Biomedical subjects
J Rajfer
Publications and source records attributed to J Rajfer.
Effects of hormones in the newborn period on the development of the penis.
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The incidence of intersexuality in patients with hypospadias and cryptorchidism.
The incidence of intersexuality was determined in 45 patients with cryptorchidism and hypospadias seen between 1952 and 1975. Including patients with ambiguous genitalia the incidence was 53 per cent. When patients with ambiguous genitalia were excluded, providing a group more representative of the patient population usually seen by the urologist, a diagnosis of intersexuality was made in 27 per cent. The concomitant occurrence of hypospadias and cryptorchidism should alert the urologist to evaluate the patient for the presence of intersexuality.
Antonie van Leeuwenhoek (1632-1723).
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Effect of in vitro ketoconazole on steroid production in rat testis.
In an attempt to confirm where in the testosterone (T) biosynthetic pathway of the rat testis ketoconazole (KTZ) inhibits T production, rat testicular mince was incubated with either 10 micrograms/ml or 100 micrograms/ml KTZ in the presence and absence of hCG (1 IU), and intratesticular pregnenolone (delta 5P), progesterone (P), 17-alpha-hydroxyprogesterone (17 alpha-HP), androstenedione (A) and testosterone (T) were assayed. In the absence of hCG, 10 micrograms/ml KTZ was sufficient to reduce intratesticular T by 80%. At this concentration of KTZ, intratesticular 17 alpha-HP (ng/g testis, mean +/- SEM) increased from 0.3 +/- 0.1 to 1.3 +/- 0.2 (p less than 0.0025), whereas intratesticular A decreased from 84 +/- 7 to 17 +/- 1 (p less than 0.005). KTZ did not inhibit the conversion of P to 17 alpha-HP. From these data it was concluded that KTZ has its inhibitory effect on testosterone biosynthesis in the rat testis primarily at the step catalyzed by the 17,20 desmolase enzyme.
Molecular pathophysiology and gene therapy of aging-related erectile dysfunction.
Erectile dysfunction (ED) is a major public health problem that seriously affects the quality of life of patients and their partners. ED is mainly associated with vascular disease, diabetes, smoking, and radical prostatectomy, and its prevalence increases significantly with aging. Vasculogenic ED, specifically corporal veno-occlusive dysfunction (CVOD), is caused by the impairment of the relaxation of the smooth muscle in the penile corpora cavernosa and occurs in 2/3 of cases, whereas the less common neurogenic ED is due to a defective nitrergic neurotransmission triggered by the sexual stimulus, either at the central hypothalamic and spinal levels or at the penile nerves. Based on animal and cell studies, neurogenic ED is assumed to be caused mainly by: (a) an insufficient synthesis of nitric oxide (NO) due to a decrease in the levels of the penile neuronal nitric oxide synthase (PnNOS) or the impairment of its regulation by protein effectors (NMDA receptor, protein inhibitor of nNOS: PIN), occurring in the neuronal bodies or nerve terminals, or (b) a loss of the cells themselves by apoptosis caused by the induction of inducible NOS (iNOS) and the production of peroxynitrite. In contrast vasculogenic ED, although may involve endothelial damage and down-regulation of endothelial NOS (eNOS), appears to be mainly caused by the relative loss of smooth muscle cells and replacement by collagen fibers (fibrosis) that impairs tissue compliance. In this case, iNOS induction may not be deleterious, but a defense mechanism preventing excessive collagen deposition. Gene therapy to the penile corpora cavernosa of cDNAs expressing PnNOS or eNOS, or counteracting PIN, has been effective in ameliorating ED in the aging rat model that exhibits both neurogenic ED and CVOD. cDNA constructs for other genes involved in the control of penile erection have also been successfully tested. Gene transfer into the penis may soon translate to the clinic as a therapy aimed to cure the underlying conditions in ED, including fibrosis, as opposed to the facilitation of erection on demand offered by the current oral therapies.
Therapeutic stimulation of penile nitric oxide synthase (NOS) and related pathways.
