Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes.

Androgens are known to influence penile erection and nitric oxide synthase (NOS) activity in cavernosal tissue homogenates. The present study was an assessment of the effects of castration and androgen replacement on the in vivo release of nitric oxide (NO), and of the simultaneously recorded intracavernosal pressure (ICP) changes elicited by electrostimulation of the cavernosal nerves (SCN) in the anesthetized rat. The extracellular levels of NO in the corpora were monitored electrochemically using porphyrin microsensors. The content of NOS isoenzymes in corporal homogenates was determined by immunoblotting. The responses of castrated rats with or without testosterone (T) implants were compared to those of intact animals. Castration virtually abolished both the NO and the ICP responses to SCN. There was a concomitant significant decrease in the content of both the neuronal (nNOS) and the endothelial (eNOS) isoenzymes in the cavernosal tissue. All these effects of castration were prevented by T replacement. The NO response to SCN was positively correlated with the levels of nNOS and eNOS, especially when the values of the two isoforms were added (r = 0.71, P < 0.001). These data suggest that the facilitatory action of androgens on penile erection involves the up-regulation of both constitutive NOS isoenzymes in the corpora cavernosa.     

Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway

Relaxation of the smooth muscle cells in the cavernosal arterioles and sinuses results in increased blood flow into the penis, raising corpus cavernosum pressure to culminate in penile erection. Nitric oxide, released from non-adrenergic/non-cholinergic nerves, is considered the principle stimulator of cavernosal smooth muscle relaxation, however, the inhibition of vasoconstrictors (that is, norepinephrine and endothelin-1, refs. 5-9) cannot be ignored as a potential regulator of penile erection. The calcium-sensitizing rho-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit myosin light chain phosphatase, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown. Using pharmacologic antagonism (Y-27632, ref. 13, 18), we examined the role of Rho-kinase in cavernosal tone, based on the hypothesis that antagonism of Rho-kinase results in increased corpus cavernosum pressure, initiating the erectile response independently of nitric oxide. Our finding, that Rho-kinase antagonism stimulates rat penile erection independently of nitric oxide, introduces a potential alternate avenue for the treatment of erectile dysfunction.     

Suppression of 125I-vasoactive Intestinal Polypeptide Binding Sites in Arteries of the Hamster Seminal Vesicle Following Castration

The presence and distribution of 125I-vasoactive intestinal polypeptide (VIP) binding sites in blood vessels supplying the hamster seminal vesicle was studied using a receptor autoradiographic technique before and following castration. 125I-VIP binding was studied in intact animals, in animals under a 15-day period of castration and in animals under the same period of castration but submitted to a further 15-day period of testosterone treatment.Our results show that, in the seminal vesicle, VIP-binding sites are localized in the gland smooth muscle coat and arterial smooth muscle. A 15-day castration period abolishes 125I-VIP binding to vascular smooth muscle but has no effect on 125I-VIP binding to the gland smooth muscle coat. Treatment with testosterone restores 125I-VIP binding to the vascular smooth muscle, completely reversing the effect of castration.Our results indicate that VIP-binding sites in the smooth muscle wall of arteries supplying the hamster seminal vesicle are under androgenic control and are more sensitive to androgen deprivation that VIP-binding sites associated to the gland smooth muscle coat.     

Lack of direct androgen regulation of PDE5 expression

It has been reported that penile PDE5 expression was under androgen regulation. However it remained unknown whether the observed change in PDE5 expression in castrated animals was under direct androgen regulation or due to changes in smooth muscle content. In the present study we showed that castration of rats caused a reduction of penile size and cavernous smooth muscle content. Immunostaining detected concomitant reduction of PDE5 and alpha smooth muscle actin (α-SMA) expression in the corpus cavernosum of castrated rats. Real-time PCR and Western blotting detected no change of PDE5 expression when normalized with α−SMA expression in castrated rats. Androgen receptor (AR) expression was increased while PDE5 expression remained unchanged in DHT-treated rat cavernous smooth muscle cells (CSMC). Prostate specific antigen (PSA) promoter activity was upregulated while PDE5A promoter activity remained unchanged in DHT-treated CSMC. Thus, PDE5 expression was not under direct androgen regulation.     

