Gene expression profiling of mouse Sertoli cell lines

The proliferation and differentiation of Sertoli cells is regulated by follicle-stimulating hormone (FSH). The molecular events following FSH stimulation are only partially known. To investigate FSH action in Sertoli cells, we established two novel FSH-responsive mouse Sertoli-cell-derived lines expressing human wild-type (WT) FSH receptor (FSHR) or overexpressing mutated (Asp567Gly) constitutively active FSHR (MUT). Gene expression profiling with commercially available cDNA arrays, including 588 mouse genes, revealed 146 genes expressed in both cell lines. Compared with the expression pattern of WT cells, 20 genes were identified as being either up- or down-regulated (>two-fold) in the MUT cells. We observed a strong differential expression of factors involved in cellular proliferation, e.g. cyclin D2 (repressed to nearly undetectable levels), proliferating cell nuclear antigen (2.5-fold repression) and Eps-8 (six-fold repression), and in genes involved in cellular differentiation, e.g. cytokeratin-18 (13-fold induction). The cDNA array results for six representative genes were confirmed by Northern blotting, which also included the parental SK-11 cell line devoid of FSHR expression. We found no further acute FSH- or forskolin-induced change in expression levels after 3-h stimulations, suggesting that the observed differences between the two cell lines is a consequence of mild, chronically increased, cAMP production in MUT cells. These results provide a platform for the further investigation of selected candidate genes in primary cultures and/or in vivo.Electronic Supplementary Material Supplementary material is available in the online version of this article at This work was supported by grants from the Deutsche Forschungsgemeinschaft (Confocal Research Group The Male Gamete: Production, Maturation, Function, grant FOR 197-3) and from the German Academic Exchange Service (DAAD) to P. Mathur     

Pharmacokinetics and pharmacodynamics of injectable testosterone undecanoate in castrated cynomolgus monkeys (Macaca fascicularis) are independent of different oil vehicles

Testosterone undecanoate (TU) dissolved in soybean oil was developed in China to improve the pharmacokinetics of this testosterone ester in comparison with TU in castor or tea seed oil. As a pre-clinical primate model, three groups of five castrated cynomolgus macaques received either a single intramuscular injection of 10 mg/kg body weight TU in soybean oil, in tea seed oil, or in castor oil (equals 6.3 mg pure T/kg body weight for all preparations). Testosterone, estradiol, luteinizing hormone, and follicle-stimulating hormone as well as prostate volume, body weight and ejaculate weight were evaluated. After injection supraphysiological testosterone levels were induced. There were no significant differences in the pharmacokinetics of the three TU preparations for testosterone and estradiol. The gonadotropin levels showed a high individual variation. Prostate volumes increased equally in all groups after administration and declined to castrate level afterwards. The results suggest that TU in soybean oil produces similar effects as TU in the other vehicles. This study in non-human primates provides no objection to testing of this new preparation in humans.     

An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.     

Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes

Although Group IV cytosolic phospholipase A2 (cPLA2) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA2 as well as evoked AA release. However, complete inhibition of phospho-ERK by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), did not completely inhibit PMA-stimulated cPLA2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA2[largely through a protein kinase C (PKC)-independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA2 due to phospho-ERK is not sufficient to evoke AA release. However, complete inhibition of ATP-induced cPLA2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP-induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA-induced responses to PKC down-regulation, prolonged treatment with PMA resulted in down-regulation of PKCalpha and epsilon in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCalpha from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA2 phosphorylation and AA release in astrocytes through both ERK1/2-dependent and ERK1/2-independent pathways.     

The beta subunit of the Escherichia coli ATP synthase exhibits a tight membrane binding property

We have developed a chromatographic procedure to analyze the association of the subunits of the Escherichia coli F1Fo-ATP synthase with the cytoplasmic membrane. Minicells containing [35S]-labeled ATP synthase subunits are treated with lysozyme, solubilized, and chromatographed on a Sepharose CL-2B column in buffer containing urea and taurodeoxycholate. ATP synthase subunits are resolved into membrane intrinsic and membrane extrinsic subunits. Interestingly, a significant amount (36%) of the F1 subunit beta fractionates with the membrane intrinsic Fo subunits. About half of this amount (19%) of beta is non-specifically bound to the membrane. Interaction of beta with the membrane is not mediated by the amino terminal portion of beta.     

