Distribution of α- and δ-tocopherols in seminal plasma and sperm fractions of men with normal and abnormal semen parameters

The aim of this study was to investigate the presence of the different isoforms of tocopherol (T) in seminal plasma (P) and in the sperm fractions of individuals with abnormal (group 1) and normal (group 2) sperm parameters; the relationships between these isoforms and conventional sperm parameters were also explored. Two vitamin E homologues, α-T and δ-T, were identified in the semen of all participants. Although α-T and δ-T concentrations were similar in the semen of the 2 groups, group 1 showed a lower α-T ratio (S/P) (0.90 vs. 1.20, P < .001) and δ-T ratio (0.86 vs 1.13, P = .007) than group 2. In addition, both T ratios were correlated with the percentage of viable cells, detected by eosin staining. These results suggested that α-T and δ-T are not homogeneously distributed in the semen fractions; in normal semen they are more concentrated in the sperm membrane, whereas in abnormal semen the damaged sperm cells may release both Ts in the plasma. To verify whether sperm membrane breakage could alter α-T and δ-T distribution between the seminal plasma and the spermatozoa, normal sperm samples were sonicated; after sonication a consistent sperm plasma membrane fragmentation, highlighted by transmission electron microscopy, and a concomitant release of α-T and δ-T were observed. In conclusion, the Ts coupled directly with the sperm membrane seem to play the main protective role in the semen, and the release of α-T and δ-T in the P fraction is probably an index of lower antioxidant power and sperm quality.     

Expression and significance of GDNF gene in recovery of spermatogenesis in mice

Objectives: To analyze the expression and significance of glial cell derived neurotrophic factor (GDNF) in the recovery of spermato genesis in mice. Methods: Adult Kunming mice were injected in traperitoneally with 2 doses of busulfan (10 mg/kg) 24 days apart so as to establish the spermatogenesis recovery modei. Testes were harvested at weeks l, 2, 3,4, 6, 8 and 10 after the second injection. Eight normai mice served as the controls. Recovery of spermatoge nesis was observed by light and electron microscopy and the GDNF mRNA measured by semi-quantitative RT-PCR and in situ hybridization. Results: After the second injection the expression of GDNF mRNA was increased significantly at week l and reached its peak at week 2. It was then decreased significantly at week 3 and reached its valley at week 4. After that it was increased gradu ally and recovered at week 10. GDNF mRNA was mainly ex pressed by the Sertoli celis. Conclusion: In the course of recovery of spermatogenesis, a high Ievel of GDNF expression     

Expression of dominant negative TGF-β receptors inhibits cartilage formation in conditional transgenic mice

Although transforming growth factor-β (TGF-β) signaling has been implicated in cartilage formation in various studies, the exact role played by TGF-β in this process remains controversial. TGF-β signals are transduced through TGF-β type II receptor (TGF-βRΙΙ) and type I receptor (ALK5). Col2a1-Cre-mediated deletion of Tgfbr2 did not cause obvious defects in long bone formation, suggesting that TGF-β signals are dispensable for normal cartilage formation in the stage of condensing mesenchymal cells and chondrocytes or that related molecules can compensate for the lack of TGF-βRΙΙ. In the present study, we established a conditional transgenic mouse in which a dominant negative form of TGF-βRII (dnTGF-βRII) is expressed in condensing mesenchymal cells and chondrocytes in limbs using the Cre/loxP system. Recombination at loxP sites and expression of dnTgfbr2 were monitored by the disappearance of LacZ expression. The conditional transgenic mice expressing dnTgfbr2 developed hypoplastic cartilage. The phenotype was much more severe than that of Col2a1-Cre-mediated Tgfbr2 conditional knockout mice, although the pattern of dnTgfbr2 expression appears similar to the pattern of Tgfbr2 deletion. These phenotypic differences suggest that the signaling through TGF-β receptors is complex in cartilage.     