Effects of castration and androgen replacement on male courtship behavior in the red-sided garter snake (Thamnophis sirtalis parietalis)

Following artificial hibernation, sexually mature male garter snakes (Thamnophis sirtalis parietalis) exhibited a decline in courtship behavior irrespective of castration, sham operation, or castration with testosterone replacement therapy. Behavior declined more rapidly in castrated animals with testosterone replacement than in castrated or sham-operated animals. In sham-operated animals, the decline in courtship was accompanied by changes in testicular weight and spermatogenic state from small spermatogenically inactive testes to large spermatogenically active testes. Serum androgen levels were more than fourfold greater in sham-operated animals than in castrated animals; cell height of the androgensensitive renal sex segment was greatest in castrated animals with testosterone replacement and least in castrated animals. These findings indicate that following artificial hibernation, male courtship behavior of T.s. parietalis is independent of the presence of the testes.     

Improved erectile function after Rho-kinase inhibition in a rat castrate model of erectile dysfunction

Androgens are reported to act as strong modulators of erectile function influencing both nitric oxide and vasoconstrictor signaling. Castration results in a depressed erectile response that is associated with a loss of nitric oxide production and increased responsiveness to constrictive agents. The increased vasoconstrictor response may be a result of an active RhoA/Rho-kinase signaling pathway. We report here results of studies designed to test the hypothesis that inhibition of the Rho-kinase pathway restores erectile function in a castrate model by relaxing the smooth muscle. Mean arterial (MAP) and corpus cavernosal (CCP) pressures were monitored during intracavernosal injection of the Rho-kinase inhibitor Y-27632. Castration reduced the maximal erectile response (CCP/MAP) by 33%, and testosterone replacement restored the response (intact, 0.736 +/- 0.040; castrate, 0.492 +/- 0.022; testosterone, 0.681 +/- 0.073). Injection of Y-27632 increased CCP in all experimental groups; it also left shifted the voltage response curve and increased the maximal CCP/MAP response (intact, 0.753 +/- 0.091; castrate, 0.782 +/- 0.081; testosterone treated, 0.894 +/- 0.033). Y-27632 dose dependently relaxed phenylephrine-stimulated cavernosal tissues. Cavernosal tissues showed increased RhoA and Rho-kinase protein levels after castration. Our data support the hypothesis that an active Rho/Rho-kinase pathway contributes to the reduced erectile response after castration due to an upregulation of RhoA/Rho-kinase protein levels and that inhibition of this pathway may serve as an effective treatment for erectile dysfunction.     

Phosphorylated endothelial nitric oxide synthase mediates vascular endothelial growth factor-induced penile erection

The objective of the present study was to evaluate whether vascular endothelial growth factor (VEGF)-induced penile erection is mediated by activation of endothelial nitric oxide synthase (eNOS) through its phosphorylation. We assessed the role of constitutively activated eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS-knockout (eNOS(-/-)) mice with and without vasculogenic erectile dysfunction. Adult WT and eNOS(-/-) mice were subjected to sham operation or bilateral castration to induce vasculogenic erectile dysfunction. At the time of surgery, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 (10(9) particle units) or with empty virus (Ad.Null). After 7 days, erectile function was assessed in response to cavernous nerve electrical stimulation. Total and phosphorylated protein kinase B (Akt) as well as total and phosphorylated eNOS were quantitatively assessed in mice penes using Western immunoblot and immunohistochemistry. In intact WT mice, VEGF145 significantly increased erectile responses, and in WT mice after castration, it completely recovered penile erection. However, VEGF145 failed to increase erectile responses in intact eNOS(-/-) mice and only partially recovered erectile function in castrated eNOS(-/-) mice. In addition, VEGF145 significantly increased phosphorylation of eNOS at Serine 1177 by approximately 2-fold in penes of both intact and castrated WT mice. The data provide a molecular explanation for VEGF stimulatory effect on penile erection, which involves phosphorylated eNOS (Serine 1177) mediation.     

Erectile function and bulbospongiosus EMG activity in estrogen-maintained castrated rats vary with behavioral context.

Electromyographic (EMG) activity in the bulbospongiosus muscles (BS) was recorded to monitor potential castration-induced alterations in muscle activity during copulation and reflexive erections. EMG recordings were made from intact male rats and from castrated rats maintained from 7 to 50 days on estradiol benzoate (300 micrograms/day) or testosterone (200 micrograms/day). Despite a 40-50% postcastration reduction in the weight of the BS and accessory sexual glands in estrogen-treated rats, the pattern of EMG activity during copulation was similar across groups. In estradiol-treated males, the EMG burst frequency during mounts and burst duration during intromissions exceeded the parameters of intact males and of castrated males maintained on testosterone. Between intromissions, and following ejaculatory patterns, estrogen-treated males displayed spontaneous muscle bursts accompanied by visually confirmed erection of the glans penis, but these males quickly lost the capacity for reflexive erections. These data demonstrate that despite castration-induced atrophy of the penile muscles and, presumably, their spinal motor nuclei, the motor output to these muscles is maintained following androgen removal. The capacity for substantial penile erection is retained during copulation long after reflexive erections have diminished.     

Androgenic maintenance of the erectile response in the rat.

Ongoing studies in this laboratory have used the castrated rat, with and without testosterone replacement, to investigate how androgens maintain the erectile response. The high intracavernosal pressures during erection depend on both an increase in the rate at which blood flows into the sinuses of the corpus cavernosum and a decrease in the rate at which blood flows out (veno-occlusion). Accordingly, our studies investigated androgenic regulation of the arterioles that regulate inflow and of the intracavernosal muscle that regulates the veno-occlusive mechanism controlling outflow. The results of these studies show that castration causes a decline in the rate of inflow and that androgen replacement reverses this decline. The decline in inflow in the castrated rats is also reversed by the administration of a nitric oxide donor drug, suggesting that the androgen may regulate inflow by increasing the synthesis of nitric oxide. Testosterone also appears to regulate outflow by controlling the sensitivity of the erectile mechanisms to norepinephrine, considered to be the principle vaso-constrictor neurotransmitter in the erectile response. Taken together, the results of these studies suggest that androgens control the erectile response by altering the synthesis and action of the neurotransmitters that normally alter the state of contraction and relaxation of smooth muscle in the erectile tissue.     

Penile erections induced by amantadine in male rats

Amantadine induces repeated episodes of penile erection and ejaculation in intact but not in castrated adult male rats. Testosterone propionate administered to castrated rats restores their responsiveness to the sexual stimulant effect of amantadine. Atropine, but not atropine methyl bromide, prevents amantadine induced penile erection.     

Etude de l’erection chez le rat: un nouveau modele physiologique

Penile erection is a muscular and vascular event mediated by the autonomic nervous system. The neurophysiology of erection remains poorly understood and controversial, requiring a suitable model for in-vitro studies of erectile function. Such a model, based in the rat whose penile innervation is very similar to man, is described here. The first study using this model considers the influence of systemic blodd pressure (BP) on penile erection. In 33 anaesthetized rats the pelvic and cavernosal nerves were identified and dissected. Supra maximal electrical stimulation was delivered over 1 minute by a train of 1 ms pulses onto the pelvic nerve (10 V, 15 Hz) or the cavernosal nerve (6 V, 10 Hz). Systemic blood pressure and intracavernosal pressure (ICP) were monitored and stored on a computer. As in previous animal models (dog, monkey), four phases of the cavernosal response to neural electrical stimulation were observed: latency, tumescence, full erection, and détumescence. In all rats electrical stimulation of either the pelvic or cavernosal nerves significantly increased intracavernosal pressure. Complete erectile response (rigidity and unfolding of the penis) was only seen with intracavernosal pressures > 95 mm Hg. Intracavernosal pressure increased proportionally with blood preessure during the full erection phase according to the equation ICP=0.94 BP ? 31 mm Hg (r=0.94 BP ? 31 mm Hg (r=0.94) for electrical stimulation of the cavernosal nerve, or the alternative aquation ICP=0.76 BP ? 21 mm Hg (r=0.73) for electrical stimulation of the pelvic nerve. The rat is a readily available model for the study of erection and present obvious advantages over existing models such as the dog, cat and monkey. Cavernosal repsonse to neural stimulation was closely related to arterial blood pressure and the two linear equations presented above should be considered further in studies modifying autonomic neurotransmission as well as in relation to the effects of pharmacological compounds with vasomotor actions on erectile function.     

去势大鼠阴茎海绵体胱硫醚γ裂解酶与硫化氢的表达

目的观察去势大鼠阴茎海绵体胱硫醚γ裂解酶（Cystathionine-γ-lyase,CSE）/硫化氢（Hydrogen sulphide,H2S）的变化,进一步探讨勃起功能障碍的发病机制。方法雄性SD大鼠72只分4组：对照组、假手术组,去势组和去势炔丙基甘氨酸（PAG,CSE阻断剂）组,检测基础条件下和阿扑吗啡（Apomorphine,APO）刺激后的海绵体内压（Intracavernous pressure,ICP）及勃起率;激光共聚焦显微镜检测CSE在大鼠勃起不同时期阴茎海绵体组织中的表达,敏感硫电极测定H2S在勃起不同时期的含量。结果与假手术组比较,去势组和去势PAG组ICP与勃起率下降（P〈0.01）;且去势PAG组较去势组ICP明显下降（P〈0.01）;阿朴吗啡刺激后,与假手术组比较,勃起前去势组和去势PAG组CSE蛋白表达降低（P〈0.01）,勃起中去势各组较假手术组CSE蛋白表达降低（P〈0.01）,且去势PAG组较去势组CSE蛋白表达明显降低（P〈0.01）;勃起后各组间CSE蛋白表达变化无差异。勃起前和勃起中去势各组较假手术组H2S含量下降（P〈0.05）,且勃起中去势PAG组较去势组H2S含量明显下降（P〈0.01）：勃起后去势组和去势PAG组较假手术组H2S含量下降（P〈0.01）。结论去势大鼠勃起功能障碍与CSE和H2S表达下降有关。     

Significance of Testosterone in Regulating Hypothalamic Content and In Vitro Release of β-Endorphin and Dynorphin

The effects of castration and testosterone replacement on hypothalamic pools of beta-endorphin and dynorphin and on the basal and corticotropin-releasing factor (CRF)-stimulated release of these peptides from hypothalamic slices in vitro were studied. The experiments were done in adult male rats. The hypothalamic content of both peptides increased significantly within 1 week of castration, and levels remained elevated for up to 4 weeks. Testosterone treatment, begun at the time of castration, prevented these increases. In addition, testosterone replacement 6 weeks after castration reversed peptide levels to normal. Basal in vitro release rates of beta-endorphin and dynorphin were significantly lower from hypothalamic slices derived from 1-week castrated animals than from intact males, and when testosterone was administered in various doses in vivo, basal release rates in vitro increased in a dose-related manner. Hypothalami from rats that had been castrated for 4 weeks, however, showed basal release rates similar to those in tissues from intact controls, a finding indicating that castration initially alters both opioid peptide synthesis and release; later, release is normalized, whereas synthesis remains elevated. CRF was found to stimulate beta-endorphin and dynorphin release from hypothalami from intact and from 1- and 4-week-castrated rats, a result indicating that castration does not alter the response of beta-endorphin and dynorphin neurons to this stimulus.     

The androgenic effect on the fine structure of the harderian gland in the male hamster

Summary A sexual dimorphism of the hamster Harderian gland at the ultrastructural level has been reported. The effect of testosterone on the fine structure of the gland from castrated male golden hamsters is reported here. Harderian glands from the following three groups of animals were examined at regular intervals up to 60 days after castration: (1) castrated; (2) castratedsham-injected, receiving 0.1 ml sesame oil per day; (3) castrated-testosterone injected, receiving 2mg testosterone propionate in 0.1 ml sesame oil per day. In groups 1 and 2, clusters of cylindrical tubules, typical of the male gland, decreased in number and disappeared almost completely 2 weeks after castration. Membranous structures, typical of the female gland, prevailed in these two groups throughout the remaining period of experiment. On the other hand, these changes were prevented in the group of castrated animals maintained on testosterone propionate. It is concluded that castration modified the ultrastructure of the male hamster Harderian gland toward the female type and that daily administration of testosterone propionate prevented this change.     

Some hormonal factors (hypophysectomy, castration and testosterone administration) modifying the course of "compensatory" muscle hypertrophy in the rat.

1. Maximum compensatory hypertrophy of the soleus and plantaris muscle in male rats is attained seven days after tenotomy of the gastrocnemius muscle (39% and 9% respectively). When tenotomy of the gastrocnemius was performed seven days ater hypophysectomy, hypertrophy in these two muscles was aproximately half that found in control animals. 2. After 81-day castration of young male rats the weight of the saleus and plantaris was reduced and hypertrophy following tenotomy of the gastrocneumius muscle did not develop. 3. Chronically castrated rats received testosterone two weeks prior to tenotomy of the gastrocnemius and a week during the muscle hypertrophy phase. Hypertrophy of the soleus in castrated rats which had received testosterone seven days after tenotomy of the gastrocnemius was 25% as compared with muscles of castrated animals. The corresponding value in the plantaris muscle was 10%. 4. These results indicate that even calf muscles of the rat, namely the soleus and plantaris muscles, are significantly affected by testosterone under these conditions, although it is not, as yet, clear whether its action is direct or indirect.     

Androgens augment renal vascular responses to ANG II in New Zealand genetically hypertensive rats

Males develop higher blood pressure than do females. This study tested the hypothesis that androgens enhance responsiveness to ANG II during the development of hypertension in New Zealand genetically hypertensive (NZGH) rats. Male NZGH rats were obtained at 5 wk of age and subjected to sham operation (Sham) or castration (Cas) then studied at three age groups: 6-7, 11-12, and 16-17 wk. Mean arterial blood pressure (MAP), heart rate (HR), and renal blood flow (RBF) measurements were recorded under Inactin anesthesia. These variables were measured after enalapril (1 mg/kg) treatment and during intravenous ANG II infusion (20, 40, and 80 ng/kg/min). Plasma testosterone was measured by ELISA. Angiotensin type 1 (AT1) receptor expression was assessed by Western blot analysis and RT-PCR. ANG II-induced MAP responses were significantly attenuated in Cas NZGH rats. At the highest ANG II dose, MAP increased by 40+/-4% in Sham vs. 22+/-1% in Cas NZGH rats of 16-17 wk of age. Similarly, renal vascular resistance (RVR) responses to ANG II were reduced by castration (209+/-20% in Sham vs. 168+/-10% in Cas NZGH rats at 16-17 wk of age). Castration also reduced MAP recorded in conscious NZGH rats of this age group. Testosterone replacement restored baseline MAP and the pressor and RVR responses to ANG II. Castration reduced testosterone concentrations markedly. Testosterone treatment restored these concentrations. Neither castration nor castration+testosterone treatment affected AT1 receptor mRNA or protein expression. Collectively, these data suggest that androgens modulate renal and systemic vascular responsiveness to ANG II, which may contribute to androgen-induced facilitation of NZGH rat hypertension.     

Receptor tyrosine kinase Ephb6 regulates vascular smooth muscle contractility and modulates blood pressure in concert with sex hormones

Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.     

