Administration of exogenous testosterone in the adult rat and its effects on reproductive organs, sex hormones and body-weight.

The present experiment was performed to observe the effects produced by high dose of a testosterone ester on the reproductive organ and body weight changes in the adult rat, and to correlate these effects with the serum hormone changes. The present study has used the benzoate ester of testosterone (Testosterone benzoate, TB) in the adult male rat (300-350 g). The aim was to co-relate the reproductive organ and body-weight changes with changes in the serum hormone levels following the administration of the ester. TB was injected i.p. for five (5) consecutive days at a dose of 100 mg/kg body-weight. The control rats were injected with vehicle (arachis oil) at the same dose. The rats were killed on the 6th, 12th, 18th, 24th and 36th days. Controls for only the 6th and 36th days were kept. Reproductive organ weight, body-weight and testosterone (T) levels in serum and testis together with serum FSH and serum LH levels were observed. The testes weights remained similar (p < 0.05) to those in the control rats until the 18th days and were reduced on the 36th day. The epididymis weights were not changed until the 36th days, while the androgen-dependent seminal vesicle and ventral prostate weights were increased (< 0.05) compared to those in the control rats. The body-weights remained unchanged at the 6th day but were significantly (p < 0.05) decreased on the 36th day. The serum testosterone (ST) concentrations were highly raised on the 6th day, came at the control level on the 18th day and were significantly decreased (< 0.05) on the 36th day. The testicular testosterone (TT) content remained significantly lower (p < 0.05) from the 6th to the 36th days post-injection. The serum LH and FSH levels also remained significantly lower (p < 0.05) throughout the treatment period. It appears that the elevated serum T levels exerted dual effect in the adult rats, namely, enhanced growth of the androgen-dependent organs and an inhibition over the hypothalamo-pituitary-testicular axis. Inhibition of the said axis was evident by the lower levels of the serum LH and FSH; probably due to this, the TT-content remained all through lower, and perhaps this low TT-content for the long period had led to the low testis weights (p < 0.05) on the 36th day. This experiment therefore, demonstrates the effects of exogenous androgen administration in the adult male rat physiology.

Thymic regression and apoptosis in the rat after treatment with the Leydig cell cytotoxin ethylene dimethanesulphonate (EDS).

Ethylene dimethanesulphonate (EDS) is an alkylating agent which is widely assumed to specifically kill Leydig cells leaving other biological systems intact. However, after EDS treatment of the male rat the thymus reversibly involutes and the gonadal regional lymph nodes are activated. In the present experiments we have demonstrated that EDS has a direct action upon the thymus both in vivo and in vitro. EDS treatment of the intact and castrated male rat and the intact female rat caused regression of the thymus by up to 50% 3 days later. Total cellularity decreased while the proliferative index increased suggesting a compensatory mechanism. Thymocytes were exposed to EDS in vitro and the response compared to the glucocorticoid methylprednisolone (P), a well characterised thymic apoptotic stimulant. EDS and P increased apoptosis in the thymocyte as characterised by the appearance of cells containing nuclei with apoptotic morphology and with DNA fragmentation visualised by a characteristic ladder after agarose gel electrophoresis. The effects of both EDS and P were time and dose dependent but, in contrast to the effects in Leydig cells, P was the most effective apoptotic stimulus (for instance 100%-P compared to 30%-EDS or 7% control/DMSO after 24 h incubation). The immunological responses of the gonadal lymph nodes were not associated with testicular regression as it was seen in the castrated rat but may be related to a direct action upon the epididymis. In conclusion, tissue specificity of the Leydig cell cytotoxin needs to be extended to the thymus and epididymis. The mode of cell death in Leydig cells and thymocytes after both glucocorticoids and EDS is apoptosis which suggests that they possess some common mechanism(s) which is responsible for the toxicity of these diverse compounds.

Testicular toxicity of the transferrin binding radionuclide 114mIn in adult and neonatal rats.

Adult (70 d) and neonatal (7 d) male rats were dosed (i.p.) with 37 MBq/kg (1 mCi/kg; approximately 1 microgram elemental indium/kg) 114mIn, a transferrin-binding radionuclide. In adults, approximately 0.25% of the injected activity localised within the testis by 48 h postinjection and remained constant for up to 63 d. In neonates, 0.06% of the activity was in the testis by 48 h, and this declined such that by 63 d only 0.03% remained. At 63 d, treated rats had reduced sperm head counts and abnormal testicular histology that was more marked in animals dosed as adults than as neonates. In vitro, uptake of 114mIn into seminiferous tubules isolated from 7-, 20-, or 70-d-old rats was compared with that of 125I. Both radionuclides were readily accumulated by the tubules. Whilst 114In uptake into 20- and 70-d tubules was inhibited by excess transferrin, uptake into 7-d tubules was unchanged. 125I uptake was not affected by excess transferrin. These data support the contention that some radionuclides may cross the blood-testis barrier by utilisation of the physiologic iron-transferrin pathway, which may lead to greater testicular damage in adult compared to neonatal animals.

