Touch imprint cytology in biopsy of the infertile testis.

OBJECTIVE: To identify different cell types in the testis by using touch imprint cytology and to compare the cytologic findings to the histopathologic diagnosis in infertile men. STUDY DESIGN: This prospective study used touch imprint preparations and included 20 infertile men. The biopsy material obtained was stained with toluidine blue, May-Grünwald-Giemsa stain and Papanicolaou stain. The cytologic results for oligospermic, normospermic and azospermic men were compared to the specific histopathologic diagnosis. The proportion of spermatogenic versus Sertoli cells was calculated. The scores were compared between three groups based on the results of the histologic biopsy: normal spermatogenesis, hypospermatogenesis and incomplete spermatogenic arrest. RESULTS: The mean ratio of the spermatogenic cells versus Sertoli cells was statistically significantly different in the three groups (P < .01). The mean ratio of spermatogenic cells to Sertoli cells was higher in cases with normal spermatogenesis than in cases with hypospermatogenesis and incomplete spermatogenic arrest, revealing a statistical difference (P<.01). This ratio was not statistically significantly different between the hypospermatogenesis and incomplete spermatogenic arrest groups. CONCLUSION: A cytologic demonstration of germinal cells by using touch imprint preparations may be a guide for histologic diagnosis.     

BCLW mediates survival of postmitotic Sertoli cells by regulating BAX activity.

Male mice deficient in BCLW, a death-protecting member of the BCL2 family, are sterile due to an arrest in spermatogenesis that is associated with a gradual loss of germ cells and Sertoli cells from the testis. As Bclw is expressed in both Sertoli cells and diploid male germ cells, it has been unclear which of these cell types requires BCLW in a cell-autonomous manner for survival. To determine whether death of Sertoli cells in Bclw mutants is influenced by the protracted loss of germ cells, we examined testes from Bclw/c-kit double mutant mice, which lack germ cells from birth. Loss of BCLW-deficient Sertoli cells occurs in the absence of germ cells, indicating that germ cell death is not required to mediate loss of Sertoli cells in BCLW-deficient mice. This suggests that Sertoli cells require BCLW in a cell-intrinsic manner for long-term survival. The loss of Sertoli cells in Bclw mutants commences shortly after Sertoli cells have become postmitotic. In situ hybridization analysis indicates that Bclw is expressed in Sertoli cells both before and after exit from mitosis. Therefore, Bclw-independent pathways promote the survival of undifferentiated, mitotic Sertoli cells. We show that BAX and BAK, two closely related death-promoting members of the BCL2 family, are expressed in Sertoli cells. To determine whether either BAX or BAK activity is required for Sertoli cell death in Bclw mutant animals, we analyzed survival of Sertoli cells in Bclw/Bax and Bclw/Bak double homozygous mutant mice. While mutation of Bak had no effect, ablation of Bax suppressed the loss of Sertoli cells in Bclw mutants. Thus, BCLW mediates survival of postmitotic Sertoli cells in the mouse by suppressing death-promoting activity of BAX.     

The second messenger pathway for germ cell-mediated stimulation of Sertoli cells.

Treatment of cultured rat Sertoli cells with FSH or dibutyryl cAMP for 30 min resulted in phosphorylation of the same Sertoli cell proteins. Different Sertoli cell proteins were phosphorylated after calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. A23187 stimulated the phosphorylation of hsp27, while TPA alone had no effect. TPA plus A23187 resulted in phosphorylation of a 14 kDa protein, in addition to hsp27. The effect of TPA plus A23187 was identical to that of germ cells on Sertoli cell protein phosphorylation. FSH-stimulated cAMP production by Sertoli cells was reduced by prior exposure of Sertoli cells to germ cells. The results indicate that germ cells stimulate Sertoli cells by the inositol trisphosphate/diacylglycerol mediated second messenger pathway. The results also suggest that the germ cell-activated pathway interacts within Sertoli cells to modulate Sertoli cell response to FSH.     

Isolation and culture of FSH responsive Sertoli cells.

Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.     

Season but not age affects Sertoli cell number in adult stallions.

