Cell-cell interactions in the control of spermatogenesis as studied using Leydig cell destruction and testosterone replacement

This review centers around studies which have used ethane dimethane sulphonate (EDS) selectively to destroy all of the Leydig cells in the adult rat testis. With additional manipulations such as testosterone replacement and/or experimental induction of severe seminiferous tubule damage in EDS-injected rats, the following questions have been addressed: 1) What are the roles and relative importance of testosterone and other non-androgenic Leydig cell products in normal spermatogenesis and testicular function in general? 2) What are the factors controlling Leydig cell proliferation and maturation? 3) Is it the Leydig cells or the seminiferous tubules (or both) which control the testicular vasculature? The findings emphasize that in the normal adult rat testis there is a complex interaction between the Leydig cells, the Sertoli (and/or peritubular) cells, the germ cells, and the vasculature, and that testosterone, but not other Leydig cell products, plays a central role in many of these interactions. The Leydig cells drive spermatogenesis via the secretion of testosterone which acts on the Sertoli and/or peritubular cells to create an environment which enables normal progression of germ cells through stage VII of the spermatogenic cycle. In addition, testosterone is involved in the control of the vasculature, and hence the formation of testicular interstitial fluid, presumably again via effects on the Sertoli and/or peritubular cells. When Leydig cells regenerate and mature after their destruction by EDS, it can be shown that both the rate and the location of regenerating Leydig cells is determined by an interplay between endocrine (LH and perhaps FSH) and paracrine factors; the latter emanate from the seminiferous tubules and are determined by the germ cell complement. Taken together with other data on the paracrine control of Leydig cell testosterone secretion by the seminiferous tubules, these findings demonstrate that the functions of all of the cell types in the testis are interwoven in a highly organized manner. This has considerable implications with regard to the concentration of research effort on in vitro studies of the testis, and is discussed together with the need for a multidisciplinary approach if the complex control of spermatogenesis is ever to be properly understood.     

Postnatal somatic cell proliferation and seminiferous tubule maturation in pigs: A non-random event

Although seminiferous tubule maturation in horses begins in the central area of the testis, this process is thought to occur randomly throughout the testis in most mammals. Studies in our laboratory revealed that the establishment of spermatogenesis may not be a synchronous event in the testicular parenchyma of pigs. The objectives of the present study were to evaluate the pattern of seminiferous cord/tubule maturation and the morphological and functional characteristics of testicular somatic cells during postnatal development in three regions of the pig testis: a) near the tunica albuginea (TA); b) in the transitional area between the seminiferous tubules and mediastinum (TR); and c) in the intermediate area (ID) between the TA and TR. Based on the diameter of seminiferous cords/tubules, nucleus size of Sertoli cells and fluid secretion, mainly at 90 and 120 d of age, seminiferous tubule maturation was more advanced in the ID and TR. The mitotic activity of Sertoli cells was higher (P < 0.05) in the TR than the ID and TA at 7 and 120 d. Except for the mitotic index of the Leydig cells, which was lower (P < 0.05) in the ID at 7, 30, and 180 d than in the TA and TR, other Leydig cell ebd points, e.g., individual cell size, nuclear volume, and cytoplasmic volume, were consistently higher (P < 0.05) in the ID, suggesting that steroidogenesis was more active in this region during the period investigated. Overall, we inferred that Leydig cells in the ID may play a pivotal role in postnatal testis development in pigs and this type of cell is likely related to asynchronous testicular parenchyma development, with the transitional area providing the primary zone for growth of seminiferous tubules.     

Expression of connexin 43 in human testis

In order to further characterize the Sertoli cell state of differentiation, we investigated the expression of connexin 43 (cx43) protein in the testis of adult men both with normal spermatogenesis and associated with spermatogenic impairment, since cx43 is first expressed during puberty. Cx43 protein was found as a single 43-kDa band on western blots of extracts of normal human testicular material. Cx43 immunoreactivity was generally present between Leydig cells. Within the normal seminiferous epithelium cx43 immunoreactivity was localized between adjacent Sertoli cells, except at stages II and III of the seminiferous epithelial cycle when primary spermatocytes cross from the basal to the adluminal compartment suggesting a stage-dependent Sertoli cell function. While testes with hypospermatogenesis and spermatogenic arrest at the level of round spermatids or spermatocytes revealed a staining pattern similar to that of normal adult testis, the seminiferous tubules showing spermatogenic arrest at the level of spermatogonia and Sertoli-cell-only syndrome were completely immunonegative. We therefore assume that severe spermatogenic impairment is associated with a population of Sertoli cells exhibiting a stage of differentiation deficiency. Accepted: 10 June 1999     

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

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.     

