The presence of ANP in rat peritoneal mast cells

Atrial natriuretic peptide （ANP） is an important component of the natriuretic peptide system. A great role in many regulatory systems is played by mast cells. Meanwhile involvement of these cells in ANP activity is poorly studied. In this work, we have shown the presence of ANP in rat peritoneal mast cells. Pure fraction of mast cells was obtained by separation of rat peritoneal cells on a Percoll density gradient. By Westem blotting, two ANP-immunoreactive proteins of molecular masses of 2.5 kDa and 16.9 kDa were detected in lysates from these mast cells. Electron microscope immunogold labeling has revealed the presence of ANP-immunoreactive material in storage, secreting and released granules of mast cells. Our findings indicate the rat peritoneal mast cells to contain both ANP prohormone and ANP. These both peptides are located in mast cell secretory granules and released by mechanism of degranulation. It is discussed that many mast cell functions might be due to production of natriuretic peptides by these cells.

Desmosterol, the main sterol in rabbit semen： distribution among semen subfractions and its role in the in vitro spermatozoa acrosome reaction and motility

Sterols are essential components of the cell membrane lipid bilayer that include molecules such as cholesterol and desmosterol, which are significantly found in the spermatozoa of several animal species. However, the presence of desmosterol in rabbit semen has never been investigated. The aims of this study were to characterize the sterol composition of subfractions of ejaculated rabbit semen and evaluate the in vitro effects of sterol on the spermatozoa acrosome reaction and motility. Two sterols, occurring prevalently in the free form （94.3%）, were identified in whole semen collected from 10 fertile New Zealand White rabbits, specifically desmosterol （58.5% of total sterols） and cholesterol （35.9% of total sterols）. Desmosterol was the predominant sterol found in all subfractions of rabbit semen, varying from 56.7% （in the prostatic secretory granules, PSGs） to 63.8% （in the seminal plasma）. Spermatozoa contained an intermediate proportion of desmosterol （59.8%）, which was asymmetrically distributed between the heads （52.0% of the total content of sterols） and the tails （81.8%）. Results showed that both desmosterol and cholesterol can be transferred from the PSGs to the spermatozoa and are equally effective in inhibiting in vitro spermatozoa capacitation at a concentration higher than 1 mg L^-1. In contrast, neither desmosterol nor cholesterol had a significant effect on spermatozoa motility. Thus, it was concluded that, the various fractions of rabbit seminal fluid differ from each other in sterol composition and quantity, probably due to their different functional properties, and these fractions may undergo significant sterol changes depending on the stage of spermatozoa capacitation.

Effects of Estrogen and/or Progesterone on Morphology of Pituitary Luteinizing Hormone Cells

Objective To identify the morphological characteristics of pituitary luteinizing hormone (LH) cells after exogenous gonadal hormone treatment Methods Effects of various doses of estrogen, progesterone and their combination on morphological parameters, including the size and shape of pituitary LH cells, the size of endocellular vacuoles, were observed and measured by immuno histochemistry and computer image analysis. Results Different kinds of gonadal hormones could recover the magnified LH cells to the normal level in ovariectomized rats. However, their final effects on the gonadotrophin levels and the cellular morphological characters of the LH cells were different. The low dose of estrogen elicited abundant hormone stored in the LH cells to an easy released status with a lot of different size of vacuoles. On the contrary, the high dose of estrogen inhibited the storage of LH, and the LH cells were filled with secretory granules and few vacuoles. The progesterone could promote the storage of LH in an uneasy released status. The administration of estrogen progesterone combination not only inhibited the storage of LH, but also the release of LH. In this group, the LH cells containing a large amount of secretory granules and a few vacuoles showed a better uniform shape compared with those administrated with high dose of estrogen. Conclusion: Different kinds of gonadal hormones could reverse the excessive secretion of LH and recover the morphological change of LH cells to the normally physiological condition.

GENERATION OF TRANSGENIC MICE FOR IN VIVO DETECTION OF INSULIN-CONTAINING GRANULE EXOCYTOSIS AND QUANTIFICATION OF INSULIN SECRETION

Insulin secretion is a complex and highly regulated process.Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation,it remains unclear how conclusions from these studies apply to living animals.That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo.When combined with genetically encoded biosensor,optical imaging is a powerful tool for visualization of molecular events in vivo.In this study,we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins,a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol.Upon exocytosis of secretory granules,the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH,while mCherry fluorescence remained constant in the process,thus allowing ratiometric quantification of insulin secretory events.Furthermore,mCherry fluorescence enabled tracking the movement of secretory granules in living cells.We validated this approach in insulin-secreting cells,and generated a transgenic mouse line expressing the optical sensor specifically in pancreaticβ-cells.The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.

Ultra-structural study of insulin granules in pancreaticβ-cells of db/db mouse by scanning transmission electron microscopy tomography

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreaticβ-cells.The main feature of type 2 diabetes(T2D)is the failure of pancreaticβ-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels.In this work,we developed and applied tomography based on scanning transmission electron microscopy(STEM)to image intact insulin granules in theβ-cells of mouse pancreatic islets.Using three-dimensional(3D)recon-struction,we found decreases in both the number and the grey level of insulin granules in db/db mouse pan-creaticβ-cells.Moreover,insulin granules were closer to the plasma membrane in diabeticβ-cells than in control cells.Thus,3D ultra-structural tomography may provide new insights into the pathology of insulin se-cretion in T2D.