Carcinoma-in-situ cells in cultured human seminiferous tubules

For the first time, early germ-cell derived tumour cells were studied in an in-vitro system of cultured seminiferous tubules. The intratubular tumour cells not only survived in culture for 7 days but were also able to multiply. Dividing tumour cells were identified in semi-thin sections and electron micrographs by morphological criteria. Additionally, mitotic activity was demonstrated by [3H]thymidine histo-autoradiography. There are numerous reports on cell lines established from solid non-seminomas, but up to now no references to seminoma cell lines or cultures of intratubular tumour cells are available. The culture of seminiferous tubules offers a tool in making carcinoma-in-situ cells accessible for experimental work.     

Microvasculature of the human testis in correlation to Leydig cells and seminiferous tubules

Summary. The microvasculature of the human testis is closely related to the Leydig cells and the seminiferous tubules. Semi-thin sections of testicular tissue serve as a basis for the computer-aided 3-D reconstruction of the microvasculature, the seminiferous tubules and the Leydig cells. After vascular perfusion with glutaraldehyde (5.5%) and paraformaldehyde (4%), it is possible by means of light and electron microscopy, to analyse the organization of the capillaries between the Leydig cells (inter-Leydig cell capillaries) as well as of those within the lamina propria (intramural capillaries). These arise from arterioles, deriving from branches of the segmental arteries. The capillaries ramify between the Leydig cells and run either semi-circumferentially around the seminiferous tubules (peritubular capillaries) or penetrate the lamina propria of the neighbouring tubules. This is the beginning of the intramural capillary which after leaving the tubular wall continues to a further capillary path. Consequently, the microvasculature of the human testis with regard to the seminiferous tubules is subdivided into afferent, intramural and efferent capillaries. Leydig cell clusters are present on both the arterial and the venous sides of the microvasculature.     

Presence of neuropeptide — Y and its C-terminal flanking peptide immuno-reactivity in the seminiferous tubules of human testis

The presence of neuropeptide Y (NPY) and its C-terminal flanking peptide (C-PON) was described by immunohistochemistry in human testes. The immunopositive material was visualized in the spermatogenic elements of the seminiferous tubules. More NPY occurred in the younger testis and more C-PON in the older ones. NPY positive material was present mainly in the spermatogonia, and in the primary spermatocytes, where C-PON also occurred. The megalospermatocytes, present in aged testis, showed C-PON immunoreactivity. Both NPY and C-PON were present in granular form in the perinuclear zone of the cells. No positive material was detected in the Sertoli cells or in the Leydig cells. It is possible that NPY and its precursor are synthetized within the testis and might play a role in the paracrine and/or autocrine regulation of spermatogenesis.     

Paracrine regulation of Leydig cells by the seminiferous tubules

Testes of adult control and unilateral cryptorchid rats were fixed by vascular perfusion. The cell profile area of peritubular Leydig cells surrounding tubules in different stages of spermatogenesis, and the cell profile area of perivascular Leydig cells were determined. The size of peritubular Leydig cells was dependent on which type of tubulus the cells were surrounding. Some peritubular Leydig cells, especially those surrounding stages VII–VIII (88.1 ± 7.1 μm², mean ± SD, n = 6 rats), were larger than perivascular Leydig cells (69.3 ± 5.9 μm²). The size of Leydig cells surrounding stages IX–XIV was similar to that of perivascular cells. In the abdominal testes no spermatogenic cycle was present and the sizes of peritubular and perivascular Leydig cells were equal (63.0 ± 5.1, vs 66.7 ± 7.3 μm², mean ± SD, n = 5 rats). It is suggested that the tubules and the spermatogenic cycle locally modulate Leydig cell activity and that Leydig cell malfunction in abdominal testes may be due to a decreased stimulatory influence from the damaged tubules.     

Transformation,migration and outcome of residual bodies in the seminiferous tubules of the rat testis

Experiments were performed to study the transformation, migration and outcome of residual bodies (RBs) in the seminiferous tubules of the rat testes. One part of the testes from adult Sprague–Dawley rats was used to generate paraffin sections to observe RBs and RB precursors through specific staining, and the other part of the testes was used to generate ultrathin sections to observe RBs under a transmission electron microscope. Deep blue particles of different sizes were observed in some seminiferous tubules through specific staining for RBs and RB precursors. These particles first appeared in the seminiferous tubules at stage I of the spermatogenic cycle, and after spermiation, the particles travelled rapidly towards the deeper region of the seminiferous epithelium and soon appeared close to the basement membrane of the seminiferous tubule. All of the particles in the tubules disappeared at stage IX. Using transmission electron microscopy, components of different electron densities were observed in the RBs on the surface of the seminiferous epithelium, all of which gradually formed in the cytoplasm of spermatozoon in later stages of spermiogenesis. After the spermatozoa were released, the RBs in the epithelium travelled quickly to the edge of the tube and were gradually transformed into lipid inclusions. These lipid inclusions ultimately became lipidlike particles. The lipidlike particles were discharged into the interstitial tissue. RBs initiate their own digestive process before their formation during spermiation in the rat testes. After spermiation, the RBs transform into lipid inclusions and finally into lipidlike particles. These lipidlike particles can be eliminated from the seminiferous tubules.     