Distributional map of the terminal and sub-terminal sugar residues of the glycoconjugates in the prepubertal and postpubertal testis of a subject affected by complete androgen insensitivity syndrome (Morris's syndrome): lectin histochemical study

In the present research we have investigated the distribution of the sugar residues of the glycoconjugates in the prepubertal and postpubertal testes of a subject with Morris's syndrome (CAIS, Complete Androgen Insensitivity Syndrome). For this purpose a battery of six horseradish peroxidase-conjugated lectins was used (SBA, PNA, WGA, ConA, LTA and UEAI). We have obtained a complete distributional map of the terminal and sub-terminal oligosaccharides in the tunica albuginea, interstitial tissue, lamina propria of the seminiferous tubules, Leydig cells, Sertoli cells, spermatogonia, mastocytes and endothelial cells. Furthermore the present study has shown that a large amount of sugar residues were detectable in the prepubertal and postpubertal testes but that some differences exist with particular regard to the Sertoli cells. The Sertoli cells and the Leydig cells of the retained prepubertal testis of the patient affected by Morris's syndrome were characterized by the presence of alpha-L-fucose, which was absent in the retained prepubertal testis of the normal subjects. Comparing the results on the postpubertal testis with those obtained on the same aged testis of healthy subjects we have demonstrated that alpha-L-fucose in the Sertoli and Leydig cells and D-galactose-N-acetyl-D-galactosamine in the Leydig cells are a unique feature of the subject affected by Morris's syndrome. D-galactose (ss1,3)-N-acetyl-D-galactosamine and sialic acid, which are present in the Leydig cells of the normal testis were never observed in the same cells of the postpubertal testis of the CAIS patient.     

Immunohistochemical study of serum albumin in normal and cadmium-treated mouse testis organs by "in vivo cryotechnique"

The in vivo injection of cadmium (Cd) was reported to induce blood-testis barrier disruption, and assumed to be an experimental model to examine junctional structures in seminiferous tubules. The purpose of this study is to investigate time-dependent changes of albumin permeability in the normal or Cd-treated mouse testis by our "in vivo cryotechnique" with immunohistochemistry, reflecting tight junctional (TJ) barriers of Sertoli cells. The albumin in the seminiferous tubules was firstly immobilized by the cryotechnique, in which normal blood circulation was always kept. The cryofixed testicular tissues were then processed for freeze-substitution, and embedded in the paraffin wax. Serial sections were immunostained by anti-mouse albumin antibody with peroxidase immunostaining, and also stained with hematoxylin-eosine (HE) for morphological observation. In normal seminiferous tubules, the immunoreaction products were localized around peritubular myoid cells and between Leydig cells, as well as in blood vessels. They were also localized as arch-like patterns around some spermatogonia in basal compartments of seminiferous tubules. Twenty-four and 48 hrs after Cd-treatment, some enlarged spaces and vesicular formations in the seminiferous epithelium were observed on the HE-stained sections. The albumin immunolocalization was detected not only in the basal compartments, but also in the adluminal compartments between Sertoli cells and germ cells. Thus, the structural disruptions of inter-Sertoli TJ barriers could be clearly demonstrated by the "in vivo cryotechnique".     

Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase in porcine testis

The immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in porcine testes was examined by applying an indirect-immunofluorescence method using an antiporcine testicular 17 beta-HSD antibody. Only the Leydig cells located in the interstitial tissue exhibited a positive immunoreaction for 17 beta-HSD: the germ cells and Sertoli cells located in the seminiferous tubules were entirely negative. These results suggest that, in porcine testis, the biosynthesis of testicular testosterone, the final step of which is the conversion of androstenedione to testosterone, takes place in the Leydig cells.     

