Whole-exome sequencing identifies novel homozygous mutation in NPAS2 in family with nonobstructive azoospermia.

OBJECTIVE: To investigate the genetic cause of nonobstructive azoospermia (NOA) in a consanguineous Turkish family through homozygosity mapping followed by targeted exon/whole-exome sequencing to identify genetic variations. DESIGN: Whole-exome sequencing (WES). SETTING: Research laboratory. PATIENT(S): Two siblings in a consanguineous family with NOA. INTERVENTION(S): Validating all variants passing filter criteria with Sanger sequencing to confirm familial segregation and absence in the control population. MAIN OUTCOME MEASURE(S): Discovery of a mutation that could potentially cause NOA. RESULT(S): A novel nonsynonymous mutation in the neuronal PAS-2 domain (NPAS2) was identified in a consanguineous family from Turkey. This mutation in exon 14 (chr2: 101592000 C>G) of NPAS2 is likely a disease-causing mutation as it is predicted to be damaging, it is a novel variant, and it segregates with the disease. Family segregation of the variants showed the presence of the homozygous mutation in the three brothers with NOA and a heterozygous mutation in the mother as well as one brother and one sister who were both fertile. The mutation is not found in the single-nucleotide polymorphism database, the 1000 Genomes Project, the Baylor College of Medicine cohort of 500 Turkish patients (not a population-specific polymorphism), or the matching 50 fertile controls. CONCLUSION(S): With the use of WES we identified a novel homozygous mutation in NPAS2 as a likely disease-causing variant in a Turkish family diagnosed with NOA. Our data reinforce the clinical role of WES in the molecular diagnosis of highly heterogeneous genetic diseases for which conventional genetic approaches have previously failed to find a molecular diagnosis.

Tunica albuginea acellular matrix graft for penile reconstruction in the rabbit: a model for treating Peyronie's disease.

OBJECTIVE: To evaluate the use of an acellular matrix graft of the tunica albuginea for functional penile reconstruction in severe cases of Peyronie's disease. MATERIALS AND METHODS: In 18 rabbits, an acellular matrix graft of the tunica albuginea was used to cover a 4 x 8 mm tunical defect, and six animals each were killed 1, 3 and 6 months later; four unoperated animals served as histological controls. Before death an erection was induced by papaverine, with the quality classified on a scale of 0-5, and cavernosography performed. After death the penis was prepared for histological study, and the cell number, collagen and elastic fibre content evaluated in the regenerated matrix, and in control specimens and four unimplanted matrices. RESULTS: Of 18 experimental animals, 11 had normal erections before death, four had slight penile deviation and three developed no erection. Failure was caused by severe postoperative haematoma, resulting in scar tissue. There was no graft rejection. Histologically there was no difference between natural and regenerated tunica. The collagen content and cell number were not significantly different in regenerated and control samples. There were significantly fewer elastic fibres in the unimplanted grafts and the 1-month group, but in later samples this difference was no longer evident. CONCLUSION: The homologous acellular matrix graft of the tunica albuginea warrants further evaluation as an alternative treatment in Peyronie's disease, despite some postoperative failures. The advantage of this orthotopic biomaterial is its rapid integration, with no rejection.

The effect of simulated birth trauma and/or ovariectomy on rodent continence mechanism. Part I: functional and structural change.

PURPOSE: We examined the changes in the lower urinary tract after delivery, intravaginal ballooning and/or ovariectomy. MATERIALS AND METHODS: The study included 10 virgin and 48 primiparous pregnant rats. Cystometry and the stress/sneeze test were performed in virgin and postpartum rats shortly after delivery and at 8 weeks before sacrifice. Half of the delivered animals underwent intravaginal balloon dilation. Four weeks later half in each group underwent ovariectomy. The rats were subdivided into group 1--delivery, group 2--delivery plus balloon inflation, group 3--delivery plus ovariectomy and group 4--delivery plus balloon inflation plus ovariectomy. Tissues from the bladder, bladder neck, urethra and levator were collected, analyzed by electron microscopy, and immunostained for caveolin-1, caveolin-3 and neuronal nitric oxide synthase. RESULTS: Higher bladder capacity was detected in postpartum than in virgin rats. Urine leakage on stress/sneeze testing increased significantly in groups 2 and 4. Electron microscopy revealed a significant decrease in sarcolemma caveolae in the smooth muscle of the bladder and urethra in groups 2 to 4. In the bladder neck in group 3 caveolae were increased in smooth muscle. In groups 2 to 4 in the smooth muscle of the bladder and urethra caveolin-1 was significantly decreased. Caveolin-3 and neuronal nitric oxide synthase in striated muscle also significantly decreased in groups 2 to 4. CONCLUSIONS: These findings suggest that birth trauma simulated by ballooning and ovariectomy may contribute to stress urinary incontinence. The alteration in smooth muscle caveolae as well as the membrane protein caveolin may have a role in functional alterations caused by birth trauma and ovariectomy.

