Canine Testis Research Articles

Toxicological effects of cadmium chloride on the canine testis following various routes of administration

The toxic effects of cadmium chloride on the canine testis were investigated. Various dosages of cadmium chloride were administered subcutaneously, intramuscularly, intravenously and intratesticularly. Single intravenous doses equivalent to 4.0 or 8.0 mg CdCl 2/kg body weight were lethal. The study showed that the canine testis is sensitive to cadmium administered intratesticularly, but relatively insensitive to the subcutaneous, intramuscular and intravenous administration of cadmium. Single intratesticular infusions of 0.2 mg CdCl 2/kg body weight caused testicular inflammation, hemorrhage, massive necrosis of central seminiferous tubules, and disorganization and degeneration of the peripheral seminiferous tubules. Leydig cell degeneration was also prominent. Atrophy of the seminiferous tubules was followed by interstitial revascularization and Leydig cell regeneration. The persistence of disorganized and degenerative seminiferous tubules was observed throughout the post-injection period (24 weeks), while signs of limited seminiferous tubule regeneration and spermatogenesis were present at 24 weeks. Epididymal damage occurred following cadmium infusion into the testes, however, it subsided by the 24th week after administration. In all cadmium-treated dogs evaluated, aspermic ejaculates were collected close to the time of sacrifice.

A scanning electron microscope study of the interstitial tissue of the canine testis

Scanning electron microscopy (SEM) is a potent tool that is especially valuable in interpreting the three-dimensional relationships of cells within tissues. This type of information is obtainable from thin sections in transmission electron microscopy (TEM) only by reconstructions of serial sections. The arrangement of the interstitial cells of the testis in relation to the capillaries and lymphatic channels, in particular, is easier to visualize in SEM than in TEM. Cytoplasmic constituents, as well as cell surface modifications, are demonstrable by this technique. The presence of droplets, presumably lipid droplets, both within and on the Leydig cells and the lymphatic endothelial cells, is quite evident. Other cytoplasmic structures are also apparent. For example, the possible functional significance of "openings" that are seen by SEM on the septa that surround lipid droplets is discussed relative to the appearance of the same area as seen in thin sections or in freeze-fracture replicas. SEM should become a very useful method for studying cytological and morphological alterations that occur in testicular tissue that is subjected to physical or chemical manipulation.

Effect of bilateral vasectomy on the structure and function of the testes

The effect of bilateral vasectomy on the testis and epididymis was studied in adult dogs. Particular attention was given to the endocrine function of the organ, and the total content of testosterone-secreting Leydig cells was quantitatively estimated. Vasectomy produces a marked degeneration of the seminiferous tubules as well as some reduction in the testicular volume. Spermatogenesis ceases completely, but the basal cells or spermatogonia remain unaffected, which can explain how spermatogenesis can occur again under favourable circumstances after vasorrhaphy. There was no fibrosis, cellular infiltration, or necrosis. No signs of regeneration are found up to eight weeks after vasectomy. Whether regeneration occurs spontaneously or only after recanalization remains uncertain. The Leydig cell content of the canine testis was 1.58 ± 0.43 ml per testis, or approximately 15 per cent of the testicular volume. The few estimates available for man are closely similar. Leydig cells become more prominent after vasectomy. Quantitatively, they increase from 1.58 ± 0.43 ml to 1.87 ± 0.70 ml per testis—a modest but definite average increase of about 20 per cent. The histopathologic picture, however, gives a more exaggerated impression because of the simultaneous collapse and degeneration of the tubules. The possible implications of the increase in Leydig cells after vasectomy and the mechanisms involved are discussed. The effect of bilateral vasectomy on the testis and epididymis was studied in adult dogs. Particular attention was given to the endocrine function of the organ, and the total content of testosterone-secreting Leydig cells was quantitatively estimated. Vasectomy produces a marked degeneration of the seminiferous tubules as well as some reduction in the testicular volume. Spermatogenesis ceases completely, but the basal cells or spermatogonia remain unaffected, which can explain how spermatogenesis can occur again under favourable circumstances after vasorrhaphy. There was no fibrosis, cellular infiltration, or necrosis. No signs of regeneration are found up to eight weeks after vasectomy. Whether regeneration occurs spontaneously or only after recanalization remains uncertain. The Leydig cell content of the canine testis was 1.58 ± 0.43 ml per testis, or approximately 15 per cent of the testicular volume. The few estimates available for man are closely similar. Leydig cells become more prominent after vasectomy. Quantitatively, they increase from 1.58 ± 0.43 ml to 1.87 ± 0.70 ml per testis—a modest but definite average increase of about 20 per cent. The histopathologic picture, however, gives a more exaggerated impression because of the simultaneous collapse and degeneration of the tubules. The possible implications of the increase in Leydig cells after vasectomy and the mechanisms involved are discussed.