In situ demonstration of both TUNEL-labeled germ cell and Sertoli cell in the cimetidine-treated rats

Cimetidine has caused dysfunction in the male reproductive system. In the rat testis, intratubular alterations and loss of peritubular tissue due to peritubular myoid cell death by apoptosis have been recently shown. Thus, the aim of this study is to evaluate which cells of the seminiferous epithelium have been affected and/or died by apoptosis after the treatment with cimetidine. For this purpose, an experimental group containing five male albino Wistar rats received intraperitoneal injections of cimetidine (50 mg/kg body weight) during 52 days. The testes were fixed with 4% buffered formaldehyde and were embedded in paraffin. For detection of DNA breaks (apoptosis) in the cells of the seminiferous epithelium, the testicular sections were treated by the TUNEL method (Apop-Tag Plus Peroxidase Kit). In the tubules affected by cimetidine, altered peritubular tissue, including the presence of TUNEL labeling in the myoid peritubular cells, were usually found. In these tubules, the seminiferous epithelium exhibited low density of germ cells and TUNEL-positive labeling in the germ cells of the basal compartment. The concomitant staining in both germ cells of the basal compartment and late spermatids suggest a sensitivity of these cells in the damaged tubules. Besides germ cells, TUNEL-positive Sertoli cells were also found in the injured seminiferous tubules. Thus, a relationship between dying germ cells and Sertoli cell damage and/or death must be considered in tubules where peritubular tissue has been affected by toxicants.     

Immunoexpression of Tyro 3 family receptors--Tyro 3, Axl, and Mer--and their ligand Gas6 in postnatal developing mouse testis.

Tyro 3 family receptors contain three members-Tyro 3, Axl, and Mer-that are essential regulators of mammalian spermatogenesis. However, their exact expression patterns in testis are unclear. In this study, we examined the localizations of Tyro 3, Axl, Mer, and their ligand Gas6 in postnatal mouse testes by immunohistochemistry. All three members and their ligand were continuously expressed in different testicular cells during postnatal development. Tyro 3 was expressed only in Sertoli cells with a varied distribution during testis development. At day 3 postnatal, Tyro 3 was distributed in overall cytoplasmic membrane and cytoplasm of Sertoli cells. From day 14 to day 35 postnatal, Tyro 3 appeared on Sertoli cell processes toward the adlumenal compartment of seminiferous tubules. A stage-dependent Tyro 3 immunoexpression in Sertoli cells was shown by adulthood testis at day 56 postnatal with higher expression at stages I-VII and lower level at stages IX-XII. Axl showed a similar expression pattern to Tyro 3, except for some immunopositive Leydig cells detected in mature testis. In contrast, immunostaining of Mer was detected mainly in primitive spermatogonia and Leydig cells, whereas a relative weak signal was found in Sertoli cells. Gas6 was strongly expressed in Leydig cells, and a relative weak staining signal was seen in primitive spermatogonia and Sertoli cells. These immunoexpression patterns of Tyro 3 family receptors and ligand in testis provide a basis to further study their functions and mechanisms in regulating mammalian spermatogenesis.     

Spermatogenesis and testicular tumours in ageing dogs

Spermatogenesis was examined in testes from 74 dogs of various breeds without clinically detected testicular disease. A modified Johnsen score system was used to determine whether spermatogenesis deteriorates with ageing. The diameter of seminiferous tubules was measured in dogs without testicular disease to examine other possible effects of ageing on tubular performance. There appeared to be no relation between age and these variables. The influence of testicular tumours on spermatogenesis was also investigated in both affected and unaffected testes. The testes of 28 dogs with clinically palpable tumours and 21 dogs with clinically non-palpable tumours were investigated. In cases of unilateral occurrence of a tumour, impairment of spermatogenesis was observed only in the affected testis of dogs with clinically detected tumours. Bilateral occurrence of tumours, whether detected clinically or non-clinically, was associated with severe impairment of spermatogenesis. The prevalence of tumours increased during ageing. Eighty-six per cent of the clinically detected and 57% of the non-clinically detected tumours were found in old dogs. Multiple types of tumour and bilateral occurrence were very common. Seminomas and Leydig cell tumours were more frequent than Sertoli cell tumours. It was concluded that spermatogenesis per se did not decrease during ageing in dogs but the occurrence of testicular tumours increased with ageing and affected spermatogenesis significantly, as reflected by a lower Johnsen score.     