Stage-specific degeneration of germ cells in the seminiferous tubules of non-obese diabetic mice

The cause of fertility problems in insulin-dependent diabetes is largely unknown. To evaluate the role of autoimmunity-associated phenomena in the testis as a possible cause of the derangement in spermatogenesis, the stage-specific apoptosis of germ cells in the insulitis phase of pre-diabetes was quantified in the testes of non-obese diabetic (NOD) mice. The seminiferous epithelium of normal BALB/c and NOD mice contained cells positive for in-situ end-labelling (ISEL) of DNA. ISEL-positive germ cells formed clusters in the seminiferous epithelium of the NOD mice in marked contrast to the seminiferous epithelium of the BALB/c mice, which contained only individual cells positive for ISEL. ISEL-positive cells were present in the basal and luminal compartments of the epithelium. Ultrastructural analysis and demonstration of externalized phosphatidyl serine confirmed that the cells were undergoing apoptosis. The ultrastructurally apoptotic cells included spermatogonia, spermatocytes and spermatids. In cytological squash preparations of segments of seminiferous tubules from NOD mice aged 17–20 weeks, the number of ISEL-positive cells/mm tubule was significantly lower in segments at stages I–II of the seminiferous epithelial wave but higher at stages III–IV in comparison to BALB/c mice. The numbers of ISEL-positive cells/mm tubule in the other stages were similar in the two strains of mice. Analysis of ³²P-3' -end labelled DNA from the testes showed that the BALB/c mice had relatively more DNA fragmentation than did the NOD mice. These data suggest that autoimmune insulitis in the NOD mice is associated with increased amounts and abnormal stage distribution of apoptosis in the seminiferous epithelium, resulting in derangement of spermatogenesis.     

Local differences in Leydig cell morphology in the adult rat testis: evidence for a local control of Leydig cells by adjacent seminiferous tubules

In order to test the hypothesis that Leydig cell function in the adult rat testis is influenced by the surrounding tubules, Leydig cell morphology was compared in different types of interstitial areas. Triangular interstitial areas surrounded by 3 cross-sectioned tubules in nearly the same stage of spermatogenesis were chosen for quantitative light microscopy. It was found that the volume density of Leydig cells in such areas was about 30%, except when the surrounding tubules were in stages IX-X or XI-XII, when it was only about 20%. This variation in total Leydig cell mass seemed to be due to a variation in Leydig cell size and not in Leydig cell number. The largest Leydig cell profile area, 118 pL 6 μm² (mean pL SE n = 6 rats), was observed when the surrounding tubules were in stages VII-VIII, i.e. just prior to sperm release. The smallest Leydig cells were seen when the surrounding tubules were in stages IX-X and XI-XII (68 pL 3 and 66 pL 4 μm²). The present results indicate that there may be a Leydig cell cycle in the adult rat testis, which is regulated by the adjacent tubules.     

Local regulation of Leydig cells by the seminiferous tubules. Effect of short-term cryptorchidism

Unilateral cryptorchism was induced in adult rats for 24 h, and its effect on testicular morphology and intratesticular testosterone concentration after hCG-stimulation were studied. In seminiferous, tubules from abdominal testes an increased number of degenerating germ cells was noted in stages XIV-III of the spermatogenic cycle and Sertoli cells contained an increased amount of lipid droplets in stages XIV-VIII. However, germ cells and Sertoli cells from tubules at other stages of the cycle appeared unaffected. In scrotal testes the size of peritubular Leydig cells varied in phase with the spermatogenic cycle. The largest cells were found adjacent to stage VII-VIII and the smallest adjacent to stage XI-XII. In abdominal testes no stage-dependent variation in the size of peritubular Leydig cells was seen. Perivascular Leydig cells were of equal size in abdominal and scrotal testes. The testicular testosterone concentration following stimulation with a low dose of hCG was significantly lower in abdominal testes. It is suggested that the seminiferous tubules locally modulate Leydig cell function and that the stage specific stimulatory influence from stage VII-VIII is rapidly lost during experimental cryptorchidism.     