Acute effect of intraventricular injection of catecholamines and indoleamines on the secretion of 17-oxosteroids by the canine testis.

Adult male dogs were injected into the third ventricle (3 rd V) with graded doses of cateeholamines and indoleamines, and changes in secretion of testicular 17-oxosteroids by the testis were investigated for up to 2-4 hr after the injection. The secretion of testicular 17-oxosteroids was not affected by intraventricular treatment with 2, 5 and 10 μg of dopamine but stimulated slightly with 50 μg. The response was transient and occurred for up to 90min after the injection. Intraventricular injection of 50 and 100 μg of noradrenaline or adrenaline had no stimulatory effect nor did it significantly alter the secretion of 17-oxosteroids by the testis for up to 120min after the injection. Furthermore, serotonin and melatonin, given into the 3 rd V with doses of 5 and 50 μg, caused little or no secretion of testicular 17-oxosteroids.

Effects of adenosine-3',5'-monophosphate and 5-hydroxytryptamine on the secretory activity of the canine testis.

An effort has been made on dogs to determine whether adenosine-3', 5'-monop'iosphate (3', 5'-AMP) and 5-hydroxytryptamine exert a direct effect on the 17-oxosteroid secretion by the testis, and to clarify whether the secretory response to 3', 5'-AMP is affected or not by the prior treatment with xnethylenedianiline, an inhi-bitor of steroidogenesis. 3', 5'-AMP (10 mg/kg) injected into the testicular tissue caused considerably increased secretion of 17-oxosteroids by the testis, although an intravenous injection of 3', 5'-AMP (20 mg/kg) was without effect, since it did not significantly alter the secretion of testicular 17-oxosteroids. The intravenous administration of 0.5 mg/kg of 5-hydroxytryptamine or intratesticular injection of total dose of 1.0 mg failed to produce a notable increase in the secretion of 17-oxosteroids in any instance. In dogs pre-treated with methylenedianiline (20 mg/kg), the 17-oxosteroid secretory response of the testis to an intratesticular injection of 3', 5'-AMP was completely abolished: no 17-oxosteroids were secreted from the testis. It is thus suggested that 3', 5'-AMP has the gonadotrophicc effect and its effect can be suppressed by pre-treatment with methylenedianiline, and that 5-hydroxytryptamine has no direct stimulatory effect on 17-oxosteroid secretion.

Effect of x-irradiation on the endocrine function of the canine testis.

Observations were made in dogs to determine whether X-irradiation affects the secretion of androgens by the testes, and their responses to exogenous gonadotrophin. The secretory activity was, under pentobarbital anesthesia, determined by measuring 17-oxosteroids in spermatic vein blood. The radiation was applied with 200 or 2, 000 R only to the testes, but it failed to stimulate or significantly alter the secretion of 17-oxosteroids in a period of up to 4 hr after exposure. However, at 200 R an increased secretion of testicular 17-oxosteroids into the spermatic vein became evident within 1-6 days, whereas after 2, 000 R it was observed within 13 days only in a few cases. During the post-irradiation period of 30 days, irradiated testes responded to pregnant mare serum in doses of 40iu/kg by producing larger amounts of 17-oxosteroids than normal, the maximum response occurring within a few days following irradiation. In connection with these experiments, enzyme histochemical studies were carried out on the activity of secondary alcohol dehydrogenase in the testicular tissue. At 200 R, the enzyme activity in the Leydig cell areas was increased considerably and attained to the maximum in a faw days after irradiation, and was then progressively reduced.

Biosynthesis and secretion of steroids by the canine testis

Radioactive pregnenolone ( 14C or 3H labeled) was infused at a constant rate via the spermatic artery of the isolated dog testis in order to study the quantitative significance of pathways used for the biosynthesis and secretion of testosterone in this organ. The amount of steroids and their content of radioactivity were estimated in spermatic venous blood during pregnenolone infusion and in the testis tissue at the end of such infusions. During continuous pregnenolone infusion the true specific radioactivity (μC/ mmole) of several steroids in spermatic venous blood plasma was different from the spermatic radioactivity of the same steroids in the infused testicular tissue. This observation supports the view that steroids may be present in different testicular compartments which are not equally supplied with infused pregnenolone and that these compartments do not add to steroid secretion into the spermatic venous blood in a homogeneous fashion. Concomitant infusion of human chorionic gonadotrophin or cyclic 3′,5′-adenosine monophosphate (cyclic AMP) with radioactive pregnenolone via the spermatic artery resulted in increased secretion of all known intermediates between pregnenolone and testosterone. The specific radioactivity of all these steroids decreased in such experiments. Thus the major effect of human chorionic gonadotrophin or cyclic AMP on the formation of testicular steroids occurred at step(s) prior to the formation of pregnenolone. The specific radioactivity of dehydroepiandrosterone decreased more than that of pregnenolone or 17α-hydroxypregnenolone, particularly in human chorionic gonadotrophin or cyclic AMP stimulated testes, and it is possible that dehydroepiandrosterone was also formed via metabolic pathways which did not use pregnenolone and 17α-hydroxypregnenolone as intermediates. Dehydroepiandrosterone appeared to be an important steroid for testosterone formation in vivo and metabolism of dehydroepiandrosterone to testosterone proceeded both via androstenediol and androstenedione. The present results clearly demonstrate that pregnenolone was converted to testosterone in the canine testis both via a. Δ 4- and a Δ 5-pathway.

Effect of human chorionic gonadotrophin on the discharge of ascorbic acid from the canine testis.

The discharge of testicular ascorbic acid into spermatic venous blood was examined in intact and methylenedianiline-treated dogs. An intravenous injection of human chorionic gonadotrophin (HCG, 20iu or 40iu/kg) to intact dogs produced a marked increase in the discharge of ascorbic acid as well as in the secretion of 17-oxosteroids from the testis within 15 minutes after the injection. However, neither of these events occurred when methylenedianiline (20mg or 60mg/kg) was given beforehand, while the administration of methylenedianiline (60mg/kg) after HCG (40iu/kg) had been injected failed to abolish an increased discharge of ascorbic acid induced by HCG, although it brought about a substantial inhibition of the increased secretion of testicular 17-oxosteroids. From these results, it is suggested that HCG markedly stimulates the discharge of testicular ascorbic acid, and ascorbic acid in the testicular tissue is closely linked with biosynthetic mechanism of androgens.

Production and secretion of 5 -dihydrotestosterone by male reproductive organs.

The androgen 5Α-dihydrotestosterone (DHT) has been isolated and identified in testicular tissue (rats, dogs) and in testicular venous blood of the canine testis. Conversion of <sup>3</sup>H-testosterone (T) to <sup>3</sup>H-DHT <i>in vitro</i> by testicular tissue from rats treated with follicle-stimulating hormone <i>in vivo</i> tended to increase. No such effect of human chorionic gonadotropin treatment could be observed. The Leydig cells of testes from mature rats contribute little to the formation <i>in vitro</i> of <sup>3</sup>H-DHT from <sup>3</sup>H-T. Both these androgens can be found in spermatic lymph and in epididymal tissue of the dog. In an isolated prostate preparation where the gland is infused via the prostatic arteries with the animal’s arterial blood at a constant rate, DHT can be found in effluent blood. The dog prostate both produces and secretes DHT. The purpose of DHT production and secretion by male reproductive organs is discussed.

Patterns of steroidogenesis in the vertebrate gonads

Over the past ten years data have been published on the biosynthesis of androgens and estrogens from Δ 5-pregnenolone in vertebrate gonads via two metabolic pathways—one pathway (A) using progesterone and 17α-hydroxyprogesterone as intermediates and another pathway (B) biotransforming Δ 5-pregnenolone to androgens and estrogens via 17α-hydroxypregnenolone, dehydroepiandrosterone, and Δ 5-androstenediol. We have investigated these problems in the canine testis and in the canine ovary in experiments in vivo. Since 17α-hydroxypregnenolone can be metabolized to 17α-hydroxyprogesterone in the gonads from both sexes, metabolic pathway B and metabolic pathway A may overlap. Thus, testosterone can be produced in the canine testis bypassing the formation of the steroid hormone progesterone. In the canine ovary androgens and estrogens can indeed be formed from Δ 5-pregnenolone via pathway B, but since progesterone is an important secretory product of the ovary, the preferred route for Δ 5-pregnenolone biotransformation in this tissue is pathway A. In the canine testis, the level of secretion of dehydroepiandrosterone and 17α-hydroxyprogesterone is much higher than that of either Δ 5-pregnenolone or progesterone. The metabolic pool of 17α-hydroxyprogesterone in the canine testis is larger than its pool of progesterone. Moreover, there may be one storage pool and one secretory pool for steroids and steroid hormones in the gonad of the male dog. These pools are not of the same size. Progesterone is rapidly converted to either 17α-hydroxyprogesterone or to Δ 5-pregnenolone by the dog testis, and the direct conversion of progesterone to testosterone (acetate) in this tissue is so small that it cannot be measured by currently available techniques. The rate of secretion of estrone and estradiol by the canine testis is minute. Since both the canine adrenal gland and the canine testis secrete the intermediates of pathway A and of pathway B into venous, effluent blood, the importance of this secretion for the allover hormonal household of the male dog will be discussed. This aspect of steroid metabolism appears to be important. It has recently been observed that when adrenal venous blood is infused via the spermatic artery of the dog, the secretion of testosterone by the infused testis is significantly increased. Thus, reutilization of steroid intermediates, secreted by the adrenal gland, can take place in vivo. Data will finally be presented on the effects of human chorionic gonadotropin (HCG), adenosine 3′,5′-monophosphate (3′,5′-AMP) and prostaglandin (PG) on testicular metabolism of Δ 5-pregnenolone to androgens. In these experiments special emphasis is placed on how HCG, 3′,5′-AMP. or PG can change testicular storage and testicular secretion of the intermediates from pathway A and from pathway B.

