Effect of secretory human calcitonin gene-related peptide recombinant AAV on penile erection in streptozotocin-induced diabetic rat / 中华男科学杂志

<p><b>OBJECTIVE</b>To determine whether recombinant adeno-associated virus-mediated overexpression of hCGRP in the corpus cavernosum can affect the continuous production of hCGRP in the penile tissue and enhance erectile responses in STZ-induced diabetic rats.</p><p><b>METHODS</b>Diabetes mellitus was induced by a single intraperitoneal injection of 60 mg/kg streptozotocin in male SD rats. VssHGCMV-hCGRP, VssCMV-GFP and rAAV solution were injected into the corporal cavernosum of STZ-induced diabetic rats, respectively. The corporal tissue was obtained from groups of 8 rats on day 5 post-injection, and the expressions of CGRP and GFP in cavernosal tissue were detected using immunohistochemistry and laser scanning confocal microscopy, respectively. Cavemosal tissue cAMP and cGMP levels were measured using radioimmunoassay. On day 5 post-injection, intracavernous pressure induced by electrostimulation of penile dorsal nerves was measured and recorded with a biological signal processing system in each group rat.</p><p><b>RESULTS</b>rAAV transduction efficiency of GFP reporter gene was measured by laser scanning confocal microscopy and was observed in the penile tissue, especially in the corporal cavernosum and the vessel 5 days after transfection with VssCMV-GFP. Immunohistochemistry showed that the CGRP increased in the corporal cavernosum. In addition, both cAMP and cGMP levels in the corpora cavernosa transfected with VssHGCMV-hCGRP were significantly increased, compared with controls [(48.4 +/- 6.5) nmol/L and (21.2 +/- 13.6) nmol/L vs (16.7 +/- 2.5) nmol/L and (0.42 +/- 0.12) nmol/L, respectively]. More importantly, 5 days after administration of VssHGCMV-hCGRP,a significant increase was observed in the erectile response to penile dorsal nerve stimulation in the diabetic rat [(60.5 +/- 4.5) mm Hg vs (22. 3 +/- 1.3) mm Hg].</p><p><b>CONCLUSIONS</b>This results demonstrate that rAAV-mediated transfer of the CGRP gene can increase production of endogenous CGRP, cAMP and cGMP in corpora cavernosa of STZ-induced diabetic rats. Moreover, overexpression of CGRP enhances ICP and the erectile response to penile dorsal nerve stimulation in the diabetic rat.</p>

Expression and effect of hCGRP recombinant adeno-associated virus in primary cultured corporal cavernosum smooth muscle cells of the rat in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>To observe the expression and effect of human calcitonin gene-related peptide (hCGRP) gene mediated by recombinant adeno-associated virus (rAAV) in primary cultured corporal cavernosum smooth muscle cells of the rat and explore the possibility of using CGRP gene for gene therapy in erectile dysfunction.</p><p><b>METHODS</b>The primary cultured corporal cavernosum smooth muscle cells of the rat were randomly divided into 4 groups and infected with recombinant virus VssHGCMV-hCGRP, VssHGCMV, VssC-MV-GFP and the untreated, respectively. CGRP-like immunoreactivity was measured by protein dot blot assay in the 24 h-culture medium, and intracellular cAMP and cGMP levels in the cultured cells were also determined using radioimmunoassay to ascertain bioactivity of transduced CGRP.</p><p><b>RESULTS</b>The exogenous gene was transferred into primary corporal cavernosum smooth muscle cells by VssHGCMV-hCGRP infection and efficiently expressed. Compared with the control group, intracellular cAMP level in the cell infected by VssHGCMV-hCGRP was significantly increased (48.7 +/- 1.1 nmol/L vs 7.8 +/- 1.4 nmol/L, P < 0.01), whereas cGMP level remained unchanged in two groups, and CGRP-like immunoreactivity was also detected in the culture medium infected by VssHGCMV- hCGRP.</p><p><b>CONCLUSION</b>The system of secretory expressing bioactive peptide rAAV mediated gene transfer may be used to express efficiently exogenous gene in corporal cavernosum smooth muscle cells and affect cAMP level in the corporal cavernosum smooth muscle cells of the rat.</p>

Analysis of Yq microdeletions in idiopathic infertile males with azoospermia and oligospermia in Shaanxi Province / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate the frequency of microdeletions in the long arm of Y chromosome of idiopathic infertile males with azoospermia and oligospermia in Shaanxi province in China and to investigate the relevance of sperm count to Y microdeletion frequencies.</p><p><b>METHODS</b>According to the sequence of sequence-tagged sits (STS) AZFa, AZFb, AZFc and SRY, 4 of the azoospermic factor regions on Y chromosome long-term supplied by GenBank, 5 sets of primers were synthesized. The Y microdeletions in AZF regions were screened by polymerase chain reaction (PCR) in 64 idiopathic cases of azoospermia and oligospermia and 20 men of known fertility.</p><p><b>RESULTS</b>No microdeletion was detected in the 20 normospermic subjects. Deletion of the AZFc/DAZ was detected in 11 individuals and one patient had both AZFb and AZFc deletion; no deletion of AZFa and SRY region was found. The frequency of Y microdeletions in the subgroups with different sperm count showed the highest value among azoospermic men (3 cases, 21.4%). The percentage progressively decreased with the deletion frequency (20.0%, 17.9% and 8.3%) in the subgroups with sperm counts of < 1 x 10(6)/ml, < (1-5) x 10(6)/ml and < (1 to approximately 10) x 10(6)/ml, respectively.</p><p><b>CONCLUSION</b>Y chromosome microdeletions are specifically associated with severe spermatogenic failure. The rate of deletion involving AZF region of the Y-chromosome is higher in infertile men with azoospermia and oligospermia. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.</p>

Intracavernous pressure responses induced by electrical stimulation of penile dorsal nerves and intracavernosal drug injection in rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate the application of intracavernous pressure (ICP) monitoring in the electrophysiologic and pharmacologic induction of penile erection.</p><p><b>METHODS</b>The penile dorsal nerves (DN) of 8 anesthetized adult male rats were isolated and the corpora cavernosa exposed. A heparinized 25-gauge angiocath (intravenous catheter) was inserted into the right corpus cavernosum to monitor the ICP and a 30-gauge needle was inserted into the left corpus cavernosum for intracavernosal drug administration. Penile erection was induced by electrical stimulation of the dorsal nerve (16 Hz frequency, 0.5 ms pulse width, 20 s duration, 4 volts) and intracavernous papaverine hydrochloride injection (0.4 mg). ICP was recorded with the SMUP-PC biological signal process system.</p><p><b>RESULTS</b>In the anesthetized rats, the baseline level of ICP was (12.3 +/- 3.1) mm Hg and the electrical stimulation of the DN significantly increased ICP[(36.4 +/- 2.3) mm Hg, P < 0.05], which slowly returned to baseline pressure after termination of the electrical stimulation. The intravavernosal administration of papaverine also induced a significant increase in ICP [(28.4 +/- 6.1) mm Hg, P < 0.05].</p><p><b>CONCLUSION</b>ICP monitoring in rats provides a useful scientific tool for in vivo studies of penile erection in experimental rat models. It is of great significance for further studying the physiology of penile erection and the pathogenesis of erectile dysfunction as well as for evaluating the efficacy of novel therapies for erectile dysfunction.</p>