Immunohistochemical identification of macrophages, lymphoid cells and HLA antigens in the human testis

The presence and distribution of macrophages, several subtypes of lymphocytes and HLA antigens in the human testis were studied using monoclonal antibodies and immunocytochemical staining. Cells identified as tissue macrophages were relatively abundant. They were dispersed individually in the interstitium and were often included in Leydig cell clusters. Occasional macrophages were observed in the tubular wall, but never in the seminiferous tubules. Very few lymphocytes were found in the testicular interstitium. These were mainly T lymphocytes. T helper/inducer and T cytotoxic/suppressor cells were extremely scarce. B1 + cells were not observed, but occasional Leu-14+ cells could be seen. NK cells were not found. The number of T lymphocytes in the tunica albuginea was considerably higher than in the interstitial tissue. HLA-ABC and HLA-DR antigens could be observed in the endothelial cells and macrophages which were both strongly positive. The myoid cells and the Leydig cells expressed these antigens only weakly. Neither of the HLA antigens was expressed in the germinal epithelium.     

Mast cells in testicular biopsies of infertile men with 'mixed atrophy' of seminiferous tubules

Mast cells in the bilateral testicular biopsies of 30 patients with a 'mixed atrophy' of seminiferous tubules were analysed. Seven biopsies from vasectomized patients served as controls. With regard to their characteristic location within testicular tissue, two groups of mast cells could be distinguished, in both control and infertile patients: 'interstitial' mast cells (located between Leydig and other interstitial cells as well as in the vicinity of blood vessels) and 'peritubular' mast cells (located in the close proximity of the tubular lamina propria or incorporated in the lamina propria itself). Morphometric data indicated a significant increase in the number and volume of mast cells in infertile patients when compared with controls. In the biopsies of infertile patients that were analysed both 'interstitial' and 'peritubular' mast cells showed a significant increase in their number and volume, although it appeared that 'peritubular' mast cells increased at a higher rate than 'interstitial' mast cells. A significant negative correlation was found between the following variables: volume and number of mast cells, testis volume and the status of spermatogenesis evaluated by Johnsen's scoring. It was concluded that the increased presence of mast cells is closely associated with an impairment of spermatogenesis.     

Lectin binding sites in human seminiferous epithelium, in CIS cells and in seminomas

The distribution of glycoconjugates in germ cells during spermatogenic differentiation, in carcinoma-in-situ (CIS) cells and in seminoma were studied by lectin histochemistry. The results show that human germ cells are rich in carbohydrate-containing compounds with specific alterations in the expression of glucosyl moieties during germ cell development. CIS cells reveal different lectin binding sites from spermatogenic cells, but the distribution of glycosubstances in CIS cells is similar to that of seminoma cells which supports the suggestion of the malignant nature of CIS germ cells.     

A study on budding protrusions of human seminiferous tubules

In order to elucidate the morphogenesis of seminiferous tubular protrusions, histometric, microscopic and electron microscopic studies were performed on the testes of 202 Japanese men, including 117 sudden deaths, 75 hospital deaths and 10 prostatic cancer cases. Protrusions usually occurred at outer convexes of multi-bending tubular portions and were divided into dome, sessile, pedunculated and multi-branched types. Aggregated Sertoli cells were present in dome-type protrusions as a major component, and spermatogenesis associated with active mitoses of spermatogonia was induced with development of protrusions. Protruding walls consisted of inner compact and outer loose layers. Distribution of lipid droplets in Sertoli cell cytoplasm in protrusions was different from those in the original tubules. The incidence of protrusions peaked in the forties and sixties, respectively, in the case of hospital and sudden death cases with underlying tubular atrophy. The findings suggest that tubular protrusions take place as a compensatory reaction for declining spermatogenesis, and therefore, probably represent a regenerative phenomenon in hypospermatogenic testes.     

Effect of testicular capsulotomy on lipid droplets in the seminiferous tubules of rats

Aim: In order to reveal the histochemical alteration that might occur during the processes of the spermatogenic disruption induced by testicular capsulotomy, the location and alteration of lipid droplets in the seminiferous tubules were observed in the present study. Methods: Osmium tetroxide was used to demonstrate the lipid droplets in the seminiferous tubules of capsulotomized and sham-operated control testes. Results: In the seminiferous tubules of the sham-operated rat testes, many small lipid droplets were located close to the basement membrane of the seminiferous tubules. But for the capsulotomized testes, the lipid droplets in the seminiferous tubules had increased in size and number, with many lipid droplets migrated towards the lumen of the tubules. Conclusion: The results indicated that a progressive fatty degeneration occurred in the seminiferous tubules after testicular capsulotomy.     