Adiponectin Modulates Oxidative Stress-Induced Autophagy in Cardiomyocytes

Diastolic heart failure (HF) i.e., “HF with preserved ejection fraction” (HF-preserved EF) accounts for up to 50% of all HF presentations; however there have been no therapeutic advances. This stems in part from an incomplete understanding about HF-preserved EF. Hypertension is the major cause of HF-preserved EF whilst HF-preserved EF is also highly associated with obesity. Similarly, excessive reactive oxygen species (ROS), i.e., oxidative stress occurs in hypertension and obesity, sensitizing the heart to the renin-angiotensin-aldosterone system, inducing autophagic type-II programmed cell death and accelerating the propensity to adverse cardiac remodeling, diastolic dysfunction and HF. Adiponectin (APN), an adipokine, mediates cardioprotective actions but it is unknown if APN modulates cardiomyocyte autophagy. We tested the hypothesis that APN ameliorates oxidative stress-induced autophagy in cardiomyocytes. Isolated adult rat ventricular myocytes were pretreated with recombinant APN (30µg/mL) followed by 1mM hydrogen peroxide (H₂O₂) exposure. Wild type (WT) and APN-deficient (APN-KO) mice were infused with angiotensin (Ang)-II (3.2mg/kg/d) for 14 days to induced oxidative stress. Autophagy-related proteins, mTOR, AMPK and ERK expression were measured. H₂O₂ induced LC3I to LC3II conversion by a factor of 3.4±1.0 which was abrogated by pre-treatment with APN by 44.5±10%. However, neither H₂O₂ nor APN affected ATG5, ATG7, or Beclin-1 expression. H₂O₂ increased phospho-AMPK by 49±6.0%, whilst pretreatment with APN decreased phospho-AMPK by 26±4%. H₂O₂ decreased phospho-mTOR by 36±13%, which was restored by APN. ERK inhibition demonstrated that the ERK-mTOR pathway is involved in H₂O₂-induced autophagy. Chronic Ang-II infusion significantly increased myocardial LC3II/I protein expression ratio in APN-KO vs. WT mice. These data suggest that excessive ROS caused cardiomyocyte autophagy which was ameliorated by APN by inhibiting an H₂O₂-induced AMPK/mTOR/ERK-dependent mechanism. These findings demonstrate the anti-oxidant potential of APN in oxidative stress-associated cardiovascular diseases, such as hypertension-induced HF-preserved EF.     

Enhancing our Understanding of Protein Structure: the Work of Jane and David Richardson

