Maturation phenotype of Sertoli cells in testicular biopsies of azoospermic men

The involvement of Sertoli cells in different spermatogenic impairments has been studied by an immunohistomorphometric technique using cytokeratin-18 (CK-18) as a marker for immature Sertoli cells. CK-18 is known to be expressed in Sertoli cells during prenatal and prepubertal differentiation and is normally lost at puberty. Forty-nine azoospermic men were included in the current study. Quantitative measurements on testicular biopsies revealed the highest CK-18 expression in the mixed atrophy biopsies (22 men), a lower expression in the Sertoli cell-only (SCO) biopsies (12 men), and minimal residual staining in the group considered as representing normal spermatogenesis (six obstructive azoospermia patients). The cytokeratin immunopositive-stained tubules were associated either with arrest in spermatogenesis or with SCO. Examination of sections from nine men with microdeletions in the AZF region of the Y chromosome revealed that these men were either negative for CK-18 expression or showed only weak residual staining. This may suggest that the spermatogenic defect in the AZF-deleted men originates in the germ cell and has no impact on Sertoli cell maturation. The cause that determined the spermatogenic defect in the other cases of male infertility with high CK-18 expression may have damaged both the Sertoli and the germ cells.     

Intermediate filaments in the Sertoli cells of the ageing human testis

The presence and distribution of intermediate filaments (vimentin, keratin, desmin) was studied in the Sertoli cells of elderly men by means of quantitative immunohistochemical methods. Sertoli cells from young men showed moderate immunogold labelling to vimentin throughout the entire cytoplasm between the cell organelles in tubules showing complete spermatogenesis. Immunogold particles were more numerous in the perinuclear cytoplasm and beneath the plasma membrane in all its faces. The testes from elderly men showed different tubule types; some showed complete spermatogenesis and a normal lamina propria, while others had spermatogenic arrest at different levels (spermatids, spermatocytes, spermatogonia). The immunohistochemical reaction to vimentin in the Sertoli cells of tubules with complete spermatogenesis (type a) was similar to that in the cells of young men. In the Sertoli cells of severely damaged tubules (type b) the immunohistochemical reaction was more intense and immunogold particles extended in similar proportions throughout the whole cytoplasm. When immunolabelling intensity was compared between the three groups of tubules, by counting the number of immunogold particles per square micrometre of cytoplasm, it was found to be significantly higher (P≤0.05) in type b tubules of elderly men than either in tubules of young men or in type a tubules of elderly men. Since the average cell surface of Sertoli cells was similar in all tubule types, these data suggest that an actual vimentin increase occurs in Sertoli cells of germ-cell-depleted tubules. Sertoli cell immunogold labelling to keratin was found neither in young men nor in type a tubules of ageing men, whereas a positive immunohistochemical reaction was observed in the Sertoli cells of type b tubules of elderly men. Immunogold particles were localized mainly in the perinuclear cytoplasm, and beneath the lateral and basal cell surfaces. The observation of vimentin increase and keratin re-expression in ageing Sertoli cells only in germ-cell-depleted tubules suggests that the changes in intermediate filaments are related to the local factors associated with completion of spermatogenesis, causing functional changes in Sertoli cells.     

The contribution of RNA-binding motif (RBM) antibody to the histopathologic evaluation of testicular biopsies from infertile men

Testicular biopsies of infertile men are often characterized by mixed histologic patterns, with different types of spermatogenic impairments being found in adjacent seminiferous tubules. RNA-binding motif (RBM) is a nuclear protein expressed exclusively in the male germ cell line. We reasoned that RBM might be a useful marker to identify germ cells in testicular sections, particularly in biopsies with mixed histologic phenotype and small focal concentrations of spermatogenesis. Testicular biopsies from azoospermic men were immunohistochemically evaluated for RBM expression. RBM expression was detectable in spermatogonia, spermatocytes, and round spermatids in biopsies of men with obstructive azoospermia and normal spermatogenesis. No specific cell staining was shown in cases of Sertoli-cell-only (SCO) syndrome. In biopsies of patients with spermatogenic disorders, all the germ cells were stained up to and including the stage level of the arrest in spermatogenesis. This approach enabled identification of small focal concentrations of spermatogenesis in a biopsy previously classified as being SCO by hematoxylin and eosin staining. Thus, RBM can be a useful immunohistochemical marker for the specific identification of germ cells and provide greater accuracy in the histopathologic evaluation of testicular biopsies.     