To evaluate the effect of age and season on Sertoli cell number per paired testes, ratio of germ cells per Sertoli cell, and daily sperm production, testes were obtained from 184 adult (4-20 yr) stallions at slaughter throughout one year. Numbers of Sertoli cells or germ cells were derived from nuclear volume density, volume of individual nuclei, and parenchymal volume. Germ cell to Sertoli cell ratios were calculated from cell numbers. Regression analysis was used to detect age-related differences in the breeding season (May-Jul) or throughout the year. A two-way analysis of variance was used to evaluate time periods (Nov-Jan, Feb-Apr, May-Jul, and Aug-Oct) and age groups (4-5.5, 6-12.5, or 13-20 yr). Paired parenchymal weight and daily sperm production per horse increased significantly with age. Neither regression nor analysis of variance revealed an effect of age on Sertoli cell number. While season contributed (p less than 0.01) to variation in Sertoli cell number per horse, there was no (p greater than 0.05) age x season interaction or age effect on Sertoli cell number. In testes obtained from adult stallions, age had no effect on the number of Sertoli cells per horse, the ratio of maturation-phase spermatids to Sertoli cells, or the ratio of all stage VIII germ cells to Sertoli cells. Given no age effect within a given season on Sertoli cell number per horse, the number of Sertoli cells in the recrudesced testis of the breeding season probably is not significantly different for a given stallion between 4 and 20 yr of age.     

Hormone interactions in the Sertoli cells.

The effects of follicle-stimulating hormone (FSH) and testosterone in rat Sertoli cells were investigated in vitro by means of isolated cell populations. The Sertoli cells selectively bind FSH, and respond to FSH stimulation with increased accumulation of endogenous cyclic AMP and secretion of androgen-binding protein (ABP). FSH binding and cyclic AMP response in the Sertoli cells change dramatically during sexual maturation. Cyclic AMP response decreases despite an increase in FSH-binding receptors per cell. Evidence has been provided for the existence of cytoplasmic and nuclear androgen receptors and chromatin acceptor-sites that specifically bind the androgen-receptor complex in the Sertoli cells. A model has been proposed for the hormonal interactions in the seminiferous tubule and the possible role of Sertoli cells in mediating the hormonal effects on spermatogenesis.     

Sertoli-germ cells interactions in the human testis.

In previous histoimmunochemical studies we reported that transferrin (TF) and insulin-like growth factor I (IGF-I) are present in the cytoplasm of the Sertoli cells of the adult human testis. Receptors for TF were found mainly in adluminal germ cells and type I receptors for IGF-I both in Sertoli and germ cells. Using electron microscopy, evidence of transfer of both TF and IGF-I from the Sertoli to the germ cells through a receptor-mediated endocytosis mechanism was also found. In this paper we report the results of the histoimmunochemical localization of alpha inhibin in the human fetal, prepubertal and adult testis. In 8- to 14-week-old fetal testes a positive immunostaining was found mainly in the interstitial cells, whereas no staining was found in the germ cords. In the prepubertal testis the immunostaining was present in the Sertoli cells but not in the interstitial cells. In the adult human testis the immunostaining was present not only in the Sertoli cells but also in the spermatocytes and in several Leydig cells. Using electron microscopy and immunogold labeling the presence of alpha inhibin immunoreactivity was found in the rough endoplasmic reticulum and in the Golgi cisternae of both Sertoli and Leydig cells. Moreover we found evidence of transfer of alpha inhibin from the Sertoli to the germ cells through receptor-mediated endocytosis.     

Spermatogenesis in Bclw-deficient mice.