Exploration for testicular remnants: implications of residual seminiferous tubules and crossed testicular ectopia

PURPOSE: Testicular remnants identified during exploration for cryptorchidism contain vascularized fibrous nodules at the termination of the vas deferens, hemosiderin, calcification, a pampiniform plexus or occasionally residual seminiferous tubules that may contain germ cells. An absent testis lacks the features of testicular remnants. To our knowledge testicular remnants have not been described in a crossed ectopic location. We reviewed orchiectomy specimens obtained at exploration for a nonpalpable testis to characterize the features of testicular remnants, including the frequency of seminiferous tubules, germ cells and crossed ectopia, as well as to clarify the diagnostic criteria for testicular remnants. MATERIALS AND METHODS: From 1990 to mid 2000 medical records and histological slides from 101 boys with nonpalpable testes who had undergone inguinal exploration and orchiectomy were reviewed. RESULTS: Of the 71 testicular remnants identified 7 (9.8%) contained residual tubules, of which 4 (5.6%) contained germ cells. In 4 boys the testis was deemed absent but 3 did not undergo laparoscopic exploration. There were 2 ectopic remnants (2.8%) on the contralateral side-the pelvis or in the scrotum. Both crossed remnants demonstrated dissociation of the testis from the vas/epididymis which remained on the correct side associated with a pampiniform plexus. No müllerian remnants were encountered. CONCLUSIONS: Adequate exploration for nonpalpable testis requires laparoscopy with visualization of the contralateral pelvic region because an ectopic remnant may be dissociated from the vas/epididymis and vessels. Identification of a pampiniform plexus, vas and spermatic vessels may not be a reliable indicator of a testicular remnant. Continued removal of testicular remnants is warranted because at least 9.8% contain residual viable tubules.     

Carcinoma-in-situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytoma

Based on evidence from morphological and histochemical studies and from clinical experience, the following hypotheses are proposed: carcinoma-in-situ (CIS) germ cells are malignant gonocytes; these CIS gonocytes have some capacity to regress into more primitive, totipotent embryonic cells which can give rise to all types of nonseminomatous germ cell tumours; the tumour germ cells of classical seminomas are malignant gonocytes derived from CIS gonocytes which have lost their ability to regress into totipotent embryonic cells; the ability of CIS gonocytes to regress into totipotent embryonic cells decreases with age, whereas the capacity to form classical seminoma cells is preserved; the transformation of CIS gonocytes into invasive tumours is dependent on factors such as gonadotrophins and/or testicular steroids; the pathogenesis of classical and spermatocytic seminoma are unrelated. As a consequence of these hypotheses an alternative nomenclature for carcinoma-in-situ, seminoma and dysgerminoma is suggested.     

Ultrastructure of the seminiferous tubules in oligoasthenoteratozoospermic men associated with varicocele

Varicocele is associated with venous reflux that may cause increased heat and interstitial pressure within the testes, with variable pathological effects on spermatogenesis. This study aimed to study the ultrastructural testicular changes in the seminiferous tubules of 20 infertile severe oligoasthenoteratozoospermia (OAT) men associated with varicocele and five patients with obstructive azoospermia without varicocele as controls. They were subjected to testicular biopsy which was evaluated by transmission electron microscopy. Ultrastructurally, the seminiferous epithelium in the testicular biopsies of infertile severe OAT men associated with varicocele was variably affected in the form of thickening of the peritubular connective tissue, vacuolation of Sertoli cell and germ cell cytoplasm, presence of degenerated and apoptotic cells among the germinal epithelium, altered spermatids and abnormal spermatozoa. It is concluded that varicocele in severe OAT men is associated with ultrastructural changes in the seminiferous tubule.     

The influence of human chorionic gonadotrophin (hCG) on the morphology of the prepubertal human undescended testis

The effect of hCG-treatment on the morphology of the undescended testis was studied in testicular biopsies from 36 prepubertal boys operated on at intervals of 1 day to 2 years after discontinuation of hormonal treatment. Immediately after treatment, mature Leydig cells were observed, and the Sertoli cells were increased in size; serum testosterone had increased to adult levels. All these changes were reversible as judged from the material taken one month or more after the last hCG injection. Based on the observations and the results of a previous study it is suggested that hCG treatment does not induce any premature onset of Sertoli cell or germ cell maturation either in the undescended or in the contralateral testis.     

Tamoxifen induced multinucleated cells （symplasts） and distortion of seminiferous tubules in rat testis

Aim: To evaluate the effect of tamoxifen citrate on male reproductive system of rat. Methods: Groups of male rats were gavaged with tamoxifen at doses of 200 mg·kg-1·d-1, 400 mg·kg-1·d-1 or 800 mg·kg-1·d-1 in 0.1 mL olive oil for 10 consecutive days. Controls were treated with 0.1 mL olive oil. Rats were anesthetized and killed on d 3, d 15 or d 35 after the last dose. Testes were collected, processed for paraffin embedding, sectioned at 5 μm thickness, stained with H&E and analyzed microscopically. Results: There was a dose-dependent increase in the occurrence of seminiferous tubular distortion with germinal cell sloughing. The highest dose increased the number of multinucleated giant cells on d 3 and d 15. Conclusion: Tamoxifen citrate induces multinucleated giant cells and germinal epithelial sloughing in a dose-dependent manner and these changes are detrimental to male fertility.