Manifestation of Y-chromosomal deletions in the human testis: a morphometrical and immunohistochemical evaluation

BACKGROUND: Deletions of the AZF (azoospermia factor) subregions on the Y chromosome are accompanied by a diverse spectrum of spermatogenic disturbances ranging from hypospermatogenesis to total depletion of germ cells causing infertility. The AZF region encodes gene products which are candidates for the genetic control of spermatogenesis. Although it is known which genes are involved, a general principle of cause and effect cannot yet be deciphered and the deletion type has non-uniform histological phenotypes. METHODS AND RESULTS: We analysed morphological parameters of testicular biopsies from 17 patients diagnosed for Y chromosome microdeletions. As control groups we analysed testes from patients with idiopathic Sertoli cell-only (SCO) syndrome (n = 11), mixed atrophy (n = 10) and complete spermatogenesis (n = 11). A detailed genetic analysis on the extension of the observed microdeletions revealed similar breakpoints in the distal and proximal region of the AZFc region, indicating a common mechanism of homologous recombination for such deletions, as has been suggested before. Morphometric parameters such as the diameter of the tubules, lumen, thickness of the lamina propria and height of the tubule epithelia were investigated. The diameter of the tubules from patients with microdeletions was found to be significantly smaller compared with patients with mixed atrophy. Considering also the size of the tubules, lumen and epithelia, a Y-chromosomal microdeletion represents an intermediate state between an idiopathic SCO and normal spermatogenesis. The immunohistochemical analysis of six different Sertoli cell markers, cytokeratin 18, vimentin, inhibin alpha subunit, 14-3-3 theta, FSH receptor and androgen receptor, revealed no impact of AZF deletion on the specific expression pattern of these genes. CONCLUSIONS: Our results suggest that, notwithstanding the deletion of a common region in the AZFc region, microdeletions of the Y chromosome lead to an intermediate status between idiopathic SCO and complete spermatogenesis, resulting in a heterogeneous histological profile regardless of the seminiferous activity. The Sertoli cell function seems not to be altered.     

Mitotic activity of Sertoli cells in adult human testis: an immunohistochemical study to characterize Sertoli cells in testicular cords from patients showing testicular dysgenesis syndrome

During puberty, normal somatic Sertoli cells undergo dramatic morphological changes due to the differentiation of immature pre-Sertoli cells in functionally active adult Sertoli cells. Sertoli cell maturation is accompanied with loss of their mitotic activity before onset of spermatogenesis and loss of pre-pubertal and occurrence of adult immunohistochemical Sertoli cell differentiation markers. Testes of infertile adult patients often exhibit numerous histological signs of testicular dysgenesis syndrome (TDS) such as microliths, Sertoli cell only (SCO) tubules, tubules containing carcinoma in situ and immature seminiferous tubules (Sertoli cell nodules). Sertoli cell tumours, however, are very rare neoplasms possibly due to the fact that the mechanism and temporal origin of neoplastic Sertoli cells underlying Sertoli cell tumourigenesis still remain unknown. To clarify the state of Sertoli cell differentiation in both immature seminiferous tubules of adult patients with TDS and Sertoli cell tumour, we compared the expression of the Sertoli cell differentiation markers vimentin, inhibin-α, anti-Muellerian-hormone, cytokeratin 18, M2A-antigen, androgen receptor and connexin43 with that of SCO tubules with hyperplasia. In addition, we demonstrated for the first time the existence of proliferating Sertoli cells by Ki67- and PCNA-immunostaining in Sertoli cell nodules of the adult human testis. Our data indicate that mitotically active Sertoli cells in Sertoli cell nodules will be arrested prior to puberty and, contrary to dogma, do not represent foetal or neonatal cells. Since all markers in Sertoli cell nodules revealed a staining pattern identical to that in neoplastic Sertoli cells, but different to that in Sertoli cells of SCO tubules with hyperplasia, it may be speculated that Sertoli cell tumours in adult men may originate from Sertoli cell nodules.     