Bclw is a death-protecting member of the Bcl2 family of apoptosis-regulating proteins. Mice that are mutant for Bclw display progressive and nearly complete testicular degeneration. We performed a morphometric evaluation of testicular histopathology in Bclw-deficient male mice between 9 days postnatal (p9) through 1 yr of age. Germ cell loss began by p22, with only few germ cells remaining beyond 7 mo of age. A complete block to elongated spermatid development at step 13 occurred during the first wave of spermatogenesis, whereas other types of germ cells were lost sporadically. Depletion of Sertoli cells commenced between p20 and p23 and continued until 1 yr of age, when few, if any, Sertoli cells remained. Mitochondria appeared to be swollen and the cytoplasm dense by electron microscopy, but degenerating Bclw-deficient Sertoli cells failed to display classical features of apoptosis, such as chromatin condensation and nuclear fragmentation. Macrophages entered seminiferous tubules and formed foreign-body giant cells that engulfed and phagocytosed the degenerated Sertoli cells. Leydig cell hyperplasia was evident between 3 and 5 mo of age. However, beginning at 7 mo of age, Leydig cells underwent apoptosis, with dead cells being phagocytosed by macrophages. The aforementioned cell losses culminated in a testis-containing vasculature, intertubular phagocytic cells, and peritubular cell "ghosts." An RNA in situ hybridization study indicates that Bclw is expressed in Sertoli cells in the adult mouse testis. Consequently, the diploid germ cell death may be an indirect effect of defective Sertoli cell function. Western analysis was used to confirm that Bclw is not expressed in spermatids; thus, loss of this cell type most likely results from defective Sertoli cell function. Because Bclw does not appear to be expressed in Leydig cells, loss of Leydig cells in Bclw-deficient mice may result from depletion of Sertoli cells. Bclw-deficient mice serve as a unique model to study homeostasis of cell populations in the testis.     

Human testicular cultures. II. Sertoli cells.

Two cell types, one epitheloid and the other fibroblast-like, were found in human testicular cultures derived from testes of patients. Ultrastructural studies indicated that, whereas the epitheloid cells were Sertoli cells, the fibroblast-like cells were fibroblasts. The Sertoli cells could maintain growth for a period of more than 4 months. In cultures derived from normal testes, only fibroblasts were observed.     

Two plasma membrane antigens of testicular sertoli cells and H-2-restricted versus unrestricted lysis by female T cells.

Sertoli cell-only seminiferous tubules of sterile XX,Sxrl-male mice served as an excellent source of pure Sertoli cells. When H-2-compatible female mice were immunized 3 times with these Sertoli cells, resulting antibodies recognized two antigens on the plasma membrane of testicular Sertoli cells. They were male-specific, but ubiquitously expressed H-Y antigen and the cell lineage-specific antigen which Sertoli cells shared with ovarian follicular cells. Doubly primed (2 or 3 times in vivo, and once in vitro) cytotoxic T cells from these females lysed target Sertoli cells in both H-2-restricted and nonrestricted manners. While H-2-restricted killings were attributable to H-Y antigen, further work is needed to identify the Sertoli follicular cell lineage-specific antigen as the cause of H-2-nonrestricted killings.     

Expression of the enkephalin precursor gene in rat Sertoli cells. Regulation by follicle-stimulating hormone

Searching for somatic cells expressing the preproenkephalin (A) gene in the testis, we have isolated Sertoli cells from the testes of 20-day-old rats. Cultured Sertoli cells contained a single species (about 1.5 kb) of preproenkephalin mRNA, and follicle-stimulating hormone (FSH) transiently increased the mRNA abundance to a maximum (about 30 molecules per cell) at 12 h. Various compounds that activate the cyclic AMP system in Sertoli cells similarly increased the abundance of preproenkephalin mRNA. Moreover, FSH increased intracellular Met-enkephalin immunoreactive peptides in Sertoli cells. Thus, the preproenkephalin gene expression in Sertoli cells is positively regulated by FSH through the cyclic AMP system.     

Sertoli cells in the testis of caecilians, Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): light and electron microscopic perspective

The caecilians have evolved a unique pattern of cystic spermatogenesis in which cysts representing different stages in spermatogenesis coexist in a testis lobule. We examined unsettled issues relating to the organization of the caecilian testis lobules, including the occurrence of a fatty matrix, the possibility of both peripheral and central Sertoli cells, the origin of Sertoli cells from follicular cells, and the disengagement of older Sertoli cells to become loose central Sertoli cells. We subjected the testis of Ichthyophis tricolor (Ichthyophiidae) and Uraeotyphlus cf. narayani (Uraeotyphliidae) from the Western Ghats of Kerala, India, to light and transmission electron microscopic studies. Irrespective of the functional state of the testis, whether active or regressed, Sertoli cells constitute a permanent feature of the lobules. The tall Sertoli cells adherent to the basal lamina with basally located pleomorphic nuclei extend deeper into the lobule to meet at the core. There they provide for association of germ cells at different stages of differentiation, an aspect that has earlier been misconceived as the fatty matrix. Germ cells up to the 4-cell stage remain in the intercalating region of the Sertoli cells and they are located at the apices of the Sertoli cells from the 8-cell stage onwards. The developing germ cells are intimately associated with the Sertoli cell adherent to the basal lamina until spermiation. There are ameboid cells in the core of the lobules that appear to interact with the germ cells at the face opposite to their attachment with the Sertoli cells. Adherence of the Sertoli cells to the basal lamina is a permanent feature of the caecilian testicular lobules. The ameboid cells in the core are neither Sertoli cells nor their degeneration products.     

