Morphological changes of Sertoli cells during the male reproductive cycle of the teleost Piaractus mesopotamicus (Holmberg, 1887).

An investigation of the histological and ultrastructural changes of Sertoli cells during the male reproductive cycle in Piaractus mesopotamicus was made. The results showed that the Sertoli cell development is closely related with germ cell maturation. Therefore, these cells may have some role in germ cell maturation during the reproductive cycle of this species, whether in forming a tissue framework for the developing spermatogenic cysts, aiding in testes reorganization for a new reproductive cycle, in addition to other possible functions discussed in the text.     

Sertoli cell proliferation in the adult testis--evidence from two fish species belonging to different orders

Germ cell survival and development critically depend on the cells' contact with Sertoli cells in the vertebrate testis. Fish and amphibians are different from mammals in that they show a cystic type of spermatogenesis in which a single germ cell clone is enclosed by and accompanied through the different stages of spermatogenesis by an accompanying group of Sertoli cells. We show that in maturing and adult testes from African catfish and Nile tilapia, Sertoli cell proliferation occurs primarily during spermatogonial proliferation, allowing the cyst-forming Sertoli cells to provide the increasing space required by the growing germ cell clone. In this regard, coincident with a dramatic increase in cyst volume and number of germ cells per cyst, in Nile tilapia, the number of Sertoli cells per cyst was strikingly increased from primary spermatogonia to spermatocyte cysts. In both African catfish and Nile tilapia, Sertoli cell proliferation is strongly reduced when germ cells have proceeded into meiosis, and stops in postmeiotic cysts. We conclude that Sertoli cell proliferation is the primary factor responsible for the increase in testis size and sperm production observed in teleost fish. In mammals, Sertoli cell proliferation in the adult testis is not observed under natural conditions. However, on the level of the individual spermatogenic cyst--similar to mammals--Sertoli cell proliferation ceases when germ cells have entered meiosis and when tight junctions are established between Sertoli cells. This suggests that fish are valid vertebrate models for studying Sertoli cell physiology.     

Role of β-catenin in post-meiotic male germ cell differentiation

Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation.     

Cell junctions in the germinal epithelium may play an important role in spermatogenesis of the catfish P. fasciatum (Pisces, Siluriformes)

We identified adhesive junctions and gap junctions between Sertoli cells, between Sertoli and germ cells and between germ cells in the testis of P. fasciatum, a catfish of commercial relevance. To investigate the role of these junctions in spermatogenesis, as well as the molecular composition of the junctions, we performed an immunohistochemistry light microscopy as well as an immunogold labelling electron microscopy study with antibodies to adhesive and gap junctions proteins. Testes that were at different stages of spermatogenesis were used. Based on our morphological studies we speculate that Sertoli–germ and germ–germ cell adhesive junctions are important for maintaining the three-dimensional structure of the germinal cysts and an organized arrangement of the germ cells inside the cysts. Connexin 32 was identified in the germ cells and in the cysts walls. Our observations also suggest that Sertoli–germ and germ–germ cells gap junctions may be involved in the mechanism of synchronous development of germ cells.     

Paracrine regulation of testicular function

Data from several experimental approaches have been reviewed and the findings clearly indicate the existence of multiple interactions between testicular cells and the potential role of these interactions in the paracrine control of testicular functions. Both testicular interstitial fluid and spent media from cultured Sertoli cells had an acute steroidogenic effect on Leydig cells, and this effect is not species specific. The secretion of this steroidogenic factor(s), which is probably a protein, is enhanced by previous FSH treatment of Sertoli cells. Coculture for 2-3 days of pig Leydig cells with homologous or heterologous Sertoli cells enhances Leydig cell specific functions (hCG receptor number and hCG responsiveness) and induces Leydig cell hypertrophy. A similar but less pronounced trophic effect is seen when Leydig cells are cultured with spent media from Sertoli cells cultured in the presence of FSH and high concentrations of insulin, but the spent media from Sertoli cells cultured in the absence of these two hormones inhibits Leydig cell specific functions. Somatomedin-C might play an important role in the positive trophic effect of Sertoli cells on Leydig cells, since this peptide is secreted by Sertoli cells and it has trophic effects on the specific function of Leydig cells. Moreover, Sertoli cells, probably through a diffusible factor and cell-to-cell contacts, control the multiplication, meiotic reduction and maturation of germ cells. In turn, the activity of Sertoli cells is modulated by the stage of neighbouring germ cells. Thus, if a normal Sertoli cell function (which depends not only on FSH but also on Leydig and myoid cell secretory products) is an absolute requirement for germ cell multiplication and maturation, these cells, in turn, cyclically regulate Sertoli cell function and through these cells the size and probably the function of Leydig cells.     