Detection of calretinin expression in abnormal immature Sertoli cells in non-obstructive azoospermia

The current study identified for the first time calretinin expression in abnormal Sertoli cells of azoospermic men who underwent testicular biopsy for sperm recovery and application of the retrieved sperm by in vitro fertilization techniques. Testicular biopsies with various spermatogenic impairments were evaluated immunohistochemically for the expression of the calretinin calcium-binding protein and the marker for immaturity of Sertoli cells, cytokeratin-18 (CK-18). Distribution of the markers was assessed in testes demonstrating a histological phenotype of mixed atrophy, Sertoli cell-only, or normal spermatogenesis (obstructive-azoospermia) and in men carrying a deletion in the azoospermia factor region located on the Y chromosome. Calretinin-immunopositive immature Sertoli cells revealed by co-localization of both markers, calretinin and CK-18, were identified in the mixed atrophy group in seminiferous tubules demonstrating spermatogenic failure. Sertoli cells expressing both markers were rarely detected in all other groups. Leydig cells in all the assessed biopsies expressed calretinin and served as a built-in control for immunoreactivity. This pattern of calretinin-selective expression in immature Sertoli cells suggests a functional relationship between calretinin expression and the degree of Sertoli cell differentiation. Disorders of Sertoli cell differentiation as indicated by calretinin and/or CK-18 expression contribute to the multifactorial mechanisms underlying spermatogenic failure.     

Reversion of the differentiated phenotype and maturation block in Sertoli cells in pathological human testis.

To study the relationship between abnormal Sertoli cell differentiation and spermatogenic impairment, we examined the expression of Sertoli cell markers normally lost at puberty, cytokeratin 18 (CK18), anti-Müllerian hormone (AMH) and M2A antigen, in three children (aged 1-2 years), 50 adults (aged 19-45 years) with obstructive or non-obstructive azoospermia or oligozoospermia, and six patients (aged 1-18 years) with 5 alpha-reductase deficiency. There was CK18 and/or AMH expression, but never M2A antigen expression, associated with spermatogonial arrest or Sertoli cell-only (SCO) syndrome in infertile men. Loss of M2A antigen suggests the transition of Sertoli cells to an adult phenotype, while CK18 and/or AMH expression may be a manifestation of de-differentiation of Sertoli cells. In 5 alpha-reductase deficiency, there was a sequential loss of CK18, M2A antigen and AMH around puberty, associated with partial spermatogenesis. The persistence of immature Sertoli cells expressing M2A antigen was associated with prepubertal seminiferous cords and SCO syndrome. Therefore, 5 alpha-reductase deficiency may prevent the maturation of Sertoli cells, resulting in impairment of spermatogenesis, and loss of M2A antigen expression coincides with a critical step in the Sertoli cell maturation. High follicle stimulating hormone concentrations due to failure of normal Sertoli cell differentiation indicate a normal development pattern of the hypothalamic-pituitary-gonadal axis.     

Altered expression of connexins 26 and 43 in Sertoli cells in seminiferous tubules infiltrated with carcinoma-in-situ or seminoma

