund’s adjuvant (CFA), a strong inflammatory stimulus, increased body mass in house sparrows, a result unprecedented in any other vertebrate. Haptoglobin (Hp), a multi-functional acute phase protein, was elevated by CFA in both species but rufous sparrows maintained more Hp than house sparrows irrespective of treatment. Lysozyme, a broadly effective antimicrobial enzyme, was reduced by CFA in both species, but not differentially so. Corticosterone was unaffected by CFA in either species, but elevated in both relative to free-living individuals.     

Chemokine‐cleaving Streptococcus pyogenes protease SpyCEP is necessary and sufficient for bacterial dissemination within soft tissues and the respiratory tract

SpyCEP is a Streptococcus pyogenes protease that cleaves CXCL8/IL‐8 and its activity is associated with human invasive disease severity. We investigated the role of SpyCEP in S. pyogenes necrotizing fasciitis and respiratory tract infection in mice using isogenic strains differing only in SpyCEP expression. SpyCEP cleaved human CXCL1, 2, 6 and 8 plus murine CXCL1 and 2 at a structurally conserved site. Mice were infected in thigh muscle with a strain of S. pyogenes that expresses a high level of SpyCEP, or with an isogenic non‐SpyCEP expressing strain. SpyCEP expression by S. pyogenes hindered bacterial clearance from muscle, and enhanced bacterial spread, associated with cleavage of murine chemoattractant CXCL1. Mice were then infected with Lactococcus lactis strains that differed only in SpyCEP expression. In contrast to the parent L. lactis strain (lacks SpyCEP), which was avirulent when administered intramuscularly, infection with a strain that expressed SpyCEP heterologously led to dramatic systemic illness within 24 h, failure to clear bacteria from muscle and marked dissemination to other organs. In the upper airways, SpyCEP expression was required for survival of L. lactis but not S. pyogenes. However, dissemination of S. pyogenes to the lung was SpyCEP‐dependent and was associated with evidence of chemokine cleavage. Taken together, the studies provide clear evidence that SpyCEP is necessary and sufficient for systemic bacterial dissemination from a soft tissue focus in this model and also underlies dissemination in the respiratory tract.     

C-terminal processing of GABARAP is not required for trafficking of the angiotensin II type 1A receptor

ObjectiveGABARAP, a small (117 aa) trafficking protein, binds to the C-terminal, cytoplasmic domain of rat angiotensin type-1A receptor (AT₁R), the predominant effector of the octapeptide angiotensin II (Ang II) (Cook et al., Circ. Res. 2008;102:1539–47). The objectives of this study were to map the interaction domains of GABARAP and AT₁R, to determine the effect of GABARAP association on AT₁R signaling activity, and to determine the importance of post-translational processing of GABARAP on accumulation of AT₁R on the plasma membrane and its signaling function.ResultsDeletion analysis identified two regions within GABARAP necessary for interaction with AT₁R in yeast two-hybrid assays: 1) a domain comprised of residues 32–51 that is nearly identical to that involved in binding and intracellular trafficking of the GABA_A receptor and 2) a domain encompassing the C-terminal 21 aa. The GABARAP interaction domain of AT₁R was delimited to the 15 aa immediately downstream of the last membrane spanning region. Overexpression of GABARAP in rat adrenal pheochromocytoma PC-12 cells increased the cell-surface expression of AT₁R and Ang II-dependent activation of the cAMP signaling pathway. Residues within AT₁R necessary for these responses were identified by mutational analysis. In PC-12 cells, GABARAP was constitutively and quantitatively cleaved at the C-terminus peptide bond and this cleavage was prevented by mutation of Gly¹¹⁶. Wild-type GABARAP and the G116A mutant were, however, equally effective in stimulating AT₁R surface expression and signaling activity.ConclusionsGABARAP and AT₁R interact through discrete domains and this association regulates the cell-surface accumulation and, consequently, ligand-induced function of the receptor. Unlike that observed with the GABA_A receptor, this regulation is not dependent on C-terminal processing and modification of GABARAP.