Decreased trabecular smooth muscle and caveolin-1 expression in the penile tissue of aged rats.

PURPOSE: Because decreased trabecular smooth muscle content is reportedly associated with vasculogenic impotence in men, we performed a rodent study to investigate the effect of aging on trabecular smooth muscle content and caveolin-1 protein expression in penile smooth muscle cells. MATERIALS AND METHODS: In 6 young (age 3 months) and 6 old (age 24 months) rats erectile function was evaluated by cavernous nerve stimulation. At sacrifice penile tissue samples were collected for Western blot analysis, Masson's trichrome staining, caveolin-1 immunostaining and electron microscopy. The percent of smooth muscle in the trabecular tissue was assessed by computer assisted image analysis. RESULTS: In the aged rats mean intracavernous pressure plus or minus standard deviation was decreased (70 +/- 8.8 versus 107 +/- 12.3 cm. water) and the latency period was increased (7.8 +/- 1.2 versus 4.5 +/- 0.5 seconds) significantly compared to values in the young rats (p <0.001). The mean ratio of trabecular smooth muscle-to-connective tissue was also significantly altered in old versus young rats (27% +/- 2.9% versus 42.1% +/- 5.1%, p <0.001). Immunostaining for caveolin-1 was noted in each group in the sarcolemma of smooth muscle cells and endothelium of trabecular sinusoids but the staining pattern was less intense and the percent of smooth muscle positive for caveolin-1 was decreased in aged versus young rats (17.9% +/- 2.5% versus 27.5% +/- 3.6%, p <0.001). Moreover, young trabecular smooth muscle cells had more caveolae in the sarcolemma on electron microscopy and a higher expression of caveolin-1 protein on Western blot analysis. In contrast, higher endothelial nitric oxide synthase protein expression was noted in the penile tissue of old rats. CONCLUSIONS: In these aged rats the decreased ratio of trabecular smooth muscle-to-collagen and the reduced expression of caveolin-1 may contribute to erectile dysfunction.

Heterologous acellular matrix graft for reconstruction of the rabbit urethra: histological and functional evaluation.

PURPOSE: In a rabbit model we evaluated urethral replacement by a free heterologous dog acellular matrix graft and compared these results with those of a homologous graft with the exclusion of antigenicity as a major goal. MATERIALS AND METHODS: In 14 male New Zealand rabbits a 0.8 to 1.1 cm. segment of urethra was resected and replaced with a tubular acellular 1.0 to 1.5 cm. (mean 1.3) urethral matrix graft placed on an 8Fr feeding tube. Seven animals received a rabbit graft, 7 received a canine graft and 3 untreated rabbits served as controls. All animals underwent urethral pressure profile determination and retrograde urethrography before 8 and 6 were sacrificed at 6 and 8 months, respectively. Grafted and normal specimens were evaluated by histological testing. RESULTS: In all animals the acellular matrix graft remained in its original position. Histological examination showed complete epithelialization and progressive vessel infiltration. At 6 months more than a third of the homologous grafts had smooth muscle bundles but the heterologous grafts had only poorly disseminated smooth muscle. Picrosirius red stain demonstrated a shift in the ratio of collagen types I-to-III with an increase in type III in the processed homologous and heterologous matrices that did not change significantly postoperatively. At 8 months the urethral pressure profile detected no difference in control and matrix grafted animals, and urethrography did not readily differentiate host from implant. CONCLUSIONS: In the heterologous matrix all tissue components were present after 6 months with no signs of rejection and even gradual improvement with time. However, regenerated smooth muscle did not equal that in normal rabbit urethra and it was not well oriented. Even after 8 months only a few disseminated smooth muscle cells were evident. Most alpha-actin positive cells were surrounding the vessels. Although function was normal, the alteration in the collagen ratio effected by matrix production indicated that the matrix collagen appeared not to have been replaced by host collagen. The increase in collagen type III may explain the lack of stricture in the grafted animals on normal retrourethrography.

The effect of adeno-associated virus mediated brain derived neurotrophic factor in an animal model of neurogenic impotence.