The expression of connexins (cx) 26 and 43 in testis infiltrated with carcinoma-in-situ (CIS) or seminoma was examined to gain insight into the relationship between aberrant gap junctional communication and spermatogenic impairment in the neoplastic testis. In uninvolved tubules with normal spermatogenesis, cx43 immunostaining was localized to the Sertoli-Sertoli junctional complex and cx26 was absent. In contrast, infiltrated tubules with spermatogonial arrest or CIS-only were negative for cx43, but displayed strong intracytoplasmic Sertoli cell staining for cx26. The Sertoli cells in these tubules re-expressed cytokeratin 18 (ck18), signifying a reversion to a less differentiated state. Western blot analysis for cx43 revealed a single immunoreactive band at 43 kD (normal spermatogenesis) and three bands at 43, 41, and 39 kD (impaired spermatogenesis with CIS or seminoma). For cx26, a doublet band at 26/28 kD (normal spermatogenesis) and an additional doublet band at 52/54 kD (impaired spermatogenesis with CIS or seminoma) were observed. The altered expression of cx26 and cx43 in Sertoli cells in testes infiltrated with CIS or seminoma suggests that a derangement in intercellular communication between Sertoli cells and between Sertoli cells and germ cells may play a role in the resulting spermatogenic impairment and possibly in the proliferation and neoplastic progression of CIS cells.     

Genetic imprinting during impaired spermatogenesis

Disorders in genetic imprinting are discussed as potential geneticrisk in assisted reproduction technology (ART), where most ofthe natural selection mechanisms are bypassed. As currentlyonly limited information about genomic imprinting in disruptivespermatogenesis is available, we analysed the imprinting stateof the paternally methylated gene H19 in various germ cell populationsderived from seminiferous tubules exhibiting impaired spermatogenesis.Different germ cell types were isolated by laser microdissectionfrom human testicular paraffin sections. Although the methylationstate of the maternally imprinted gene SNRPN was investigatedby methylation-specific PCR (M-PCR) to establish the isolationmethod, methylation of H19 was analysed by a single-strand conformation-basedmethod. Contamination by somatic Sertoli cells was excludedbecause of Sertoli cell-specific vimentin immunohistochemistrybefore germ cell laser microdissection. We demonstrate correctgenetic imprints for H19 even in spermatogonia selected fromseminiferous tubules exhibiting spermatogenic arrest at thelevel of spermatogonia, providing no evidence for incorrectgenomic imprinting in spermatozoa from infertile men used forICSI.     

Testis of the lizard Mabuya carinata: a light microscopic and ultrastructural seasonal study

Histomorphology and ultrastructure of the testis during breeding and nonbreeding phases of the reproductive cycle of the lizard Mabuya carinata are studied. Observations of the ultrastructural features of the testis during breeding and nonbreeding phases of the reproductive cycle reveal a prenuptial type of spermatogenesis and a clearcut discontinuous spermatogenic cycle. Seminiferous tubules are enlarged and there is active spermatogenesis as shown by the presence of all the stages of spermatogenesis (spermatogonia to spermatids) and spermatozoa during the breeding phase (November). During the nonbreeding phase (April) only spermatogonia and Sertoli cells are seen in the shrunken seminiferous tubules. Leydig cells and Sertoli cells show distinct changes in the morphological appearance with hypertrophy of the cells in breeding phase and atrophy of the cells in the nonbreeding phase of the reproductive cycle. The present study suggests that Sertoli cells and Leydig cells functions are synchronous in the lizard M. carinata.     

DOG1 immunohistochemical staining of testicular biopsies is a reliable tool for objective assessment of infertility

Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.     

Misregulation of histone acetylation in Sertoli cell-only syndrome and testicular cancer

In many species, including humans, chromatin remodelling during spermiogenesis is initiated with a marked increase in histone acetylation in elongating spermatids. We have investigated whether this process is disturbed when spermatogenesis is defective or in human testicular tumours. For this purpose, the presence of highly acetylated histone H4 was detected on testicular sections from men with a severe impairment of spermatogenesis of several origins, as well as in different types of testicular tumours. In most tubules devoid of germinal cells (including SCO, Sertoli cell only syndromes) or lacking spermatocytes and spermatids, the Sertoli cells' nuclei showed a global increase in histone H4 acetylation. A similar observation was made in the peritumoral seminiferous tubules of testicular tumour tissues, whenever they were lacking germinal cells, with carcinoma in situ (CIS) cells being hypoacetylated. The global hyperacetylation of elongating spermatids during spermatogenesis could be part of an intercellular signalling pathway involving Sertoli cells and germinal cells, which could be disturbed in cases of severe spermatogenesis impairment, as well as in tubes surrounding germ cells in testicular tumours.     