The advent of a novel oral treatment for erectile dysfunction appears to have reduced the urgency for seeking alternative therapeutic innovations. However, the currently available therapies can be viewed as simply palliative, requiring on-demand access and having a significant failure rate. Therefore, additional approaches for patients unresponsive to any type of medical treatment require investigation. The modulation of the synthesis of nitric oxide (NO), the main mediator of penile erection, is an attractive target for such an approach. Models for human erectile dysfunction related to aging, diabetes and endocrine disorders have been characterized in the rat. The affected animals exhibit a reduced erectile response to electrical stimulation of the cavernosal nerve and/or a loss of spontaneous erectile reflexes. In virtually all cases, the content and/or enzyme activity of penile NOS is significantly reduced, or NO synthesis may be insufficient to overcome the relatively poor compliance of the penile corpora cavernosa smooth muscle (aging). This led to the proposal that the stimulation of penile NO synthesis may be a viable therapy for erectile dysfunction. The upregulation of penile NOS activity may be achieved by: a) increase in NOS substrate concentration, b) stimulation of NOS activity through related pathways or c) blockade of endogenous NOS inhibitors. We have shown that approach a ameliorated the erectile dysfunction of aging rats by elevating intracavernosal NOS substrate through long-term oral administration of high doses of L-arginine. Evidence supporting approach b is based on our recent discovery that the main stimulating pathway for NOS, the NMDA receptor, is present in the penis. However, NMDA receptor antagonists unpredictably trigger cavernosal relaxation in vitro in a NO-independent pathway. Finally, in the case of approach c we have found that a protein inhibitor of NOS (PIN) is expressed in the corpora cavernosa. Penile NOS content may be pharmacologically increased by: d) induction of NOS expression, and e) gene therapy with NOS cDNA. Approach d applied to the penis ameliorated erectile dysfunction in aging rats, but may be clinically unfeasible because of the risk of side effects. Our laboratory cloned the inducible and neuronal NOS isoforms expressed in the penis (PiNOS and PnNOS, respectively), and showed that approach e, based on gene transfer of PiNOS into the rat corpora cavernosa improved the erectile response. Since PnNOS is a nNOS variant different from the one expressed in the CNS, gene therapy with PnNOS is promising in combination with viral vectors and tissue-specific promoters. The aim is to achieve a long-lasting increase in penile NOS that is responsive to sexual stimulation. Following validation in animal models, oral L-arginine, intracavernosal antagonists of NMDA receptor and PIN, and intracavernosal or systemic PnNOS, may eventually translate to clinical trials.
Presence of NMDA receptor subunits in the male lower urogenital tract.
Some sexual responses in the male rat, specifically penile erection, are controlled by neural circuits in the brain and spine that are stimulated by the binding of excitatory amino acids (EAAs) to the postsynaptic N-methyl-D-aspartate receptor (NMDAR). In the hypothalamus, EAA/NMDAR interaction triggers the activation of neuronal nitric oxide synthase (nNOS) to produce nitric oxide (NO). The local synthesis of this neurotransmitter in the penile nerve terminals causes corpora cavernosal relaxation and erection. During sexual activity, NO is assumed to participate in seminal emission and ejaculation in the prostate and to inhibit voiding reflexes in the bladder. This study aimed to determine in vitro whether NMDAR is present in these organs and whether it affects the tone of tissue strips through an NO-dependent mechanism. We obtained penile, urinary bladder, and ventral prostate tissues from adult male rats and homologous surgical tissues from human male patients. We detected the NMDAR protein by Western blot and determined the binding of the NMDA antagonist, 3H-CGP. The NMDAR messenger ribonucleic acid (mRNA) was detected by reverse transcription/polymerase chain reaction and identified by cloning and sequencing. The in vitro response to NMDAR antagonists was measured in tissue strips that were precontracted with bethanechol or electrical field stimulation (in rat and human bladder), phenylephrine (in human corpora cavernosa), or norepinephrine (in human prostate). The NMDAR2B protein; ligand-binding activity; and NMDAR1, 2A, and 2B mRNAs were detected in all tissues studied. We found an NMDAR1 variant in rat prostate and penis and in human prostate that is larger than its cerebellar counterpart, but it encodes a 767-amino acid truncated protein (NMDAR1-T). The in vitro contraction of tissue strips was inhibited by NMDAR antagonists against the following sites: polyamine (with ifenprodil); ion channnel high affinity (with dizocilpine); ion channel low affinity (with memantidine, dextrometorphan, and ketamine); and, in an NO-independent, nonadrenergic-noncholinergic pathway that was only partially affected by EAAs. We conclude that, in vitro, all the essential NMDAR subunits are present in the lower urogenital tract, a novel variant of subunit 1 is expressed, the tissues bind an NMDAR ligand, and the NMDAR antagonists induce relaxation of tissue strips. Further work is necessary to determine whether the NMDAR subunits form a fully active receptor and participate in the control of organ tone that is relevant to male sexual activity.
Expression of inducible nitric oxide synthase in smooth muscle cells from rat penile corpora cavernosa.