PURPOSE: We tested the hypothesis that transfecting penile tissue with brain derived neurotrophic factor may facilitate neural recovery and erectile capability after cavernous nerve injury. MATERIALS AND METHODS: Of the 34 Sprague-Dawley rats used 10 underwent sham operation and 24 underwent bilateral cavernous nerve freezing and intracavernous injection of adeno-associated virus-LacZ (12) or adeno-associated virus-brain derived neurotrophic factor (12). Erectile function was assessed by cavernous nerve electrostimulation at 4 and 8 weeks, and samples of penile tissue and the major pelvic ganglia were evaluated histologically. RESULTS: In the brain derived neurotrophic factor group mean maximal intracavernous pressure plus or minus standard deviation was significantly higher than in the LacZ group at 4 and 8 weeks (58.5 +/- 11.7 cm. water versus 28.4 +/- 5.5 and 61.3 +/- 12.5 versus 37.7 +/- 7.9, respectively). In addition, in the brain derived neurotrophic factor group reduced nicotinamide adenine dinucleotide phosphate diaphorase staining and neuronal nitric oxide synthase immunostaining revealed significantly more positive nerve fibers in the dorsal nerves and cavernous tissue than in the LacZ group at each time point and the percent of neuronal nitric oxide synthase positive neurons in the major pelvic ganglia was also significantly greater. Moreover, in the LacZ group most neurons showed a light staining pattern with irregular contours and numerous vacuoles in the cytoplasm. CONCLUSIONS: Intracavernous injection of adeno-associated virus-brain derived neurotrophic factor may prevent the degeneration of neuronal nitric oxide synthase containing neurons in the major pelvic ganglia and facilitate the regeneration of neuronal nitric oxide synthase containing nerve fibers in penile tissue, thus, enhancing the recovery of erectile function after bilateral cavernous nerve injury.

Mapping visual cortex in monkeys and humans using surface-based atlases.

We have used surface-based atlases of the cerebral cortex to analyze the functional organization of visual cortex in humans and macaque monkeys. The macaque atlas contains multiple partitioning schemes for visual cortex, including a probabilistic atlas of visual areas derived from a recent architectonic study, plus summary schemes that reflect a combination of physiological and anatomical evidence. The human atlas includes a probabilistic map of eight topographically organized visual areas recently mapped using functional MRI. To facilitate comparisons between species, we used surface-based warping to bring functional and geographic landmarks on the macaque map into register with corresponding landmarks on the human map. The results suggest that extrastriate visual cortex outside the known topographically organized areas is dramatically expanded in human compared to macaque cortex, particularly in the parietal lobe.

Effect of a Chinese herbal medicine mixture on a rat model of hypercholesterolemic erectile dysfunction.

PURPOSE: We examine the effect of a Chinese herbal medicine mixture on erectile function in a rat model of hypercholesterolemic erectile dysfunction. MATERIALS AND METHODS: In this study 32, 3-month-old Sprague-Dawley rats were used. The 8 control animals were fed a normal diet and the remaining 24 were fed 1% cholesterol diet for 4 months. After 2 months herbal medicine was added to the drinking water of the treatment group of 16 rats but not the cholesterol only group of 8. Of the 16 rats 8 received 25 mg./kg. per day (group 1) and 8 received 50 mg./kg. per day (group 2) of Chinese herbal medicine mixture. Serum cholesterol levels were measured at 2 and 4 months. At 4 months erectile function was evaluated with cavernous nerve electrostimulation in all animals. Penile tissues were collected for electron microscopy, and to perform Western blot for endothelial nitric oxide synthase, neuronal nitric oxide synthase, basic fibroblast growth factor (bFGF) and caveolin-1. RESULTS: Serum cholesterol levels were significantly higher in animals fed the 1% cholesterol diet compared to controls at 2 and 4 months. Nevertheless, there was no significant difference among group 1 (145 +/- 30 mg./dl.), group 2 (157 +/- 20) and the cholesterol only group (143 +/- 15). Systemic arterial pressure was not significantly different between the animals that were fed the 1% cholesterol diet and the controls. During electrostimulation of the cavernous nerve peak sustained intracavernous pressure was significantly lower in the cholesterol only group (50 +/- 23 cm. H2O) compared to the control group. Conversely erectile function was not impaired in the herbal medicine treated rats. Electron microscopy showed many caveolae with fingerlike processes in the cavernous smooth muscle and endothelial cell membranes in control and treated rats but not in the cholesterol only group of rats. Western blot did not show a difference among groups in protein expression for endothelial nitric oxide synthase and neuronal nitric oxide synthase in penile tissue but caveolin-1 and bFGF protein expression was significantly higher in groups 1 and 2 than in the cholesterol only and control groups. CONCLUSIONS: Rats developed erectile dysfunction after being fed a 1% cholesterol diet for 4 months. Although serum cholesterol levels were similar in the cholesterol only rats and those treated with Chinese herbal medicine mixture, erectile response was significantly better in the treated group. The mechanism of the herbal medicine is unknown. High levels of bFGF and caveolin-1 expression in the treated group may protect the cavernous smooth muscle and endothelial cells from the harmful effect of high serum cholesterol.