Immunohistochemical analysis of connexin43 expression in infertile human testes

Connexin43 (Cx43) is abundantly expressed in mammalian testes and implicated in the regulation of cell-to-cell interaction between germ cells and Sertoli cells, which is essential to the normal process of spermatogenesis. In the present study, we investigated the relation between Cx43 expression and the degree of spermatogenesis in infertile human testes. Immunohistochemical analysis of Cx43 was performed on testicular biopsies from 29 patients with azoospermia (n=23) and severe oligospermia (n=6), who gave informed consent to this experiment. The degree of testicular spermatogenesis was evaluated by Johnsen score. In the interstitium, immunostaining for Cx43 was localized to some focal parts of plasma membrane between neighboring Leydig cells. In seminiferous tubules with normal spermatogenesis, Cx43 expression was found between Sertoli cells and germ cells. However, Cx43 expression in maturation arrest was decreased and located mainly in the basal compartment of seminiferous tubules. Finally, there was a significant positive correlation between histological score of spermatogenesis and intensity of Cx43 (p=0.0294). These data suggest that the alteration of Cx43 expression may be involved in spermatogenic impairment, and that the communication between Sertoli cells and germ cells through Cx43 may be important for maturation of spermatogenesis.     

Immunohistochemical analysis of the vimentin filaments in Sertoli cells is a powerful tool for the prediction of spermatogenic dysfunction

The close interaction between male germ cells and Sertoli cells, a type of somatic cell found in the seminiferous tubules of mammalian testis, is essential for the normal progression of spermatogenesis in mammals. Vimentin is an intermediate filament protein that primarily provides mechanical support, preserves cell shape, and maintains the nuclear position, and it is often used as a marker to identify Sertoli cells. Vimentin is known to be involved in many diseases and aging processes; however, how vimentin is related to spermatogenic dysfunction and the associated functional changes is still unclear. In a previous study, we reported that vitamin E deficiency affected the testes, epididymis, and spermatozoa of mice, accelerating the progression of senescence. In this study, we focused on the Sertoli cell marker vimentin and explored the relationship between the cytoskeletal system of Sertoli cells and spermatogenic dysfunction using testis tissue sections that caused male reproductive dysfunction with vitamin E deficiency. The immunohistochemical analysis showed that the proportion of the vimentin-positive area in seminiferous tubule cross-sections was significantly increased in testis tissue sections of the vitamin E-deficient group compared with the proportion in the control group. The histological analysis of testis tissue sections from the vitamin E-deficient group showed that vimentin-positive Sertoli cells were greatly extended from the basement membrane, along with an increased abundance of vimentin. These findings suggest that vimentin may be a potential indicator for detecting spermatogenic dysfunction.     

Role of the spermatogenic–Sertoli cell interaction through cell adhesion molecule-1 (CADM1) in spermatogenesis

Endocrine and local secretory factors have long been known to be required for spermatogenesis. Evidence has been accumulating in recent years indicating that direct contact between spermatogenic and Sertoli cells is also required for spermatogenesis. Cell adhesion molecules of various types have been found in the mammalian testis that are expressed in spermatogenic and/or Sertoli cells and involved in homophilic and/or heterophilic binding. We have cloned a novel cell adhesion molecule, cell adhesion molecule-1 (CADM1), also known as immunoglobulin superfamily 4A or spermatogenic immunoglobulin superfamily, from the mouse testis. CADM1 belongs to the immunoglobulin superfamily and is composed of three immunoglobulin-like domains, a transmembrane domain, and a short intracellular domain. In the seminiferous epithelium, CADM1 is expressed in intermediate spermatogonia through to early pachytene spermatocytes as well as in elongating spermatids—but not in round spermatids, mature spermatozoa, or Sertoli cells. One of the heterophilic binding partners of CADM1 has proven to be a poliovirus receptor, another member of the immunoglobulin superfamily that is expressed in Sertoli cells. Knockout mice for CADM1 develop male infertility due to defective spermatogenesis. These findings suggest that cell adhesion molecules between spermatogenic and Sertoli cells play essential roles in spermatogenesis.     