Structural characteristics of immature rat Sertoli cells in vivo and in vitro.

The structural properties of pelleted prepubertal Sertoli cells (pre-culture pelleted cells) from 19-day-old rats and of similar cells cultured for 7 days were compared with Sertoli cells from the intact animal (testis tissue from 19- and 26-day-old rats, the in vivo groups). Sertoli cells from freshly isolated pellets and those cultured for 7 days were similar in cell and nuclear volumes to their in vivo counterparts. Cell volumes, organelle volumes, and organelle volume densities of newly isolated Sertoli cells were similar to those of sectioned cells taken from the 19-day-old in vivo group, indicating that the procedure for isolation does not grossly alter Sertoli cells. Mean height of cells cultured for 7 days was significantly lower than that of cells from intact animals at 19 and 26 days of age. In vivo, Sertoli cells of 26-day-old animals displayed increased organelle volumes and organelle surface areas compared with those from 19-day-old animals; volume densities and surface densities remained relatively constant, indicating that in vivo, organelle growth is in proportion to growth of the cell. Most organelle volume and surface densities were not significantly different when 19-day-old in vivo cells and pre-culture pelleted cells were compared. Many organelle volume and surface density values were significantly less in cells grown in culture for 7 days as compared to freshly isolated pelleted cells. After 7 days of culture, most Sertoli cell organelles were significantly less in both volume density and surface density, as compared to the in vivo cell groups (19 or 26 day). This indicates that in vitro the organelles do not develop in proportion to the growth of the cell. After 7 days in culture, the absolute volumes and surface areas of the organelles remained generally unchanged as compared to cells from 19-day-old animals. The data show that Sertoli cells grow in volume in vitro like their in vivo counterparts; however, their subcellular features, although well maintained, do not develop in proportion to the cell. This suggests that short-term cultures are a more ideal system in which to study biochemical responses. Also, cultured prepubertal Sertoli cells are most appropriately used to study prepubertal Sertoli cell function. This is the first study to quantify developmental changes in Sertoli cell structure in vivo as well as to compare them with cellular changes occurring in vitro.     

Developmental regulation of Sertoli cell aromatase activity and plasminogen activator production by hormones, retinoids and the testicular paracrine factor, PModS.

Testicular peritubular cells produce a paracrine factor termed PModS that has dramatic effects on Sertoli cell function in vitro. The current study was designed to examine the actions of PModS and hormones on Sertoli cell aromatase activity and plasminogen activator production at various stages of pubertal development. Sertoli cells were isolated from 10-, 20-, and 35-day-old rats (ages correspond to prepubertal, midpubertal, and late-pubertal stages of development). Aromatase activity was found to be high and hormone-responsive in prepubertal Sertoli cells and to decline and be nonresponsive to hormones in late-pubertal Sertoli cells. FSH was the only hormone found to influence aromatase activity and estrogen production. PModS alone was not found to affect aromatase activity at any of the developmental stages examined. Interestingly, PModS was found to suppress the ability of FSH to stimulate aromatase activity and estrogen production in midpubertal Sertoli cells. Results imply that PModS may promote Sertoli cell differentiation to a more adult stage of development that is less responsive to FSH in stimulating aromatase activity. In contrast to aromatase activity, plasminogen activator production was found to increase during pubertal development. Production of Sertoli cell tissue-type plasminogen activator (tPa) was stimulated by FSH at each of the developmental stages examined, whereas production of urokinase-type plasminogen activator (uPa) was influenced by FSH only in prepubertal Sertoli cells. Insulin also stimulated uPa and tPa production by prepubertal Sertoli cells, and retinol significantly suppressed uPa production and the ability of FSH to stimulate tPa production by midpubertal Sertoli cells.     