Analyis of Protamine mRNA in sperm of men,rats and mice

Aim: To investigate the existence of the protamine mRNA insperm of men, rats and mice. Methods: By means of RT-PCRtechniques, protamine cDNA fragments were obtained from totalRNA of the mature sperm of men, rats and mice. Results:mRNA of protamine gene was present in the mature sperm of men,rats and mice. The protamine cDNA obtained by RT-PCR in ratsperm with an abnormal head was much less in number than that inthe normal rat sperm. Conclusion: mRNA in the sperm mightrepresent the condition of corresponding gene expression duringspermatogenesis. (Reprod Contracep 2001; 21: 200 - 5)     

Distribution and property of nerve fibers in human long bone tissue

Objective： To observe the distribution of the nerve fibers in the bone tissue and the entry points of these fibers into the bone. Methods： The adult tibia was used for the ground sections which were afterwards made into the slice sections by decalcification in ethylenediamine tetraacetic acid （EDTA）. The ground sections were stained in silver and the slice sections were stained in silver and haematoxylin and eosin （HE） respectively. Then, the samples of the transmission electron microscope and the atomic force microscope were made and observed. Results ： In the human long bone tissue, many nerve fibers were distributed in the membrane, cortical bone, cancellous bone and marrow. The nerve fibers entered the bone from the nutrient foramen, and passed through the nutrient canal, Haversian＇ s canal and Volkmann＇ s canal, and finally into the bone marrow. In the nutrient canal, the nerve fibers, mainly the medullary nerve fibers, followed the blood vessel into the bone. In the cortical bone, the nerve fibers also followed the blood vessels and were mainly distributed along Haversian＇ s canal and Volkmann＇ s canal. In the bone trabecular and bone marrow, there were many nerve fiber endings arranged around the blood vessels, mainly around the tunica media of medium-size arteries in the marrow and around capillary blood vessels, and a few scattered in the bone marrow. There were sporadic nerve endings in epiphyseal plate and no nerve fibers permeated epiphysis to diaphysis. No distribution of nerve fibers could be found in cartilaginous part. Conclusions： There are many nerve fibers in bone and the nerve passageway is nutrient foramen, Volkman＇ s canal, Haversian＇ s canal and bone marrow.     

Correlation of cell apoptosis with brain edema and elevated intracranial pressure in traumatic brain injury

Traumaticbraininjury(TBI) isamongthemostseriousinjuries, frequentlycausingdeathorpermanentdisability. ItiscloselyrelatedwiththehottesttopicsabouthowtoreducetheneurologicalimpairmentwhichmayresultfromTBI.Necrosisinthedamagedregionhadbeenconventionallythoughttobetheonlydeathmodeoftheinjuredneuronsuntilrecently. Atpresent, alotofevidencehasindicatedthatapoptosismightbeanothermainmodeofcelldeathafteraheadtrauma.1 Inthisresearch, animalmodelofTBIwasmadewithadultrabbit, apoptoticcellsweredetecte…     

Effect of mild hypothermia on partial pressure of oxygen in brain tissue and brain temperature in patients with severe head injury

OBJECTIVE: To study the changes of partial pressure of oxygen in brain tissue (P(bt)O(2)) and brain temperature (BT) in patient s in acute phase of severe head injury, and to study the effect of mild hypothermia on P(bt)O(2) and BT. METHODS: The P(bt)O(2) and the BT of 18 patients with severe head injury were monitored, and the patients were treated with mild hypothermia within 20 hours after injury. The rectal temperature (RT) of the patients was kept on 31.5-34.9 degrees C for 1-7 days (57.7 hours+/-28.4 hours averagely), simultaneously, the indexes of P(bt)O(2) and BT were monitored for 1-5 days (with an average of 54.8 hours+/-27.0 hours). According to Glasgow Outcome Scale (GOS), the prognosis of the patients was evaluated at 6 months after injury. RESULTS: Within 24 hours after severe head injury, the P(bt)O(2) was significantly lower (9.6 mm Hg+/-6.8 mm Hg, 1 mm Hg=0.133 kPa) than the normal value (16-40 mm Hg). After treatment of mild hypothermia, the mean P(bt)O(2) increased to 28.7 mm Hg+/-8.8 mm Hg during the first 24 hours, and the P(bt)O(2) was still maintained within the range of normal value at 3 days after injury. The BT was higher than the RT in the patients in acute phase of severe head injury, and the difference between the BT and the RT significantly increased after treatment of mild hypothermia. Hyperventilation (the partial pressure of carbon dioxide in artery (P(a)CO(2)) approximately 25 mm Hg) decreased the high intracranial pressure (ICP) and significantly decreased the P(bt)O(2). CONCLUSIONS: This study demonstrates that P(pt)O(2) and BT monitoring is a safe, reliable and sensitive diagnostic method to follow cerebral oxygenation. It might become an important tool in our treatment regime for patients in the acute phase of severe head injury requiring hypothermia and hyperventilation.     