Pharmacologic treatment of erectile dysfunction

Penile erection occurs in response to cavernous smooth muscle relaxation, increased blood flow to the penis, and restriction of venous outflow. These events are regulated by a spinal reflex relying on visual, imaginative, and olfactory stimuli generated within the central nervous system (CNS) and on tactile stimuli to the penis. Drugs can have a facilitatory or inhibitory effect either on the nerves regulating this reflex or on the cavernous smooth muscle. A balance between contractile and relaxant factors governs flaccidity/rigidity within the penis. Drugs that raise cytosolic calcium either prevent or abort erection. Conversely, drugs that lower cytosolic calcium relax smooth muscle and can initiate penile erection. Efficacy in treating erectile dysfunction (ED) with phosphodiesterase inhibitors, especially type 5; alpha-adrenergic-receptor antagonists; and dopamine agonists exploit these mechanisms within the penis or CNS. Recent advances in our understanding of the pharmacology of penile erection are being translated into effective therapies for ED.     

Pituitary luteinizing hormone level in the male house musk shrew, Suncus murinus L., following castration and testosterone treatment

The effect of castration and replacement therapy with testosterone propionate (TP) on the pituitary LH concentration and contents in the house musk shrew was investigated by using an in vitro bioassay for LH, the Rat Interstitial Cell Testosterone assay. The concentration and contents of LH increased slightly 10 days after castration, but decreased progressively thereafter to about a half of the pre-operation level by 90 days after the operation. The replacement with TP (100 micrograms/day) for 7 days significantly depressed LH contents when it was begun 10 days after castration, while the same treatment started immediately after or 30 days after the operation did not significantly affect the pituitary LH level. The feedback mechanism between the gonad and the pituitary may be slightly different in the shrew from that in other mammals. TP replacement, started immediately after castration, completely inhibited the decrease in the weight of male accessory sex organs in castrated shrews. In castrated animals when more than 10 days had elapsed after the operation, however, the decreased weight of the organs could not be fully restored by the TP replacement for 7 days.     

Electron microscopic and morphometric studies of the main kidney segment of male and female rats following castration and testosterone substitution

The effect of testosterone on the 3 segments of the renal proximal tubule (S1, S2, S3) of male and female rats was studied by electronmicroscopic and morphometric methods. Only light, granulated and dark lysosomes as well as microbodies (peroxisomes) and dictyosomes (Golgi zones) were investigated. After castration the area density of light lysosomes in the S1 segment increases in males whereas it decreases in females; therefore the sex different pattern of light lysosomes, that is to be seen in normal animals, is reversed. The absolute size and number of light giant lysosomes is also elevated in castrated males in comparison to normal animals as well as to animals substituted by testosterone. - Dark lysosomes of the S1 segments are more numerous in castrated females and less numerous in castrated males than in normal animals. - The distinct sex difference in dark lysosomes of the S2 segment which is demonstrable in normal animals disappears after castration the area density of dark lysosomes increasing in castrated females and decreasing in castrated males. The three species of lysosomes in the S1 segments show no longer a sex difference after substitution with testosterone: substituted males develop the same pattern as normal animals and substituted females are almost comparable with normal males. However, the sex difference in dark lysosomes of the S2 segment is more pronounced after testosterone treatment. - The characteristic pattern of light lysosomes in the S1 and S2 segments as well as the change of the sex different lysosomal pattern after castration and substitution with testosterone, respectively - especially in S1 - seem to be caused by testosterone which results in an inhibition of resorption. Only after castration a sex difference appears in dark lysosomes of the S3 segment (males show more dark lysosomes than females). This sex difference is reversed by testosterone treatment. There are more numerous lysosomes with an non-homogeneous matrix in both sexes after castration which are seldom to be seen in normal and substituted animals. The area density of microbodies shows sex differences in all 3 segments of normal animals. While no significant changes in S1 and S2 are to be seen after castration and substitution, there is a pronounced decrease of the area density of microbodies in S3 of males after castration, so that no sex differences are then available.(ABSTRACT TRUNCATED AT 400 WORDS)