Changes in the rat lymphoid tissues after Leydig cell destruction by ethane-1,2-dimethanesulphonate.

Since ethane-1,2-dimethanesulphonate (EDS) causes Leydig cell destruction which may elicit an immune response the lymphoid tissues of sexually mature male rats were examined after an intraperitoneal injection of EDS. The changes observed in the testicular regional lymph nodes confirm an immune response while those observed in the thymus may indicate involvement in the immunological response or be a toxic reaction to EDS.

Testicular effects of the Leydig cell toxicant ethane dimethanesulphonate given to neonatal rats.

Neonatal rats were injected with either 50 mg/kg ethane dimethanesulphonate (EDS) or vehicle on days 1 to 5 inclusive or on day 1 alone. Studies were made on days 6, 28, and 63 of testicular structure; related endocrinologic parameters were measured in the day 1 to 5 treated animals only. Leydig cells and their activities were identified by cell counts using sections stained for 3 beta-hydroxysteroid dehydrogenase, hCG binding to LH receptors in testicular homogenates, and assays of intratesticular testosterone, plus pituitary and/or serum concentrations of testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH). Given on days 1 to 5, EDS reduced Leydig cell populations estimated by morphometry and 125I-HCG binding, and testicular and body weights between days 6 and 63, and permanently retarded the development of the seminiferous epithelium. Decreases of serum and intratesticular testosterone occurred with homeostatic rises in FSH and LH. Injection on day 1 reduced Leydig cell numbers only on day 6 although body weight remained retarded. The data illustrate the susceptibility of the developing rat testis to the cytotoxicant EDS; whether this is related to withdrawal of androgen production or nonspecific cytotoxicity remains to be evaluated.

Growth and development of male and female rats treated with the Leydig cell cytotoxin ethane dimethane sulphonate during the suckling period.

The growth inhibiting effects of the Leydig cell cytotoxin ethane dimethane sulphonate (EDS) were investigated after treatment of both male and female neonatal rats. Pups were injected daily from day 4 to day 15 of age with EDS (50 mg/kg body weight sc), vehicle or water, or were not injected, according to a within-litter control experimental design. The rats were killed for necropsy at 40 and 80 days. The timing of attainment of the developmental milestones that appear during the suckling period was unaffected by treatment, but vaginal opening was 2 days later in EDS-treated females. After a delay of 9-10 days after the start of treatment the growth in body weight of both male and female EDS-treated rats was substantially and progressively depressed, such that by 80 days male and female EDS-treated rats showed body-weight deficits of 18 and 25%, respectively. The food intake of EDS-treated rats at 10 wk of age was less than that of controls, but was appropriate or more than appropriate for metabolic body size. As expected, testes were vestigial in EDS-treated males and the accessory male sexual organs were very small. Ovary and uterus weights were appropriate for body weight in EDS-treated females. At 80 days, liver weight was high for body weight in EDS-treated rats of both sexes. Possible mechanisms for the effect on body growth are discussed. Depressed food intake is discounted. Endocrine involvement, perhaps some anomaly of growth hormone release, or a more generalized toxic effect seem more likely.

Inhibition of testicular and somatic development after treatment of the postnatal rat with the Leydig cell cytotoxic ethylene dimethanesulphonate.

The Leydig cell cytotoxic ethylene dimethanesulphonate (EDS) was administered s.c. daily (50 mg/kg) to male rats aged 5-16 days. Apart from loss of weight and that the eyelids unfused earlier, no gross toxicity was observed during treatment. On day 17 testis weights, serum testosterone concentrations, testicular serum testosterone content and 125I-labelled human chorionic gonadotrophin (hCG) binding to testicular homogenates were reduced. Serum LH and FSH concentrations were elevated. The testes did not recover from EDS treatment and at 63 and 120 days were minute (less than 2% of control), and the prostate and seminal vesicles were small although not completely atrophied. In addition, body weights were substantially reduced. Serum and testicular testosterone and 125I-labelled hCG binding to testicular homogenates were reduced but not absent. Serum LH and FSH concentrations were increased. Light microscopy of the adult testes showed that EDS treatment inhibited the development of the seminiferous tubules. Most of the tubules were devoid of germ cells and Sertoli cells were rare. Occasionally tubules also contained spermatogonia and spermatocytes but no signs of spermiogenesis. The testes were composed mainly of closely packed interstitial tissue with no lymphatic space. The interstitial cells resembled Leydig cells and stained for 3 beta-hydroxysteroid dehydrogenase. Histochemically identified Leydig cells were absent during treatment but reappeared when treatment was withdrawn.(ABSTRACT TRUNCATED AT 250 WORDS)

Accelerated recovery of Leydig cells of the immature rat testis after administration of the cytotoxic ethylene dimethanesulphonate.

A single injection of ethane-1,2-dimethanesulphonate (EDS; 100 mg/kg) selectively destroys Leydig cells in the testis of the adult rat; however, unconfirmed reports indicate that Leydig cells in the immature rat are not affected. In this study the effect of EDS was examined 2 days after treatment of rats aged 20, 25 or 35 days. There was a large reduction in the in-vitro binding of 125I-labelled human chorionic gonadotrophin (hCG) to the homogenates of testes from EDS-treated immature rats. EDS reduced the testosterone content of the testes at all ages studied, but 2 days after injection had only significantly lowered the serum testosterone concentration of 25- or 35-day-old animals. Light microscopic examination of the testis of the 22-day-old rat, 2 days after treatment with EDS, indicated that there were still many cells staining for 3 beta-hydroxysteroid dehydrogenase. The interstitium also contained numerous atypical cells which did not stain for 3 beta-hydroxysteroid dehydrogenase. Electron microscopy of testes from the 22-day-old EDS-treated rat showed that Leydig cells were still present in the interstitium together with macrophages and fibroblast-like cells. Six days after EDS treatment of 20-day-old rats, but not 35-day-old rats, there was an increase in the binding of 125I-labelled hCG to testis homogenate to 70% of control value. Testicular testosterone content 6 days after treatment of the 20-day-old rat had risen to 50% of the control testis value.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal development of the testis after administration of the Leydig cell cytotoxic ethylene-1,2-dimethanesulphonate to the immature rat.

Male rats were injected with 50 mg ethylene-1,2-dimethanesulphonate/kg from Day 5 to Day 16 after birth and control rats received injections of the same volume of vehicle. Testes were studied at various times from Day 6 to Day 108 using histochemistry, light and electron microscopy. Fine structural degenerative changes were observed in the Leydig cells and seminiferous tubules of EDS-treated animals as early as Day 6. By Day 11 no Leydig cells could be detected and the interstitia of EDS-treated testes contained large numbers of fibroblast-like cells which formed peritubular collars 3-5 cells thick; the tubules contained Sertoli cells with heterogeneous inclusions and large numbers of lipid droplets. A small number of Leydig cells was found at Day 14 and their numbers increased so that, in animals of 28 days and older, large clusters of Leydig cells were present between severely atrophic tubules. These tubules contained Sertoli cells with few organelles; germinal cells were not observed after 28 days in EDS-treated animals. These results show that EDS destroys the fetal population of Leydig cells postnatally and this mimics the well documented effect of EDS on adult Leydig cells. The seminiferous tubules were permanently damaged by EDS in the present experiments. Tubular damage could have been due to a direct cytotoxic effect of multiple injections of EDS on the tubule before the blood-testis barrier develops or due to withdrawal of androgen support secondary to Leydig cell destruction.

Study using in-vivo binding of 125I-labelled hCG, light and electron microscopy of the repopulation of rat Leydig cells after destruction due to administration of ethylene-1,2-dimethanesulphonate.

Gonadotrophin binding to rat Leydig cells after a single administration of ethylene dimethanesulphonate (EDS) (75 mg/kg i.p.) was followed by using intratesticular microdoses of 125I-labelled hCG, whilst corresponding morphological changes in the testicular interstitium were studied with light and electron microscopy. No discernible effect on 125I-labelled hCG binding compared with controls was observed until 24 h after treatment. Between 24 and 32 h a sharp decline in binding occurred which was correlated with extensive Leydig cell destruction. By 48 h the 125I-labelled hCG binding was negligible and no morphologically recognizable Leydig cells were found at this time. The specific binding remained low until 21 days after treatment and then a marked increase occurred to give nearly normal levels by 49 days. This was consistent with a generalized repopulation of the interstitium with Leydig cells, seemingly the result of differentiation of fibroblast-like precursor cells.

Congenital vertebral anomalies and the possible aetiological relationship with spina bifida.

The frequency and distribution of hemivertebrae, fused vertebral bodies, fused vertebral arches and fused ribs were recorded in 376 children and young adults with either spina bifida cystica or occulta, or solitary vertebral body anomalies, or multiple vertebral body anomalies. An attempt was made to find evidence to support the hypotheses that multiple vertebral anomalies and anencephaly-spina bifida are aetiologically related, and that solitary vertebral body anomalies are sporadic and developmentally distinct. Statistical analysis of the distribution of the vertebral and rib defects partly supported these hypotheses.

Are congenital vertebral anomalies and spina bifida cystica aetiologically related?