Osmic acid staining of myelin sheath in normal and regenerated peripheral nerves

Objective: To introduce a practical, economical, and time-saving method to stain (with osmic acid) the myelin sheath in normal and regenerated peripheral nerves. Methods: A total of 12 Sprague Dawley rats, weighing 250-320 g (mean =276 g±38 g), were divided into two groups: a normal nerve group (n=6) and a regenerated nerve group (n=6). In the normal nerve group, the ventral and dorsal roots of L4 to L6 and their sciatic nerves were harvested for histological analysis. While in the regenerated nerve group, the right sciatic nerves were severed and then repaired with an epineurial microsuture method. The repaired nerves were harvested 12 weeks postoperatively. All the specimens were fixed in 4% paraformaldehyde and transferred to 2% osmic acid for 3-5 days. Then the specimens were kept in 75% alcohol before being embedded in paraffin. The tissues were cut into sections of 3μm in thickness with a conventional microtome. Results: Under a light microscope, myelin sheaths were clearly visible at all magnifications in both groups. They were stained in clear dark colour with a light yellow or colorless background, which provided high contrast images to allow reliable morphometric measurements. Morphological assessment was made in both normal and regenerated sciatic nerves. The ratios of the myelin area to the fibre area were 60. 28%±7. 66% in the normal nerve group and 51. 67%±6. 85% in the regenerated nerve group, respectively (P<0.01). Conclusions: Osmic acid staining is easy to perform and a very clear image for morphometrical assessment is easy to obtain. Therefore, it is a reliable technique for quantitative evaluation of nerve morphology.     

Expression of DNApolβ and ferritin in gallbladder malignancy and its clinical significance

DNApolβ和FT与肿瘤发生、进展及预后等密切相关[1,2].作者应用EnvisionTM免疫组化法研究胆囊腺癌、慢性胆囊炎和胆囊结石组织DNApolβ和FT表达水平,探讨两者表达的临床病理意义.     

Expression of DNApolβ and ferritin in gallbladder malignancy and its clinical significance

DNApolβ和FT与肿瘤发生、进展及预后等密切相关[1,2].作者应用EnvisionTM免疫组化法研究胆囊腺癌、慢性胆囊炎和胆囊结石组织DNApolβ和FT表达水平,探讨两者表达的临床病理意义.     

Changes of interleukin-1 β, tumor necrosis factor α and interleukin-6 in brain and plasma after brain injury in rats

Objective: To study the changes of interleukin-1 β(IL-1β), tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) levels in brain and plasma after brain injury and to assess the relationship between the cytokine levels and injury severity in rats. Methods. A total of 51 male Wistar rats, weighing 280-340g, were anesthetized with chloral hydrate(400 mg/kg body weight) through intraperitoneal injection and fixed on a stereotaxic instrument. Severe brain injury was created in 16 rats (severe injury group) and moderate brain injury in 18 rats (moderate injury group) by a fluid percussion model, and cytokine levels of IL-1β, TNFα and IL-6 were measured with biological assay. And sham operation was made on the other 17 rats (control group). Results: In the control group, the levels of IL-1β,TNFα and IL-6 were hardly detected in the cortex of the rats, but in the ipsilateral cortex of the rats in both injury groups, they increased obviously at 8 hours after injury.The increasing degree of these cytokines had no significant difference between the two injury groups. The levels of IL-6 in the plasma of all the rats increased slightly, whereas the levels of IL-1β and TNFα were undetectable. Conc|usions: The increase of IL-1β, TNFα and IL-6 levels is closely related to brain injury. The increased cytokine levels in the central nervous system are not parallel to those in the peripheral blood. It suggests that inflammatory cytokines play important roles in the secondary neural damage after brain injury.     

Multimodality imaging of β-cells in mouse models of type 1 and 2 diabetes

OBJECTIVE

β-Cells that express an imaging reporter have provided powerful tools for studying β-cell development, islet transplantation, and β-cell autoimmunity. To further expedite diabetes research, we generated transgenic C57BL/6 “MIP-TF” mice that have a mouse insulin promoter (MIP) driving the expression of a trifusion (TF) protein of three imaging reporters (luciferase/enhanced green fluorescent protein/HSV1-sr39 thymidine kinase) in their β-cells. This should enable the noninvasive imaging of β-cells by charge-coupled device (CCD) and micro-positron emission tomography (PET), as well as the identification of β-cells at the cellular level by fluorescent microscopy.

RESEARCH DESIGN AND METHODS

MIP-TF mouse β-cells were multimodality imaged in models of type 1 and type 2 diabetes.

RESULTS

MIP-TF mouse β-cells were readily identified in pancreatic tissue sections using fluorescent microscopy. We show that MIP-TF β-cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed a progressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing proinsulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse β-cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, providing a new experimental model of type 1 diabetes. Mice fed a high-fat diet to model early type 2 diabetes displayed a progressive increase in their pancreatic bioluminescent signals, which were positively correlated with area under the curve–intraperitoneal glucose tolerance test (AUC-IPGTT).

CONCLUSIONS

MIP-TF mice provide a new tool for monitoring β-cells from the single cell level to noninvasive assessments of β-cells in models of type 1 diabetes and type 2 diabetes.The expression of imaging reporter genes in β-cells has provided powerful tools for studying β-cell development, embryonic and adult stem cell differentiation into β-cells, transdifferentiation, and islet transplantation (1–13). Bioluminescent reporters, such as firefly luciferase, emit photons that can be detected with high sensitivity using a cooled charge-coupled device (CCD). We and others have demonstrated human and rodent islets can be genetically engineered ex vivo to express luciferase so that the islets can be monitored following their transplantation by CCD and that CCD signal corresponded with the implanted β-cell mass (1,2). Several laboratories have generated transgenic mice that express luciferase in their β-cells and shown that the CCD signal from the pancreas is correlated with β-cell mass (3–5). Moreover, the transgenic islets can be monitored after transplantation (3,4,6). Fluorescence imaging and bioluminescence imaging (BLI) are highly useful for research purposes in small animals, but they are not yet applicable to imaging internal organs in large animals because of the limited transparency of tissues to photons.Positron emission tomography (PET) imaging is used clinically to assess various disorders of the heart and brain and for detecting various cancers (7,8). Islets that were engineered ex vivo to express a PET can be noninvasively monitored by microPET after implantation into the liver (9,10). The microPET signal reflects β-cell mass, and islets can be monitored long-term after implantation (11). Radiolabeled ligands of vesicular monoamine transporter (VMAT2) can image β-cells in BB rats, although VMAT2 may not be a sensitive biomarker of β-cell mass in humans (12–14). Mouse pancreatic β-cells are inherently difficult to detect by microPET because of the small size of mice, the long thin shape of the pancreas and its proximity to the probe excretion pathway—which eliminates the vast majority of the tracer and obscures the weaker signals from the pancreas, and the resolution of microPET (1–2 mm³). Recent advances in PET reporter genes and PET probes with better clearance kinetics and biodistribution, however, have allowed progress towards microPET imaging of mouse β-cells in situ (14).Previous imaging studies of β-cells have used animal models in which the β-cells express a single reporter gene. We have developed a series of trifusion imaging reporters consisting of a bioluminescent reporter (e.g., luciferase), linked by a few alanine residues to a fluorescent reporter gene (e.g., enhanced green fluorescent protein [EGFP]), which in turn is linked to a PET reporter (e.g., herpes simplex virus [HSV] thymidine kinase) (15). All three reporters are expressed as a single fusion protein, allowing noninvasive CCD and PET imaging, as well as fluorescent microscopic analysis of tissue sections or fluorescence-activated cell sorter (FACS) isolation of single cells expressing EGFP (15,16). Using an insulin promoter to drive the expression of a trifusion reporter in transgenic mouse β-cells should enable the longitudinal monitoring of β-cells in the same mice by both CCD and microPET. After killing, fluorescent microscopy can identify β-cells at the cellular level and facilitate correlation of β-cell number and mass with the CCD and microPET signals. Because all three reporters are linked together in a fusion protein, the magnitude of signals from one imaging reporter should predict the magnitude of the signals from the other imaging reporters. Here, we provide proof-of principle of the utility of mice expressing a trifusion imaging reporter specifically in their β-cells in mouse models of type 1 diabetes and type 2 diabetes.     

Crosstalk of TGF-β and Estrogen Receptor Signaling in Breast Cancer

Estrogen receptor-α (ERα) and transforming growth factor (TGF)-β signaling pathways are major regulators during mammary gland development, function and tumorigenesis. Predominantly, they have opposing roles in proliferation and apoptosis. While ERα signaling supports growth and differentiation and is antiapoptotic, mammary gland epithelia cells are very sensitive to TGF-β—induced cell cycle arrest and apoptosis. Their regulatory pathways intersect, and ERα blocks TGF-β pathway by multiple means, including direct interactions of its signaling components, Smads. However, relatively little is known of the dysfunction of their interactions in cancer. A better understanding would help to develop new strategies for breast cancer treatment.