Glycosyl phosphatidylinositol-anchored ceruloplasmin is expressed by rat Sertoli cells and is concentrated in detergent-insoluble membrane fractions.

The copper-binding protein, ceruloplasmin, is both a serum component and a secretory product of Sertoli cells. Studies on serum ceruloplasmin have demonstrated it to be a ferroxidase that is essential for iron transport throughout the body. We report here that a glycosyl phosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed by Sertoli cells. Sertoli cell GPI-anchored proteins were selectively released by phosphatidylinositol-specific phospholipase C and were analyzed by Western blotting. A 135-kDa band was identified as ceruloplasmin by multiple antibody recognition and by amino acid sequence analysis. The presence of the GPI anchor on ceruloplasmin was confirmed by Triton X-114 phase partitioning experiments and by recognition with an antibody to the GPI anchor. GPI-anchored ceruloplasmin was enriched in detergent-insoluble glycolipid-enriched membrane microdomains (DIGs) of Sertoli cells. This is the first report of GPI-anchored ceruloplasmin in Sertoli cells and the first study of GPI-anchored ceruloplasmin in DIGs. We suggest that GPI-anchored ceruloplasmin may be the dominant form expressed by Sertoli cells and that Sertoli cell DIGs may play a role in iron metabolism within the seminiferous tubule.     

Comparative aspects of spermatogenic cell metabolism and Sertoli cell function in Xenopus laevis and mammals.

Due to the relative dearth of data regarding somatic cell-germ cell interactions in the testes of non-mammalian chordates, functional homologies between Sertoli cells from diverse organisms have been difficult to assess. However, recent developments in non-mammalian testis cell and organ culture techniques have provided experimental approaches to compare Sertoli cell-germ cell interactions in different vertebrates. Data from in vitro analyses of Sertoli cell-germ cell interactions are presented to suggest that Sertoli cells from rodents and the frog Xenopus laevis have similarities in supporting energy metabolism and glutathione metabolism in spermatogenic cells. Comparative in vitro analyses of Sertoli cell functions should provide further insights into the evolution of cell-cell interactions in the testes.     

Light and electron microscopic localization of ATPase in normal and degenerating testes of Syrian hamsters.

The distribution of Mg++-activated ATPase was determined with light and electron microscopy in normal and degenerating seminferous tubules. In the normal animals ATPase was localized in the interface between spermatids and Sertoli cells, in association with the cytoplasmic filaments contained within Sertoli cell processes, and in the lymphatic endothelium. ATPase activity increased in degenerating tubules as observed by light microscopy. Electron microscopic investigations of the degenerating tubules which contained only spermatogonia and Sertoli cells revealed reaction product on the outer surface of the Sertoli cell processes and within the interface between adjacent Sertoli cells. Reactaction product was also observed in the Sertoli cell processes between the cytoplasmic filaments and the cell membrane. Where filaments were absent in Sertoli cell processes, no reaction product was observed. These electron microscopic studies indicate that the increase in ATPase activity in testicular degeneration is probably a relative increase due to a loss of the germinal elements of the tubular epithelium and subsequent apposition of the Sertoli cell processes. We speculate that the ATPase activity localized within the Sertoli cell processes may be involved in providing an energy source for filament motility.     

Intermediate-sized filaments present in Sertoli cells are of the vimentin type.

The cytoplasmic structure of Sertoli cells of rat testes has been studied by electron microscopy of ultrathin sections. Sertoli cells contain numerous intermediate-sized (7-11 nm) filaments which form a meshwork extending throughout the whole cytoplasm. Often the frequency of such filaments appears especially high in juxtanuclear and cortical regions, including the apical recesses containing the spermatids. Examination of frozen sections of testes by indirect immunofluorescence microscopy using guinea pig antibodies to prekeratin and vimentin has shown the absence of intermediate-sized filaments of the cytokeratin type in all cells of the testes but the presence of filaments of the vimentin type in Sertoli cells as well as in cells of the interstitial space. These results show that the intermediate-sized filaments, abundant in Sertoli cells, are of the vimentin type. In addition we conclude that the "germ epithelium" differs from others true epithelia by the absence of cytokeratin filaments and typical desmosomes and, in Sertoli cells, the presence of vimentin filaments, suggestive of a mesenchymal character or derivation.     

Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfide-linked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein. Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis.