Heritable testicular hypoplasia in Nguni (Bos indicus) bulls: vascular characteristics and testosterone production.

The biased unilateral occurrence of heritable gonadal hypoplasia was investigated by examining the gross- and microanatomy of the testicular artery and vein, testicular blood flow and testicular testosterone secretion in normal Nguni bulls and in Nguni bulls showing unilateral left, unilateral right and bilateral hypoplasia of the testis. A high incidence of branching of the testicular artery was found ipsilateral to hypoplastic testes. The branching occurs a short distance from the dorsal aorta: one branch proceeds to the testis, the other to the ipsilateral kidney. The association between arterial branching to the kidney and ipsilateral hypoplasia of the testis held for both unilaterally left and unilaterally right hypoplastic bulls. Variations in the anatomy of the testicular vein occurred in both normal and hypoplastic bulls but there was no specific association between the variations and ipsilateral hypoplasia. The lumen diameter of the testicular artery or branch correlated with testis mass. Wall thickness of the artery ipsilateral to hypoplastic testes was not different from that in normal bulls, discounting hyperplasia of the endothelium. Total blood flow to the testis correlated with testis mass. The secretion rate of testosterone from hypoplastic testes was lower than that of normal testes but there was no difference when compared on a unit mass basis.     

Three-dimensional organisation of the vasculature of the rat spermatic cord and testis

Summary The vascular architecture of the rat testis and spermatic cord was studied by a corrosion cast technique combined with scanning and transmission electron microscopy, and light microscopy. The casts preserve the endothelial impressions of the vessels and enable them to be differentiated into the various vascular components. Frequent arterio-arterial anastomotic arcades and occasional arterio-venous anastomotic channels are seen. A well defined hexagonal pattern of intertubular and peritubular vessels surround the seminiferous tubules. Prominent large endothelial nuclei protrude into the arterial lumina at branching sites, but their functional significance is not known. The outermost vascular layer of the testis consists of large veins, venules, and capillaries, but lacks any arterial branches; it also contains loosely arranged veno-venous anastomotic networks. We have named this vascular layer the sub-albugineal venous plexus. The testicular artery increases in luminal diameter as it approaches the testis. The periarterial capillary plexus, which lies between the pampiniform plexus and the testicular artery, is drained by two types of venules.     

Ultrastructural localisation of alkaline phosphatase in the intertubular tissue of the testis in the domestic fowl

Alkaline phosphatase activity in the intertubular tissue of the testes of the domestic fowl was examined using an ultracytochemical technique based on the lead capture method. In the interstitial tissue, the Leydig cells, transitional cells and the fibroblasts displayed enzyme activity on their cell membranes. Vacuoles located in the transitional cells were lined by reaction products of enzyme activity, whereas the vacuoles representing extracted lipid droplets and present mainly in the Leydig cells were free of enzyme activity. In the peritubular tissue the cell processes of fibroblasts showed enzyme activity on the cell membranes and in pinocytotic vesicles. Cell processes lying adjacent to blood vessels showed pronounced activity. In the blood vessel itself some activity was present in the basement membrane and the endothelium. The surface of the red blood cell showed moderate activity. The possible role of alkaline phosphatase in the transfer of hormone from the Leydig cells to the seminiferous tubules and from the seminiferous tubules to the interstitium is discussed. The myoid cells and their processes were devoid of enzyme activity.     

Spermatogenesis, testicular composition and the concentration of testosterone in the equine testis as influenced by season

The influence of season on spermatogenesis, testicular composition and the concentration of testosterone in the equine testis was evaluated using testes from 45 stallions. Testes were obtained through a commercial abbatoir during September, December-January, March and July. The weights of the testes, the tunica albuginea and testicular parenchyma and the proportion of the testicular parenchyma occupied by seminiferous tubules or interstitial tissue were similar during each season. How ever, diameter of the seminiferous tubules was greater in July than during other months of the study. In addition, the concentration of testosterone within the testicular parenchyma was twice as great during July as during the fall and winter, and this period of peak testicular testosterone concentrations was associated with spermatozoal production rates, which were 65% greater than those observed in September.     

Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX----XY chimaeric mouse testes

The distribution of XX and XY cells in XX----XY chimaeric mouse testes was analysed by enzyme marker analysis of separated testicular tissues and by in situ DNA marker analysis of air-dried testicular cells and testis sections. XX cells contributed to the Leydig cells, the peritubular cells and the vascularized connective tissue of the tunica albuginea. The Sertoli cells, on the other hand, appeared to be exclusively XY. These results indicate that during the development of the testis, Sertoli cell differentiation is triggered by cell-autonomous activity of the Y chromosomal testis-determining gene Tdy. Subsequent steps in testis differentiation may be a consequence of Sertoli cell activity.     

Functional arterio-venous anastomoses between the testicular artery and the pampiniform plexus in the spermatic cord of rams

Blood flow to the testis, haemoglobin oxygen saturation and testosterone concentration in arterial and venous testicular blood vessels were studied in Texel rams in the breeding and non-breeding season. Blood flow in the proximal and distal testicular artery was measured electromagnetically. The mean flow in the proximal testicular artery was 18.5 ml/min and in the distal testicular artery 7.5 ml/min, and there was no detectable seasonal influence. Haemoglobin oxygen saturation and testosterone concentration were measured in the saphenous artery and vein, the distal testicular artery and vein, and in the proximal testicular vein. The haemoglobin oxygen saturation in the proximal testicular vein was significantly higher than in the distal testicular vein in both seasons. The mean testosterone concentration was significantly lower in the proximal testicular vein than in the distal testicular vein in both seasons. Based on haemoglobin oxygen saturation and testosterone data, it was calculated that between 28 and 46% of the testicular arterial blood was bypassing the testis and was directly flowing through arterio-venous anastomoses towards the pampiniform plexus in the spermatic cord of conscious rams. In anaesthetized rams 55 and 64% of the blood was flowing directly from the testicular artery to the pampiniform plexus based on blood flow data. Transfer of testosterone and oxygen by passive diffusion from the testicular artery to the pampiniform plexus and vice versa in the spermatic cord was not detected.     

Characterization of rat testicular guanylate cyclase during development.

The biochemical characteristics of rat testicular guanylate cyclase were investigated and the activity and subcellular distribution of the enzyme was determined during testicular development. Examination of the effects of metal ions, nucleotides, detergents and other in vitro activators on the activity of guanylate cyclase revealed that the testicular enzyme is similar in most respects to guanylate cyclase isolated from other mammalian tissues. Changes in the total activity of guanylate cyclase during testicular development paralleled changes in the tissue concentration of cyclic GMP; i.e. guanylate cyclase activity and tissue cyclic GMP were highest during the early stages of development. Subcellular fractionation revealed that the activity of the soluble form of guanylate cyclase was best correlated with tissue cyclic GMP. Biochemical analysis of the soluble enzyme prepared from testes of neonatal and adult rats did not reveal any significant differences in the characteristics of the enzyme during ontogeny with the exception of a 2.5 fold increase in V noted in the neonatal testis. The results of this study are consistent with a molecular mechanism that allows independent regulation of the different forms of guanylate cyclase.     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9 B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9 B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9 B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

Summary The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

Expression, activity, and subcellular localization of testicular hormone-sensitive lipase during postnatal development in the guinea pig

The present work reports on testicular hormone-sensitive lipase (HSL), the biological significance of which has been documented in male fertility. The HSL protein levels and enzymatic activity were measured, respectively, by densitometry of immunoreactive bands in Western blots, performed with antibodies against recombinant rat HSL, and by spectrophotometry in seminiferous tubules (STf) and interstitial tissue (ITf) enriched fractions generated from neonatal, pubertal, and adult guinea pig testes. In addition, HSL was studied in subcellular fractions obtained from STf isolated from adult testes and in epididymal spermatozoa (Spz). A 104-kDa HSL protein was detected in STf and ITf, the expression and activity of which increased with testicular development. Three immunoreactive bands of 104, 110, and 120 kDa were detected in the lysosomal subfraction, and two bands of 104 and 120 kDa were detected in Spz. The HSL activity was positively correlated with free (FC) and esterified (EC) cholesterol ratios in STf and ITf, but not with triglyceride (TG) levels, during testicular development. Immunolabeling localized HSL to elongated spermatids and Sertoli cells, where its distribution was stage-dependent, and within the cells lining the excurrent ducts of the testis. The findings of the 104- and 120-kDa HSL immunoreactive bands and of HSL activity in Spz as well, as the detection of the 104-, 110-, and 120-kDa immunoreactive bands in lysosomes, suggest that part of HSL may originate from germ cells and be imported in Sertoli cells. The HSL protein levels and enzymatic activity in ITf and STf were positively correlated with serum testosterone levels during development. To the best of our knowledge, this study is the first to contribute insights regarding the impact of HSL on FC:EC cholesterol ratios and TG levels in the interstitial tissue and tubules in relation to serum testosterone levels during postnatal development, and regarding the immunolocalization of the enzyme in regions of the male gamete consistent with spermatozoa-oocyte interaction.     

Effect of sex steroids on the male reproductive organs of the monitor lizard Varanus monitor (Linn.).

Exogenous sex steroids were administered to adult males of the monitor lizard Varanus during the retrogressive and inactive phases of their annual reproductive cycle. Androgen treatment renews spermatogenetic activity and causes an increase in the number of Leydig cells of the testis during the retrogressive phase; during the inactive phase the testicular response to androgen is only slight. In either reproductive phase oestradiol treatment has an inhibitory action on the germ tubules. Progesterone has no effect on the testis in the inactive phase. The vasa deferentia are well developed during the retrogressive phase and thus the effect of androgens is not appreciable. However, during the inactive phase testosterone highly stimulates the deferent ducts. In the inactive phase oestrogen and progesterone also seem to stimulate slightly the deferent tubules; progesterone increases the interstitial tissue of the deferent ducts. Renal sexual segments hypertrophy and become secretory by androgen treatment in either phase of the reproductive cycle, whereas oestrogen and progesterone have no effect. The hemipenes are also stimulated by androgen treatment.     

Evaluation of the safety and efficacy of testicular biopsies in llamas

Evaluation of the reproductive function of Lama glama is generally considered to be a challenging task due to the difficulty of obtaining representative semen samples. One method that has been proposed for evaluation of testicular function in these animals is histologic examination of testicular needle biopsies. This study was undertaken to examine the safety and efficacy of using needle biopsies to assess testicular function in this species. One randomly selected testicle from each of 16 sexually mature llamas was biopsied with a 14-gauge self-firing biopsy instrument. The llamas were evaluated over a 6-week period with thermography for temperature changes of the scrotum. At the end of the 6-week trial, the llamas were castrated and sections of each testis were fixed in Bouin's solution for histologic examination. Immediately prior to castration, an additional biopsy was taken from each testis to compare the tissue obtained via biopsy with sections from the corresponding testis obtained after castration. A qualitative grading scale was used to compare the seminiferous tubules from each testis. No difference was found between the biopsied and the nonbiopsied testes (P = 0.69). The percentage of normal tubules between the biopsied and the nonbiopsied sides also did not differ (P = 0.70). Furthermore, the percentage of normal seminiferous tubules did not differ between the needle biopsy samples and the corresponding tissue samples obtained at castration (P = 0.48). The number of round seminiferous tubules counted in each biopsy section ranged from 3 to 67. There was no significant difference in the thermographic images of the scrotum between the biopsied and the nonbiopsied testes. This study supports testicular biopsies as a safe and useful procedure in the evaluation of testicular function.     

Characterization of rat testicular guanylate cyclase during development

The biochemical characteristics of rat testicular guanylate cyclase were investigated and the activity and subcellular distribution of the enzyme was determined during testicular development. Examination of the effects of metal ions, nucleotides, detergents and other in vitro activators on the activity of guanylate cyclase revealed that the testicular enzyme is similar in most respects to guanylate cyclase isolated from other mammalian tissues. Changes in the total activity of guanylate cyclase during testicular development paralleled changes in the tissue concentration of cyclic GMP; i.e. guanylate cyclase activity and tissue cyclic GMP were highest during the early stages of development. Subcellular fractionation revealed that the activity of the soluble form of guanylate cyclase was best correlated with tissue cyclic GMP. Bichemical analysis of the soluble enzyme prepared from testes of neonatal and adult rats did not reveal any significant differences in the characteristics of the enzyme during ontogeny with the exception of a 2.5 fold increase in V noted in the neonatal testis. The results of this study are consistent with a molecular mechanism that allows independent regulation of the different forms of guanylate cyclase.     

Intercellular transport of steroids in the infused rabbit testis

Tritiated-pregnenolone and tritiated-testosterone were infused via the testicular artery into the rabbit testis in situ, in order to determine if steroids can pass the "blood-testis barrier". After various periods of infusion (5-60 minutes) the testis were frozen cryostat sections were cut and freeze-dried. Interstitium and tubules were isolated by micro dissection and radioactivity per mcg dry weight was measured in both tissue compartments. Radioactive steroids could be isolated from the interstitial tissue as well as from the seminiferous tubules. Levels of radioactivity in the testes after infusion of tritiated-pregnenolone varied from 4 to 50% of the infused dose and were found to be dependent on the type of perfusion medium and the duration of the perfusion. Separation and identification of the radioactive steroids after infusing tritiated prognenolone showed that pregnenolone and testosterone were the major radioactive steroids in both interstitium and seminiferous tubules. After infusion with tritiated-testosterone, both tritiated-testosterone (77%) and tritiated-androstenedione (23%) were dound in the seminiferous tubules. It is concluded that steroids can be transported from the Leydig cells to the seminiferous tubules.     

Subcutaneous grafts of fresh or cryopreserved testis in castrated rats and mice: comparison of the histologic aspects of the grafts

In the castrated rat, only testis taken in one to two week-old donors observed three months after sub-cutaneous isograft contain a well developed interstitial tissue and some seminiferous tubules with germinal cells. On the contrary in castrated mice, testicular grafts taken in adult animals show some Leydig cells and degenerating seminiferous tubules. These grafts permit the restoration of androgenic activity in previously castrated recipients.     

Spatial topography of the excurrent duct system in the bovine testis

Summary The rete testis of the bull is situated within an axial mediastinum and consists of approximately 30 longitudinally arranged, anastomosing rete channels. At the cranial testicular pole all rete channels empty into a common space, the area confluens reds, which is subdivided by small septa and narrow chordae retis. The area confluens always contains numerous spermatozoa and is connected with the bulbous initial portions of the efferent ductules by short, often tortuous rete tubules. Since the connection between rete and efferent ductules is situated within the tunica albuginea, the bovine excurrent duct system is not provided with an extratesticular rete as in many other mammals.Straight testicular tubules merge from all directions to connect with superficial rete channels, but the inlets are not evenly distributed. In the periphery each straight tubule begins with a cup-like structure followed by a narrow stalk region and a heavily folded portion opening either immediately into a rete channel or into a tube-like lateral rete extension.In close contiguity to the rete testis lie extremely coiled arterial portions connecting the centripetal and the centrifugal branches of the testicular artery. Since intrinsic musculature is scarcely developed in the mediastinum, and transport of rete content relies primarily on massage due to external pressure changes, the pulsatile blood flow through these coiled arteries may influence conveyance processes within the rete testis.An intimate spatial association between area confluens reds and adjacent large, thin-walled lymph vessels may facilitate a transfer of androgens into the fluid of the rete testis.Supported by the Stiftung zur Förderung der wissenschaftlichen Forschung an der Universität Bern     

Endogenous steroid production in cellular and subcellular fractions of rat testis after prolonged treatment with gonadotropins.

Steroid production and enzyme activities were examined in preparations of whole testis tissue, isolated interstitial tissue and seminiferous tubules obtained from adult rats with intact pituitaries receiving daily subcutaneous injections of 100 I.U. human chorionic gonadotropin for 5 days and from control animals. After human chorionic gonadotropin administration testosterone concentrations were increased in total homogenates of whole testis tissue, interstitial tissue and seminiferous tubules. The testosterone production from endogenous precursors was enhanced only in total homogenates of whole testis tissue and interstitial tissue obtained from testes of human chorionic gonadotropin-treated rats. The production of testosterone in the corresponding homogenates of isolated seminiferous tubules was very low. The specific activity of 3 beta-hydroxysteroid dehydrogenase was increased in total homogenates of whole testis tissue, isolated interstitial tissue and seminiferous tubules. No effect was observed on the specific activities of marker enzymes such as cytochrome c oxidase, monoamine oxidase, steroid sulfatase and lactate dehydrogenase, whereas the specific activities of carboxyl esterase were decreased in homogenates of whole testis tissue and interstitial tissue. Total activity of monoamine oxidase was increased in homogenates of interstitial tissue of tests from human chorionic gonadotropin treated rats. After the same prolonged human chorionic gonadotropin treatment the concentration of pregnenolone was increased in mitochondrial fractions of whole testis tissue, interstitial tissue and seminiferous tubules, and the amount of protein isolated in the mitochondrial fraction of interstitial tissue increased by 40%. Steroid production (estimated as pregnenolone) from endogenous precusors by mitochondrial fractions of whole testis tissue and interstitial tissue were increased after human chorionic gonadotropin treatment, for whole testis from 580 pmol/mg mitochondrial protein per h to 1420 pmol/mg per h; and for interstitial tissue from 2665 pmol/mg per h to 7050 pmol/mg per h. The production of pregnenolone in mitochondrial fractions obtaine from isolated seminiferous tubules was very low and contributed hardly at all to the total pregnenolone production in mitochondrial fractions of whole testis tissue from normal rats as well as from human chorionic gonadotropin-treated rats.     

