Thiol-ene micropillar array electrospray ionization platform for zeptomole level bioanalysis.

A micropillar array electrospray ionization (µPESI) platform fabricated from thiol-enes with 56 individual polyethylene glycol coated µPESI chips for bioanalytical mass spectrometry is introduced. Bioanalysis capability is shown by measurement of a protein, a protein digest and a cell lysate sample. The thiol-ene polyethylene glycol (PEG) coated µPESI chip allows the use of a wide range of aqueous-organic solvent compositions and provides a detection limit at 60 zeptomole level (6 × 10-20 mol) for a peptide standard.

Hormonal and photoperiodic modulation of testicular mRNAs coding for inhibin/activin subunits and follistatin in Clethrionomys glareolus, Schreber.

Photoperiodic and hormonal modulation of mRNAs for testicular inhibin/activin subunits and follistatin were studied in a seasonally breeding rodent, the bank vole (Clethrionomys glareolus). Photoperiod-induced testicular regression had no effect on the relatively low steady-state levels of follistatin mRNA. Inhibin alpha (I alpha) and beta B (I beta B) mRNA levels were significantly higher in regressed than in active gonads, but inhibin beta A was undetectable. The effect of gonadotropin administration on testicular weight and mRNA concentrations differed between the sexually active and quiescent voles. Neither FSH (1.2 U/kg; s.c. for 5 days) nor hCG (600 IU/kg; s.c. for 5 days) affected testicular weight in sexually active voles, whereas both gonadotropins significantly increased testicular weight in photo-regressed individuals. FSH had no effect on I alpha or I beta B mRNA concentrations in the active testes, whereas excessive hCG challenge induced a decrease in the steady-state levels of these mRNAs. FSH induced an increase in I alpha mRNA concentrations in the regressed gonad, whereas both gonadotropins concomitantly down-regulated I beta B mRNA levels. In conclusion, the high expression of I alpha and I beta B mRNA in the regressed testis imply autocrine and paracrine roles for inhibin/activin in the quiescent gonad of seasonal breeders. Inhibin alpha-subunit expression is at least partly under the control of FSH in the bank vole testis.

Photoperiod-induced changes in androgen receptor expression in testes and accessory sex glands of the bank vole, Clethrionomys glareolus.

Photoperiodic modulation of androgen levels and androgen receptor (AR) expression in testes and accessory sex glands were studied in a seasonally breeding rodent, the bank vole. Juvenile voles subjected to long photoperiod (20L:4D) for 6-8 wk attained sexual maturity, which was associated with a prominent increase in testicular testosterone (T) levels and weight of testes and accessory sex glands. Pubertal development in short photoperiod-treated (6L:18D for 6-8 wk) juveniles was arrested, and subsequently reproductive regression set in with a marked decrease in testicular T levels and gonadal weight. In sexually active voles, strong AR immunostaining was detected in nuclei of epithelial, smooth muscle, and stromal cells of the epididymis, prostate, and seminal vesicles. In active testes, AR was present in nuclei of Sertoli cells, peritubular cells, Leydig cells, and vascular smooth muscle cells. In juveniles, strong to moderate nuclear immunoreactivity was encountered in epithelial and stromal cells of the epididymis and prostate, whereas a weaker reaction was discerned in seminal vesicles. In juvenile testes, AR was localized to vascular smooth muscle cells, peritubular, and interstitial cells. In sexually regressed animals, nuclear staining was almost absent in accessory sex glands, whereas in testes, moderate immunostaining was retained in all other cell types except the Sertoli cells. Western blots of active and regressed testes indicated a marked photoperiod-induced down-regulation of immunodetectable AR in the regressed gonad.

Hormonal and photoperiod-induced changes in testicular interleukin-1-like factor in bank voles, Clethrionomys glareolus.

Testicular interleukin-1-like factor (tIL-1) is a cytokine secreted presumably by Sertoli cells in several mammalian species. The function of this cytokine is unknown: tIL-1 may control meiosis, act as a mitogen for spermatogonia or have both of these functions. The present investigation was conducted to assess tIL-1 activity and its hormonal control in a seasonally breeding photoperiodic mammal during testicular maturation and photoperiod-induced regression. Testicular maturation in long photoperiod (20 h light:4 h dark) was accompanied by the appearance of tIL-1 activity at the age of 32-39 days which increased as full sexual maturity was reached. No significant tIL-1 activity was detected when pubertal development was inhibited or testicular regression induced by subjecting juvenile and adult bank voles to a short photoperiod (6 h light: 18 h dark) for 6 to 8 weeks. Administration of human chorionic gonadotrophin (hCG; 60 IU kg-1) increased tIL-1 activity in sexually mature as well as regressed testes. In the photoregressed voles FSH (1.2 U kg-1) administration, which induced a three-fold increase in testicular weight and stimulated spermatogenesis, did not induce detectable concentrations of tIL-1. Administration of FSH followed by hCG increased tIL-1 activity significantly in the atrophic testis, but this was probably due to hCG, since FSH treatment alone was without effect. In conclusion, in accordance with the proposed role of tIL-1 as a germ-cell mitogen and a meiosis-promoting factor, tIL-1 activity correlated positively with spermatogenic activity during testicular maturation and photoperiod-induced regression.(ABSTRACT TRUNCATED AT 250 WORDS)

Early steroid metabolism by chick blastoderm in vitro.

Yolk free blastoderms of chick embryo were incubated 3 or 22 hours with labeled pregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone and estradiol-17 beta. Metabolites and unconverted substrates were found both in the incubation medium and in the cells. Enzymes responsible for identified conversions were: 17 alpha-hydroxylase, 17-20-desmolase, delta 5 3 beta- and 3 alpha-hydroxysteroid dehydrogenase, 17 beta-hydroxysteroid dehydrogenase and 5 alpha- and 5 beta-reductase. The results suggest that the steroid metabolizing enzyme activities found may reflect a more general ability of early embryonic cells.

Lack of correlation between mesenchymal cell death and morphogenesis after different extents of apical ectodermal ridge/rim ectoderm removal in the chick embryo wing bud.

The removal of the apical ectodermal ridge (A.E.R.) subsequently causes distal deletion defects in the limb. There have been contradictory reports as to the appearance of cell death in the mesenchyme after A.E.R. removal, as well as to its morphogenetic significance. In our study the A.E.R./ rim ectoderm removal was varied to test whether different degrees of cell death would correlate with different degrees of distal deletions. From the right wing bud of stage 19 and 20 (HH) embryos the rim ectoderm was removed in four ways: all of the rim, the anterior third, the middle third (most of the A.E.R.), or its posterior third. The removal of all or of the anterior third caused a definite band of subwound mesenchymal cell death to appear. There was little or no cell death after removal of the middle or posterior thirds. Removal of the anterior third caused no distal deletion defects, and only a few were noted after removal of the posterior third. The proximo-distal level of the distal deletions, however, was the same after removal of all of the rim or only its middle third. As there was no difference in the degree of distal deletions after the removal of all or of the middle third of the rim but a definite difference in the mesenchymal cell death patterns we conclude that cell death is not part of the mechanisms of the distal deletion defect. Our findings also suggest that cell death does not play a role in the A.E.R.-mesenchyme reciprocal interaction that controls limb proximo-distal morphogenesis.