Meiotic Chromosome Alterations produced by Progesterone

ADMINISTRATION of progestins has been shown to suppress spermatogenesis in intact animals and humans, but the mechanisms involved have not been fully defined1–3. Using an adaptation of the method of Eik-Nes4 we have shown that perfusion of the canine testis with progesterone causes alterations in the meiotic chromosomes—chiefly sticky degeneration and improper spreading with clumping of the chromosomes.

Steroid metabolism of a Sertoli cell tumour of the testis of a dog with feminization and alopecia and of the normal canine testis.

SUMMARY The metabolism of [7α-3H]pregnenolone and [4-14C]dehydroepiandrosterone (DHA) by a Sertoli cell tumour of the testis from a dog with signs of feminization has been studied in vitro and compared with the metabolism of the normal canine testis. The tumour formed oestrone and oestradiol-17β from DHA thus providing direct evidence for the synthesis of oestrogen by this type of neoplasm. Relative or complete inactivity of several enzyme systems involved in the synthesis of testosterone was found in the tumour tissue, and the conversion of either precursor to testosterone was considerably less than in the normal testis. Suggestive evidence is presented for the occurrence of steroid-specific 17α-hydroxylase and 3β-hydroxysteroid dehydrogenase-isomerase systems in canine testicular tissue. The formation of sulphates of pregnenolone and DHA was shown both in normal and in neoplastic tissues and, in addition, the tumour either formed the sulphate of 17α-hydroxypregnenolone or caused the 17α-hydroxylation of pregnenolone sulphate.

Conversion of 17α-hydroxypregnenolone to 17α-hydroxyprogesterone by the canine testis in vivo

17α-Hydroxypregnenolone labeled with tritium in position 7 has been infused at a constant rate by the left spermatic artery of anesthetized dogs. Testosterone, δ 4-androstenedione, dehydroepiandrosterone and 17α-hydroxyprogesterone containing tritium were isolated from the venous blood of the left testis. 1. 1. 17α-Hydroxypregnenolone is capable of being converted to 17α-hydroxyproegsterone by the canine testis in vivo. 2. 2. When the testis is stimulated with human chrionic gonadotropin there is an increased conversion of 17α-hydroxypregnenolone to 17α-hydroxyprogesterone as well as an increase in testosterone production. 3. 3. Infusion of large amounts of unlabeled pregnenolone may also increase the conversion of 17α-hydroxypregnenolone to 17α-hydroxyprogesterone.

Metabolism in vivo of steroids by the canine testes

The formation of testosterone in vivo has been investigated in anesthetized dogs by infusing radioactive steroids via the left spermatic artery and isolating radioactive testosterone in venous blood obtained from the left spermatic vein. The data from such experiments indicate that the formation of 17α-hydroxypregnenolone and the metabolism of this steroid to testosterone is a major route in the biosynthesis in vivo of testosterone from pregnenolone by the canine testis. 1. 1. When an equal amount of [4 −14C]progesterone and 17α-hydroxy[7α- 3H]pregnenolone were infused at a constant rate by the spermatic artery, the concentration of 3H in isolated spermatic vein testosterone was from 3–8 times that of 14C. 2. 2. When [7α- 3H]prenenolone was infused by the left spermatic artery for 5 min, more [ 3H]17α-hydroxypregnenolone than [ 3H]progesterone was found in the left testis at the end of this infusion. 3. 3. The problem of preferred metabolism to testosterone in vivo by testicular tissue is discussed.