Jane and David Richardson have worked together since 1964, and their research has made strides in enhancing our understanding of protein structure. Two of the crystal structures they solved are staphylococcal nuclease and copper, zinc superoxide dismutase, both of which were published in the Journal of Biological Chemistry Classic papers reprinted here.A High Resolution Structure of an Inhibitor Complex of the Extracellular Nuclease of Staphylococcus aureus. I. Experimental Procedures and Chain Tracing (Arnone, A., Bier, C. J., Cotton, F. A., Day, V. W., Hazen, E. E., Jr., Richardson, D. C., Richardson, J. S., and, in part, Yonath, A. (1971) J. Biol. Chem. 246, 2302–2316)The Crystal Structure of Bovine Cu²⁺,Zn²⁺ Superoxide Dismutase at 5.5-Å Resolution (Thomas, K. A., Rubin, B. H., Bier, C. J., Richardson, J. S., and Richardson, D. C. (1974) J. Biol. Chem. 249, 5677–5683)From an early age, Jane Shelby Richardson was interested in astronomy; she came in third in the Westinghouse Science Talent Search by calculating the orbit of the Sputnik satellite from her own observations. Wanting to continue her science education, she enrolled at Swarthmore College as a math, physics, and astronomy major. However, she became interested in philosophy and ended up switching majors. It was at Swarthmore that she met David Richardson. Both Jane and David graduated in 1962, and they married a year later. David went on to the Massachusetts Institute of Technology to work on a doctorate in chemistry while Jane went to Harvard University to get a degree in philosophy. Opting out after a year with a Master''s degree, Jane tried teaching high school science and in 1964 settled in to a job as a technician in the lab where David was doing graduate research.Open in a separate windowJane and David RichardsonWhen David was accepted at MIT, he decided to join the laboratory of F. Albert Cotton, who was working on small molecule inorganic chemistry and crystallography. Having become intrigued by biochemistry, David wanted to solve the structure of a protein and settled on staphylococcal nuclease upon the suggestion of Nobel laureate and Journal of Biological Chemistry (JBC) Classic author Christian Anfinsen (1). At that point, only two protein structures had been solved, those of hemoglobin and myoglobin. The methodology of protein crystallography was still in its infancy, so David and Jane along with Ted Hazen tested crystal growth conditions one at a time in batch (2), modified a small molecule x-ray diffractometer (controlled by punched paper tape) to take good protein data, wrote all their own software, and first learned what amino acids look like in 2 Å electron density when their structure was solved.After working for 7 years, they determined the structure of the nuclease in 1969, as reported in the first JBC Classic reprinted here, tying with Eisenberg and Dickerson''s cytochrome c for the 10th new protein structure. The 2 Å resolution staphylococcal nuclease structure showed the arrangement of β and helical features, the presence of disordered terminal tails and a partially disordered loop, and the geometry of binding a Ca²⁺ and the thymidine 3′,5′-diphosphate inhibitor at the active site. As envisioned by Anfinsen, this relatively simple single-domain protein did indeed become an important model system for the study of protein folding, first at NIH and later at Johns Hopkins.Shortly after this feat, David was offered a faculty position in biochemistry at Duke University. He and Jane established a lab there and started working on the structure of copper,zinc superoxide dismutase, which was a favorite enzyme at Duke (its function was discovered by JBC Classic author Irwin Fridovich (3), the sequence was solved in JBC Classic author Robert L. Hill''s lab (4), and a number of other people in the department have worked on it). Superoxide dismutases catalyze the dismutation of superoxide into oxygen and hydrogen peroxide, making them an important antioxidant defense in nearly all cells exposed to oxygen.By 1972, David and Jane were able to get crystals of the bovine superoxide dismutase with strong diffraction out to 2 Å, published in a short communication in the JBC (5). Two years later, they solved the structure of the enzyme at 5.5 Å, as reported in the second JBC Classic reprinted here. From their data, they concluded that each subunit of the dimeric enzyme had extensive β structure surrounding a central hydrophobic core, and they were able to determine a probable location for the zinc ion from a mercury for zinc substitution. They traced the polypeptide chain from further data at 3 Å, showing that the protein''s dominant structural feature was an 8-stranded barrel of antiparallel β-pleated sheet and that the copper and zinc were only 6 Å apart at the active site, sharing a His ligand. The β-sheet topology was compared with immunoglobulins, nuclease, and others, leading to definition of the “Greek key” β-barrel fold (6). At 2 Å resolution, the overall structure and active site were described in detail, including electrostatic guidance of substrate (7).In the 1980s the Richardsons started exploring synthetic biochemistry and computational biology and helped open up the field of protein de novo design. In the 1990s they pioneered molecular graphics for personal computers by developing the kinemage system of molecular graphics and the Mage program to display them on small computers, and they developed all-atom contact analysis to measure goodness of fit inside proteins and in interactions with surrounding molecules. The Richardson lab currently studies structural motifs in RNA as well as proteins, as part of the RNA Ontology Consortium, collaborates in development of the Phenix crystallographic software system, and hosts the MolProbity web service for validation and accuracy improvement of protein and RNA experimental structures.In addition to the accomplishments above, Jane is widely known for her creation of ribbon drawings to schematize protein 3D structures, first published in Advances in Protein Chemistry in 1981 (8). The drawings stemmed from Jane''s realization that a general classification scheme could be developed from the recurring patterns of structural motifs within the “folds” of proteins. She created ribbon drawings of those folds, making a uniform set of conventions for drawing the 75 protein structures that had been solved at that time, including the superoxide dismutase shown in Fig. 1. Computer versions of the ribbon representation have since become one of the most common ways of visualizing proteins.Open in a separate windowFIGURE 1.Hand-drawn ribbon schematic of the Cu,Zn-superoxide dismutase subunit, by Jane Richardson.Jane and David remain in the Department of Biochemistry at Duke University, where Jane is a James B. Duke professor and David is a professor. Although Jane does not have a Ph.D. degree, she has been given three honorary doctorates, from Swarthmore College, the University of North Carolina Chapel Hill, and the University of Richmond. In 1985 she was awarded a MacArthur Fellowship for her work in structural biology, and she was elected to the National Academy of Sciences and the American Academy of Arts and Sciences in 1991 and to the Institute of Medicine in 2006. In 2010, Jane was elected president of the Biophysical Society. She also is the co-recipient of the Protein Society''s Amgen Award with David (1995) and the Biophysical Society''s Emily M. Gray Award (2001). David is the founding director of the Structural Biology and Biophysics Graduate Training Program at Duke and also has received numerous honors including Science Digest''s 100 Best Innovations of 1985, a BioTechnology Winter Symposium Special Achievement Award (1995), and the Duke University Gordon Hammes Teaching and Mentoring Award (2009).     

Blood clotting and coagulation factors: the work of Yale Nemerson. 1980, 1982

After developing a blood disorder, Yale Nemerson became interested in hematology. This led to his lifelong study of thrombogenic tissue factor and to his contributions to developing the modern theory of blood coagulation. The two Classic papers reprinted here detail some of Nemerson's studies on coagulation factors IX and VII.