Homologous acellular matrix graft for urethral reconstruction in the rabbit: histological and functional evaluation.

OBJECTIVE: To evaluate urethral replacement by a free homologous graft of acellular urethral matrix in a rabbit model. MATERIALS AND METHODS: In 30 male New Zealand rabbits, a 0.8 to 1.1 cm. segment of the urethra was resected, replaced with an acellular matrix graft of 1.0 to 1.5 cm. (mean 1.3 cm.), and placed on an 8F feeding tube. Additionally 4 animals underwent sham operation. At varying intervals before sacrifice (from 10 days to 8 months), the animals underwent urodynamic evaluation and retrograde urethrography (for which 4 untreated rabbits served as control). The grafted specimens were prepared for evaluation histologically and by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: In all animals, the acellular matrix graft remained in its original position. Histological examination showed complete epithelialization and progressive vessel infiltration. At 3 months, smooth muscle bundles were first observed infiltrating the matrix at the end-to-end anastomosis; after 6 months, the smooth muscle bundles had grown into one-third of the matrix. Urodynamics did not detect any difference between the control and matrix-grafted animals in bladder volume, leak-point pressure and residual volume. RT-PCR detected an increase in IGF mRNA in the graft between week 3 and month 6 and in HB-EGF mRNA after day 10 through month 3. TGF-alpha mRNA was not detected; TGF-beta mRNA was unchanged from normal urethral tissue. By 8 months, the host and implant could not be differentiated by urethrography. CONCLUSION: The acellular urethral matrix allows single-stage urethral reconstruction. All tissue components were seen in the grafted matrix after 3 months, with further improvement over time; however, the smooth muscle in the matrix was less than in normal rabbit urethra and was not well oriented. RT-PCR revealed the importance of time-dependent growth factor influences during regeneration.

The effect of pregnancy and delivery on the function and ultrastructure of the rat bladder and urethra.

OBJECTIVE: To examine the effect of pregnancy and delivery on the function and ultrastructure of the bladder and urethra in rats. Material and methods The study comprised six virgin and 18 pregnant rats; both groups underwent cystometry (at the 19th day of gestation, and 2 days and 6 weeks after parturition). Tissues from the bladder and urethra were collected for electron microscopy, western blotting and immunostaining for caveolin-1 and caveolin-3. RESULTS: The bladder capacity was greater and the modified leak-point pressures lower in pregnant and 2-day postpartum rats than in virgin and 6-week postpartum rats. The residual volume was significantly higher in the pregnant group. Electron microscopy showed more sarcolemmal caveolae in the smooth muscle cells of both the bladder and urethra of virgin rats than in the other groups. Lipid droplets and subsarcolemmal mitochondria accumulated in pregnant and 2-day postpartum rats. Caveolin-1 protein was detected in the cytoplasmic membrane of urethra and bladder smooth muscle cells. Caveolin-3 was detected in the membrane of striated muscle in the intrinsic sphincter. Western blotting showed increased caveolin-1 protein expression in the bladder and urethra of 2-day postpartum rats; in contrast, levels of caveolin-1 were lower in pregnant rats than in virgin and 6-week postpartum rats. CONCLUSION: s During pregnancy there was a significant decrease in sarcolemmal caveolae and caveolin-1 in the smooth muscle cells of the rat bladder and urethra. The changes in caveolae and the membrane protein caveolin may play a role in the functional changes associated with pregnancy and after delivery.

The effects of colchicine on a Peyronie's-like condition in an animal model.