Nitric oxide (NO), the main mediator of penile erection, is assumed to be synthesized in the penis by the neuronal constitutive nitric oxide synthase (nNOS). However, nNOS has not been identified in the penile smooth muscle, the target of NO action. The other NOS isozymes, the inducible NOS (iNOS) and the endothelial NOS (eNOS) have not been reported in any penile tissue. The smooth muscle vascular and trabecular tissue from rat corpora cavernosa is represented in vitro by cell cultures designated RPSMC. To determine whether iNOS can be expressed in penile smooth muscle, RPSMC were treated with different lymphokines and/or bacterial lipopolysaccharide (LPS). The selected inducer, LPS/interferon, elicited at 48 hours up to a 50-fold increase in nitrites in the medium; the nitroarginine methyl ester (L-NAME), aminoguanidine, actinomycin D, cycloheximide, transforming growth factor-beta1 (TGF-beta1), and dexamethasone, but was resistant to nifedipine and platelet-derived growth factor AB (PDGF-AB). iNOS induction increased with cell passage. The [3H]L-arginine/citrulline measurement of NO synthesis with intact cells confirmed these results. Incubations of soluble and particulate fractions showed that the cytosol contained most of the activity (Km = 43 microM), which was partially inhibited by ethyleneglycal-bis-tetraacetic acid (EGTA). The 4.4-kb iNOS mRNA peaked at a late period (24-30 hours) and remained high for up to 72 hours. iNOS mRNA induction was strongly inhibited by actinomycin D and dexamethasone, partially inhibited by TGF-beta1, inhibited slightly by PDGF-AB, and unaffected by nifedipine. These results show that iNOS can be expressed in RPSMC in a cell passage-dependent fashion that has so far not been reported for other cell lines, and that the induction reaches much higher levels than in rat or human vascular smooth muscle cells. The expression pattern is also distinctive for the penile cells in time course of induction, Ca2+ dependence, response to certain agents, and mRNA stability.
The role of nitric oxide in erectile function.
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Prevention of seminiferous tubular atrophy in a naturally cryptorchid rat model by early surgical intervention.
In an attempt to determine whether the seminiferous tubular atrophy of the cryptorchid testis is preventable by early surgical correction of the cryptorchid state, aberrantly developed gubernacula destined to result in a cryptorchid testis in the Long-Evans cryptorchid (LE/ORL) rat were surgically reimplanted to the bottom of the scrotum on day 10 to 12 of age. Testis descent was monitored and the changes in testicular histology and in the volumes of the seminiferous tubules and Leydig cells were examined at day 60. As expected, normal testis descent occurred on or about day 25. Compared to untreated undescended testes at day 60, relative seminiferous tubular volumes (volume: % +/- SEM) were significantly increased by early surgical reimplantation of the gubemacula (89 +/- 1 vs. 66 +/- 3; P < 0.01). Absolute seminiferous tubular volumes (microliter +/- SEM) were also significantly increased by early surgical intervention when compared to undescended nontreated testes (893 +/- 27 vs. 170 +/- 12; P < 0.01). The testes of the surgically corrected cryptorchid animals were similar in all respects to those found in the descended testes of the sham-operated controls. Relative Leydig cell volume (% +/- SEM) was increased in the untreated cryptorchid testes compared to the surgically corrected testes (5.2 +/- 0.6 vs. 1.2 +/- 1.0; P < 0.05). Relative Leydig cell volumes in the surgically corrected testes were not significantly different from those found in the sham-operated descended controls. A modest but significant (P < 0.05) increase in absolute Leydig cell volume was also noted in the cryptorchid testes when compared both to normal controls or surgically corrected cryptorchid testes. From these observations, we conclude that early gubernaculopexy reverses the histologic changes normally seen in the cryptorchid rat testis to a relatively normal histologic architecture. These data provide experimental evidence to support the value of orchiopexy in the treatment of cryptorchidism.
Transforming growth factor-beta1 (TGF-beta1) in penile and prostate growth in the rat during sexual maturation.
The goal of this study was to determine whether transforming growth factor-beta1 (TGF-beta1) may contribute to the arrest of penile growth and the down-regulation of androgen receptors (AR) that occur during sexual maturation in the rat penis. For this purpose, body, penis, and prostate weights were obtained from male rats of increasing ages, and penis and prostate TGF-beta1 concentrations were determined by a sandwich enzyme-linked immunosorbent assay. The cytosol fraction was obtained from the shafts and glandes of immature (19-day-old) and adult (90-day-old) rat penises, and ARs were measured by a western blot assay. The effect of exogenous TGF-beta1 on penile growth was examined in vivo in two groups of immature rats (21 and 27 days old) implanted with miniosmotic pumps delivering either human TGF-beta1 or vehicle only directly into the corpora cavernosa for 6 days. The penises, prostates, and testes were weighed, and the AR content was estimated by western blot. The growth rate of the penis declined after 8 weeks of age, whereas the ventral prostate growth rate increased until 14 weeks of age and then slowed down. The content of penile AR protein decreased seven-fold in the adult rats compared to the immature animals. Penile TGF-beta1 concentration increased nearly three-fold from the 19-day-old rats to a peak at 60 days of age and then decreased over the next 4 months to the initial levels. In contrast, TGF-beta1 concentration in the prostate was not significantly affected by age and remained below the lowest penile values in all age groups. Transforming growth factor-beta1 given locally to the penis reduced penile shaft weight by 38 and 22% in two groups of immature rats, while the weights of the penile glans, testis, and ventral prostate remained unaffected. Androgen receptor content was higher in the glans than in the shaft and was not changed by TGF-beta1 treatment. These results suggest that the increase of TGF-beta1 levels in the penis may reinforce growth arrest caused by the down-regulation of penile ARs, whereas the maintenance of a high content of ARs and a low TGF-beta1 concentration may allow prostate growth to continue.