The germ cell development strategy and seasonal changes in spermatogenesis and Leydig cell morphologies of the spiny lizard Sceloporus mucronatus (Squamata: Phrynosomatidae)

The viviparous lizards of the Sceloporus genus exhibit both seasonal and continuous spermatogenesis. The viviparous lizard Sceloporus mucronatus from Tecocomulco, Hidalgo, México, exhibits seasonal spermatogenesis. This study demonstrates the relationship between changes in testis volume, spermatogenesis activity, and Leydig cells during the male reproductive cycle of S. mucronatus. A recrudescence period is evident, which starts in the winter when testicular volume is reduced and climaxes in February, when the greatest mitotic activity of spermatogonia occurs. The testicular volume and Leydig cell index increase gradually during the spring with primary spermatocytes being the most abundant cell type observed within the germinal epithelium. In the summer, the secondary spermatocytes and undifferentiated round spermatids are the most abundant germinal cells. The breeding season coincides with spermiogenesis and spermiation; testicular volume also increases significantly as does the Leydig cell index where these cells increase in both cytoplasmic and nuclear volume. During fall, testicular regression begins with a significant decrease in testicular volume and germinal epithelium height, although there are remnant spermatozoa left within the lumen of the seminiferous tubules. During this time, the Leydig cell index is also reduced, and there is a decrease in cellular and nuclear volumes within these interstitial cells. Finally, during quiescence in late fall, there is reduced testicular volume smaller than during regression, and only spermatogonia and Sertoli cells are present within the seminiferous tubules. Leydig cells exhibit a low index number, their cellular and nuclear volumes are reduced, and there is a depletion in lipid inclusion cytoplasmically.     

Sertoli and Leydig cells of the human testis express neurofilament triplet proteins

 Using RT-PCR, western blot and enzyme and fluorescence immunocytochemical techniques, the three isoforms of neurofilament proteins (NFPs), namely NF-L (NFP-68 kDa), NF-M (NFP-160 kDa) and NF-H (NFP-200 kDa) were found in Sertoli and Leydig cells of human testes. RT-PCR showed specific for the three NFP fragments in testicular tissue, in isolated seminiferous tubules and in isolated Leydig cells. In protein preparations from the same testicular components, western blot analysis detected bands with molecular weights characteristic for NF-H, NF-M and NF-L. Application of immunofluorescence and immunoenzyme methods on cryostat and paraffin sections resulted in differences in the staining pattern in Sertoli cells and Leydig cells. In these cells, the NFPs showed predominantly a perinuclear location from which bundles emerge that were directed towards the basal, apical and lateral extensions of the Sertoli cells as well as the periphery of Leydig cells. NF-H coexists with vimentin-type filaments as seen by dual staining and staining of conseccutive serial sections of material embedded in paraffin. In Sertoli cells, vimentin and NF-H showed distinct dynamic changes depending on the stage of spermatogenesis and some structural variations of seminiferous tubules. Although in some tubules both vimentin and NF-H immunoreactivity was present at high levels, in the Sertoli cells from most individuals an inverse relationship in the staining intensity of vimentin and NF-H was observed. The strongest NF-H immunoreactivity was detected in Sertoli cells associated with stage 3 spermatids, whereas vimentin immunoreactivity was most abundant in association with stage 5 spermatids. The leydig cells did not show functional changes of the NFP immunoreactivity. The results obtained provide new evidence for the heterogeneous phenotype of human Sertoli cells and raise the question of their exact nature and origin. Accepted: 17 November 1998     

Variations in testicular androgen receptors and histology of the lamb testis from birth to puberty

Changes in testicular androgen receptor numbers were studied in lambs from 25 to 100 days of age. During this period, cytoplasmic receptors increased from 5 to 80 pmol/testis and nuclear receptors from 1 to 12 pmol/testis, while the total volume of Leydig cells increased 7-fold. The total number of Sertoli cells doubled between 25 and 40 days of age. From 40 days onward their number remained constant while their cellular and nuclear sizes increased by a factor of 3 and 1.5 respectively. Cytoplasmic receptor concentration was positively correlated with the number of Sertoli cells per section of seminiferous tubule, and negatively correlated with the number of germinal cells per cross section. One explanation for these results could be that Sertoli cells are the main androgen target cells in lamb seminiferous tubules.     

Morphologic study of the testes from spontaneous unilateral and bilateral abdominal cryptorchid boars

Macroscopical and histological characteristics were examined in both testes from three healthy boars, three boars with unilateral abdominal cryptorchidism on the right side, and three boars with bilateral abdominal cryptorchidism. Abdominal cryptorchidism, unilateral and bilateral, provoked a significant decrease of the weight and volume of the ectopic testes. The scrotal testis of the unilateral cryptorchid boars showed an increase in its volume and weight. Cryptorchidism also induced abnormalities in the histological structure of seminiferous tubules, lamina propria, and interstitial tissue of the abdominal testes. The number of seminiferous tubules decreased; the seminiferous epithelium was constituted by few spermatogonia with an atypical pattern and by abnormal Sertoli cells. The lamina propria showed a variable degree of thickening and collagenization. The interstitial tissue was very developed but displayed a decrease in the Leydig cell population. These abnormalities were more critical in bilateral cryptorchidism than in unilateral cryptorchidism. The scrotal testis of the unilateral cryptorchid boars showed normal appearance, but a decrease of the number of seminiferous tubules was observed. Moreover, the seminiferous tubules showed impaired spermatid maturation. The alterations observed in the abdominal testes of the unilateral and bilateral cryptorchid boars were attributed to defective proliferation and differentiation of Sertoli cells and Leydig cells. The anomalies in the scrotal testis of the unilateral cryptorchid boars were due to disturbances in the Sertoli cell activity.     

Structural and functional abnormality of ectopic testes in rats

Some males of a mutant strain of King-Holtzman rats exhibit an anomalous heritable defect manifested as either unilateral or bilateral ectopic testes. In the adult, these testes contain seemingly immature Sertoli and Leydig cells, seminiferous tubules greatly reduced in diameter, and exhibit arrested spermatogenesis. Thus, the affected testis is essentially sterile. An inability to produce normal amounts of testosterone and androstenedione by these gonads is probably a reflection of changes that have been effected in their Leydig cells. Thus, this study suggests that abnormal function of the Leydig and Sertoli cells and seminiferous tubule failure in these mutant animals result from the physiologically cryptorchid condition.     

Long-term effects of irradiation before adulthood on reproductive function in the male rhesus monkey.

Today, many patients, who are often young, undergo total body irradiation (TBI) followed by bone marrow transplantation. This procedure can have serious consequences for fertility, but the long-term intratesticular effects of this treatment in primates have not yet been studied. Testes and epididymides of rhesus monkeys that received doses of 4-8.5 Gy of TBI at 2-4 yr of age were studied 3-8 yr after irradiation. In all irradiated monkeys, at least some seminiferous tubule cross-sections lacked germ cells, indicating extensive stem cell killing that was not completely repaired by enhanced stem cell renewal, even after many years. Testes totally devoid of germ cells were only found in monkeys receiving doses of 8 Gy or higher and in both monkeys that received two fractions of 6 Gy each. By correlating the percentage of repopulated tubules (repopulation index) with testicular weight, it could be deduced that considerable numbers of proliferating immature Sertoli cells were killed by the irradiation. Because of their finite period of proliferation, Sertoli cell numbers did not recover, and potential adult testis size decreased from approximately 23 to 13 g. Most testes showed some dilated seminiferous tubules, indicating obstructed flow of the tubular fluid at some time after irradiation. Also, in 8 of the 29 irradiated monkeys, aberrant, densely packed Sertoli cells were found. The irradiation did not induce stable chromosomal translocations in spermatogonial stem cells. No apparent changes were seen in the epididymides of the irradiated monkeys, and the size of the epididymis adjusted itself to the size of the testis. In the irradiated monkeys, testosterone and estradiol levels were normal, whereas FSH levels were higher and inhibin levels lower when testicular weight and spermatogenic repopulation were low. It is concluded that irradiation before adulthood has considerable long-term effects on the testis. Potential testis size is reduced, repopulation of the seminiferous epithelium is generally not complete, and aberrant Sertoli cells and dilated tubules are formed. The latter two phenomena may have further consequences at still longer intervals after irradiation.     

Expression pattern of asialoglycoprotein receptor in human testis

During acute or chronic hepatitis B virus (HBV) infection, the virus can invade the male reproductive system, pass through the blood–testis barrier and integrate into the germ line, resulting in abnormal spermatozoa. However, the pathway remains unclear. The asialoglycoprotein receptor (ASGR), a potential receptor for HBV, is mainly distributed in hepatocytes. We have examined the distribution of ASGR in human testis and found it in the seminiferous tubules and interstitial region but its enrichment in human testis is much lower than that in liver. By multiple immunoenzyme histochemistry staining, ASGR was precisely co-localized with vimentin (Sertoli cell marker) but not proliferating cell nuclear antigen (spermatogonial cell marker) in testis tissue. ASGR was expressed in human Leydig cells, stromal cells in the seminiferous tubules and Sertoli cells but seldom in spermatogonial cells. Therefore, ASGR could provide HBV with access to the luminal compartment of human testis. The mechanism by which HBV invades germ cells remains unknown.     

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.