PURPOSE: We have developed an animal model of Peyronie's disease by injecting transforming growth factor beta (TGF-beta) into the rat penis. Our objective is to study the effects of colchicine on the Peyronie's condition in an animal model. MATERIALS AND METHODS: Thirty-six adult male Sprague-Dawley rats received TGF-beta injections into the tunica albuginea and were divided into two groups (n = 18 each). Rats in the first group were divided into three subgroups (n = 6 each). Each rat in the three subgroups received the following: Subgroup 1 received colchicine, subgroup 2 received ibuprofen, and subgroup 3 received regular water. The rats were euthanized after 6 weeks. Rats in the second group were also divided into three subgroups. These rats received the same treatments as the rats in the first group, but treatments began 6 weeks after TGF-beta injection. These rats were euthanized after 12 weeks. Tunical tissue samples were collected and examined using Hart and trichrome stains, electron microscopy (EM), and western blot analysis for TGF-beta detection. RESULTS: In the first group, the colchicine-treated rats exhibited less collagen deposition and less elastic fiber fragmentation than the untreated or ibuprofen-treated rats. EM confirmed the results and showed normal distribution and shape of both collagen and elastic fibers in the colchicine-treated group. In the second group, the colchicine-treated rats exhibited less crowding of the collagen fibers. However, the elastic fibers remained fragmented and scarce. Western blot analysis showed significant down-regulation of TGF-beta expression (5/6) in the colchicine-treated group after 6 weeks. Down-regulation was observed in only 1/6 in both ibuprofen and non-treated groups. After 12 weeks 2/6, 1/6, and 1/6 rats displayed down regulation in the colchicine treated, ibuprofen treated, and non-treated groups, respectively. CONCLUSION: Early colchicine treatment may suppress a Peyronie's like condition in the rat animal model.

Possible molecular mechanisms of cryoablation-induced impotence in a rat model.

OBJECTIVES: Cryoablation of the prostate has been reported to induce impotence as a consequence of cavernosal nerve injury. This study is designed to investigate the early and late effects of cavernosal nerve cryoablation on growth factor expression and erectile function in a rat model. METHODS: Forty male rats were divided into two groups (n=20 each). The first group underwent unilateral cavernosal nerve freezing (experimental group). Before their euthanization at 1 and 3 months (10 rats each), erectile function was assessed by electrostimulation of the cavernous nerves. The second group served as the control and was killed at the same time points. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to identify protein and gene expression of nerve growth factor (NGF), transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) in the rat penis and pelvic ganglia. RESULTS: Electrostimulation of the frozen nerve after 3 months revealed a significantly higher maximal intracavernosal pressure and a shorter latency period than in the 1-month group. At 3 months, immunoblot showed upregulation of NGF, TGF-alpha, and the precursor form of IGF-1 protein expression in the penile tissue; RT-PCR showed downregulation of NGF gene expression in the pelvic ganglia of the frozen side. CONCLUSIONS: The results show that erectile function decreased at 1 month and then partially recovered 3 months after cavernosal nerve freezing. This alteration in erectile function was associated with differential gene and protein expression of the growth factors (NGF, TGF-alpha, EGF, and IGF-1). Further studies are required to elucidate the potential role of these growth factors in the prevention and treatment of cryoablation-induced impotence.

Curve matching on brain surfaces using Frenet distances.

This paper describes methods for diffeomorphic matching of curves on brain surfaces. Distances between curves are defined by Frenet representation via speed, curvature, and torsion. The curvematching algorithm is based on bipartite graph matching, with weights defined by the Frenet distance over diffeomorphic maps of one curve onto the other (Sedgewick [1983]: Algorithms). We follow Khaneja ([1996]: Statistics and Geometry of Cortical Features) and define fundus curves on the brain surfaces as extremal curvature lines generated using dynamic programming. Examples are shown for fundus curve matchings on macaque brain surfaces.

mRNA and protein expression of nitric oxide synthase and adrenoceptor alpha 1 in young and old rat penile tissues.

OBJECTIVE: To investigate alterations of neuronal structure and the mRNA expression of nitric oxide synthase (NOS) and adrenoceptor alpha 1 in young and old rat penile tissues, and assess the importance of these factors in erectile dysfunction (ED) associated with ageing. MATERIALS AND METHODS: NOS was determined in the pelvic ganglia of 32 rats using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. Adrenergic neurons were identified by histofluorescence staining. The gene expression for NOS and for adrenoceptor alpha 1 was investigated in corporal tissue by differential reverse transcriptase-polymerase chain reaction (RT-PCR) using specific oligonucleotide primers. b-actin was used as an internal standard in each tube to quantify the baseline gene expression. RESULTS: The following changes occurred in old rats: (i) an increase of lipofuscin and a decrease in both bNOS-containing neurons and fluorescent adrenergic neurons in the pelvic ganglia; (ii) significantly less expression of NOS mRNA than in young rat penile tissues; and (iii) no significant alteration in adrenoreceptor alpha 1 mRNA expression. CONCLUSIONS: The fewer adrenergic and NOS-containing neurons and neurotransmitters in pelvic ganglia from old rats may explain the enhanced penile smooth muscle tone previously reported in older impotent men. Furthermore, lower mRNA expression of NOS may be associated with structural and functional changes associated with ageing penile tissue.