Effect of surgery and efferent duct ligation on testicular blood flow and testicular steroidogenesis in the rat

A moderate reduction in testicular blood flow was observed in both testes 24 h after unilateral efferent duct ligation without any corresponding change in testosterone secretion as indicated in the peripheral blood, in testicular venous blood, or in testicular tissue fluid. At 21 days a pronounced unilateral decrease in blood flow was associated with the extensive degeneration of tubules in the testis on the ligated side. These changes were also associated with decreased testosterone secretion by the testis on the ligated side, although Leydig cell function was not abolished since testosterone in the tissue increased rather than decreased. It is therefore concluded that testicular blood flow may play an important role in the changes of testosterone secretion that follow unilateral efferent duct ligation.     

Regulation of testicular and Sertoli cell gamma-glutamyl transpeptidase by follicle-stimulating hormone

Hormonal deprivation achieved by hypophysectomy or gonadotropin-releasing hormone (GnRH)-antagonist treatment of immature rats resulted in markedly lower testicular gamma-glutamyl transpeptidase (GGT) activity than in the testes of age-matched controls. When begun 15 days after hypophysectomy, follicle-stimulating hormone (FSH) treatment significantly increased testicular GGT above that in testes from hypophysectomized controls in a time- and dose-dependent manner. In contrast, testosterone propionate had only a small effect. Testicular GGT was higher in adult hypophysectomized rats treated with FSH from the time of surgery than in untreated hypophysectomized rats; testosterone propionate treatment had no effect. GGT activity in Sertoli cells isolated from GnRH antagonist-treated or hypophysectomized immature rats was also lower than in cells from control rats. FSH treatment from the day of hypophysectomy resulted in Sertoli cell GGT values equivalent to those from intact controls. These data indicate that FSH regulates GGT activity in rat testis and Sertoli cells.     

Morphometric studies on hamster testes in gonadally active and inactive states: light microscope findings

This study provides quantitative information on the testes of seasonally breeding golden hamsters during active and regressed states of gonadal activity. Seminiferous tubules occupied 92.5% of testis volume in adult gonadally active animals. Leydig cells constituted 1.4% of the testicular volume. The mean volume of an individual Leydig cell was 1092 microns 3, and each testis contained about 25.4 million Leydig cells. The volume of an average Sertoli cell nucleus during stage VII-VIII of the cycle was 502 microns 3. A gram of hamster testis during the active state of gonadal activity contained 44.5 million Sertoli cells, and the entire testis contained approximately 73.8 million Sertoli cells. Testes of the hamsters exposed to short photoperiods for 12-13 wk displayed a 90% reduction in testis volume that was associated with a decrease in the volume of seminiferous tubules (90.8% reduction), tubular lumena (98.8%), interstitium (72.7%), Leydig cell compartment (79.3%), individual Leydig cells (69.7%), Leydig cell nuclei (50.0%), blood vessels (85.5%), macrophages (68.9%), and Sertoli cell nuclei (34.1%). The diameter (61.1%) and the length (36.8%) of the seminiferous tubules were also decreased. Although the number of Leydig cells per testis was significantly lower (p less than 0.02) after short-photoperiod exposure, the number of Sertoli cells per testis remained unchanged. The individual Sertoli cell in gonadally active hamsters accommodated, on the average, 2.27 pre-leptotene spermatocytes, 2.46 pachytene spermatocytes, and 8.17 round spermatids; the corresponding numbers in the regressed testes were 0.96, 0.20, and 0.04, respectively. The striking differences in the testicular structure between the active and regressed states of gonadal activity follow photoperiod-induced changes in endocrine function and suggest that the golden hamster may be used as a model to study structure-function relationships in the testis.