Experimental allergic orchitis in mice

Summary Experimental allergic orchids was induced in (C57BL/6J × A/J)F₁ mice by two injections of syngeneic testicular homogenate emulsified with adjuvants immediately followed by intravenous injection of pertussis vaccine, at a 2 week interval.Histologically, in the initial stage there was occasional focal degeneration and desquamation of both spermatogonia and Sertoli cells within limited parts of the seminiferous tubules, in the peripheral region of the testis. No inflammatory change was present. In some cases, however, inflammatory reaction in the rete testis and focal lymphocytic infiltration in the interstitium were also observed. Subsequently, marked infiltration of lymphocytes, monocytes, and polymorphs were found not only in the testes, but also in rete testis and epididymis. In later stages the inflammatory reaction gradually subsided, but the testes became atrophic due to progression of spermatogenic arrest. Many tubules were lined only with monolayers of Sertoli cells, surrounded by hyperplastic Leydig cells in the interstitium. At 5 months after the 2nd immunization, there was still variable depression of spermatogenesis and hyperplasia of Leydig cells with scattered fibrous scars in the seminiferous tubules, although good regeneration of germ cells appeared in some tubules.Immunological studies revealed that lymphocytes obtained from mice bearing developed orchitis showed a significantly enhanced response in the mixed culture with syngeneic testicular cells, and suggest that cellular immunity plays an important role in the induction of experimental allergic orchitis in mice.     

Effects of dietary zinc deficiency on protein secretory functions of the mouse testis.

The effects of dietary zinc deficiency on testicular protein secretion, mainly that by Sertoli cells, were examined by electron microscopy and two-dimensional polyacrylamide gel electrophoresis of [35S] methionine-labeled secretory proteins from mouse testes. Zinc deficiency caused a significant decrease in the gonadosomatic index and a distinct increase in deoxyribonucleic acid concentration. Sertoli cells maintained normal fine-structural features; junctional complexes among Sertoli cells continued to divide seminiferous tubules into basal and adluminal compartments in the zinc-deficient mouse testes. Severe atrophic changes were observed in spermatogenic cells after meiotic division in the adluminal compartment, but not in spermatogonia located in the basal compartment. Zinc replacement treatment caused spermatogenesis to recover normally. Although total protein secretion was not affected by zinc deficiency, one polypeptide spot appeared due mainly to the loss of its target spermatogenic cells. The present study indicates that zinc is indispensable for spermatogenic cells after meiosis and that testicular protein secretory functions can be preserved in the absence of zinc.     

The testis in adreno-leukodystrophy.

Testicular tissue from 7 juvenile and 6 adult patients with adreno-leukodystrophy (ALD) or its adrenomyeloneuropathic (AMN) variant was examined by light and electron microscopy. The seminiferous tubules of the men exhibited hypocellularity, maturation arrest, or Sertoli cells and spermatogonia only. Ultrastructural examination of these specimens revealed vacuolation of Sertoli cell endoplasmic reticulum. Germ cells also demonstrated vacuolation and necrosis, accompanied by slight tubular atrophy and thickening of the tunica propria. Fibrosis or proteinaceous fluid was observed in the interstitium. The tissue of 5 men and 2 boys contained Leydig cells with cytoplasmic striations, which were detectable with the light microscope. A decrease in the number of Leydig cells was noted in some men. All specimens displayed pathognomonic lamellas and lamellar-lipid profiles in mature, immature, or precursor Leydig cells at the ultrastructural level. The Leydig cell demonstrated the primary morphologic defect in the ALD testis; damage to Sertoli cells appeared to be the initial lesion of seminiferous tubules.