Radiological records of 104 patients with multiple vertebral anomalies without apparent spina bifida and 112 infants with spina bifida cystica were surveyed and the incidences of hemivertebrae and of rib, vertebral body, and vertebral arch fusions were recorded. The distributions of these four anomalies along the vertebral axis were found not to be statistically different between the two age groups. This is additional evidence to support the hypothesis that multiple vertebral anomalies and anencephaly-spina bifida cystica are aetiologically related.

Renal polycystosis in the rat induced by prednisolone tertiary butyl acetate.

A single i.m. injection of 66 mg/kg prednisolone tertiary butyl acetate given on the 1st day of life produced glomerular degeneration and collecting duct and proximal tubule cysts in rat kidneys. There was evidence of delayed nephrogenesis leading to persistance of the neogenic zone.

Vertebral body widths and heights in normal infants and in those with myelomeningocele.

Measurements were made of vertebral body widths and heights from post-mortem radiographs of 20 newborn children with myelomeningocele and 10 "normal" children. The only statistically significant difference was reduction of vertebral body widths at T11 in infants with myelomeningocele. There was no evidence of shortening of the lumbosacral region as proposed by previous workers.

A radiological study of vertebral and rib malformations in children with myelomeningocele.

Postmortem radiographs from 112 infants with thoracolumbosacral or lumbosacral myelomeningoceles were surveyed for associated vertebral and rib malformations. Sixty-four exhibited a variety of anomalies including hemivertebrae, fused vertebral bodies, fused vertebral arches, fused ribs, absence of ribs and absence of vertebral bodies. Anomalies were commonly associated with the cranial end of a dysraphic spina bifida region; in addition hemivertebrae and rib fusion exhibited peak incidences centred around the seventh cervical and fourth thoracic vertebra respectively. Fusion of vertebral arch elements increased in frequency throughout the thoracic region towards T10. The twelfth pair of ribs were absent in 20% of cases. Apparent absence of vertebral bodies from the caudal end of the vertebral column was seen in 24% of the radiographs.

A synergistic interaction between the teratogenic effect of trypan blue and dietary deficiency in the rat.

There was an increased incidence, compared to controls, of exencephaly and microphthalmia in the offspring of rats fed a vitamin D deficient diet and injected with trypan blue on day 9 of gestation. Oral vitamin D did not reverse the effect.

The autonomic innervation of the human male and female bladder neck and proximal urethra.

Neurohistochemical and electron microscopic techniques have been used to determine the autonomic innervation of smooth muscle in the normal human male and female bladder neck and proximal urethra. Numerous cholinergic nerves supply the bladder neck and female proximal urethra, although nerves of the noradrenergic type rarely are observed in these regions. In contrast the male proximal urethra is supplied richly with noradrenergic nerves, indicating that the region is under direct sympathetic control and functions to prevent vesical reflux of ejaculate. The sympathetic innervation of the human bladder neck and female proximal urethra is different from that in other species, a finding that emphasizes the need for caution when results obtained from experimental animals are applied to the human. The sex difference is the innervation of the human proximal urethra has been discussed, particularly in relation to autonomic blocking agents currently used in the treatment of bladder neck and urethral dysfunction.

The distribution of endocrine-like cells in the human male and female urethral epithelium.

Histochemical and electron microscopic techniques have been used to study the nature and distribution of fluorescent, endocrine-like cells in the urethra of the human male and female. The confinement of such cells to specific regions of the urethra is discussed in relation to the embryological development of this part of the urinary tract.

Heterotopic dorsal-root ganglion cells around the spinal cord in children with spina bifida aperta.

Sixty-three of 95 cords from children with spina bifida aperta had associated heterotopic ganglion cells, usually located in dorsal nerve roots posterior or lateral to the cord. Heterotopic cells were more common in cords with complete or partial diplomyelia, and the largest number was found in the upper sacral region. It is proposed that the heterotopic position of the cells is due to abnormal and delayed migration of neural crest cells during primary neurogenesis. The findings lend support to the concept that in children with myelomeningocele the cord lesion is not due to secondary rupture.

The embryogenesis of trypan-blue induced spina bifida aperta and short tail in the rat.

The study of litters from 140 Wistar-derived rats injected with trypan blue during gestation leads to conclusion that a myelocele can result from faulty closure of the neural plate and that this is accompanied in many cases by blebs in the paraxial mesoderm. Haematomata usually underlie the open neural plate at an early stage and they form by extravasation of blood from the dorsal aortae into the blebs. Local ventral deflection of the notochord beneath a myelocele probably results from delayed separation of the notochord from the hindgut. Complete failure of separation and abnormality induced in the tail-bud could result in sacral agenesis and/or a short tail. All these malformations may result from varying severity of the action of the trypan blue at different developmental stages.