KCNQ1/KCNE1 channels during germ-cell differentiation in the rat: expression associated with testis pathologies

KCNQ1/KCNE1 channels are responsible for the Jervell-Lange-Nielsen cardiac syndrome, which is also characterized by congenital deafness. KCNQ1/KCNE1 is crucial for K+ transport in the inner ear. We show that KCNQ1 and KCNE1 are associated in testis and that their expression is closely regulated during development. Both genes were expressed in undifferentiated germ cells in 21-day-old rats and mostly confined to basal immature germ cells in adulthood. Leydig and Sertoli cells were negative. KCNQ1 and KCNE1 were also studied in various germ-cell pathologies. First, in spontaneous unilateral rat testis atrophy, hematoxylin-eosin analysis revealed massive germ-cell aplasia with only Sertoli cells and groups of interstitial Leydig cells. In these samples, KCNQ1 and KCNE1 were not expressed. In human seminoma samples characterized by a proliferation of undifferentiated germ cells, KCNQ1/KCNE1 protein levels were higher than in healthy samples. Our results demonstrate that the expression of KCNQ1 and KCNE1 is associated with early stages of spermatogenesis and with the presence of undifferentiated healthy or neoplastic germ cells. The presence of a K+ rich-fluid in the seminiferous tubule suggests that KCNQ1/KCNE1 is involved in K+ transport, probably during germ-cell development.     

Observations on rat sertoli ectoplasmic (‘junctional’) specializations in their association with germ cells of the rat testis

The ectoplasmic (‘junctional’) specialization, a subsurface modification of the Sertoli cell that is often seen facing germ cells, was studied in relation to the development and maturation of these germ cells. This structure is composed of sub-surface bundles of filaments and more deeply placed endoplasmic reticulum. The data indicate that these subsurface modifications of Sertoli cells are reutilized in a cyclic fashion, being transferred from their position facing late spermatids to one opposing less mature germ cells. Ectoplasmic specializations appeared to function mechanically in grasping the heads of the spermatids which are undergoing the elongation and maturation phases of spermiogenesis rather than in actually attaching Sertoli cells to these germ cells. It is postulated that the ectoplasmic specialization imparts rigidity to that area of the Sertoli cell that surrounds the head region of the germ cell, forming a recess and a mantle by which the germ cell may be moved toward the base or toward the surface of the seminiferous epithelium. The observed linkage of microtubules to the cisternae of the complex provided a morphological basis for the changes in the cytoarchitecture of the Sertoli cell, which must accompany these movements.     

An ultrastructural study of the effect of a diabetogenic dose of alloxan on the secretory ameloblasts of the rat incisor

Sertoli cells of the ground squirrel (Spermophilus lateralis), a seasonal breeder, were examined by light and electron microscopy and their structure, particularly the organization of the cytoskeleton, was related to events that occur in the seminiferous epithelium during spermatogenesis. Among the events considered and described are the apical movement of elongate spermatids, withdrawal of residual cytoplasm from germ cells, transport of smooth endoplasmic reticulum (SER) between the base and apex of the Sertoli cells, and sperm release. These events are dramatically evident in this species because the seminiferous epithelium is thin, i.e., there are few germ cells, and both the germ cells and Sertoli cells are large. Sertoli cells of the ground squirrel have a remarkably well developed cytoskeleton. Microfilaments occur throughout the cell but are most evident in ectoplasmic specializations associated with junctions. Intermediate filaments occur around the nucleus, as a layer at the base of the cell, and adjacent to desmosome-like junctions with germ cells. Intermediate filaments, together with microtubules, are also abundant in regions of the cell involved with the transport of SER, in cytoplasm associated with elongate spermatids, and in processes that extend into the residual cytoplasm of germ cells. Our observations of ultrastructure are consistent with the hypothesis that Sertoli cell microtubules are involved with the movement of germ cells within the seminiferous epithelium, and further implicate these structures as possibly playing a role in the retraction of residual cytoplasm from germ cells and the intracellular transport of SER. The abundance and organization of intermediate filaments suggest that these cytoskeletal elements may also be involved with events that occur during spermatogenesis.     

The amphibian testis as model to study germ cell progression during spermatogenesis

Testicular morphology of vertebrate testis indicates requirement of local control. In urodeles, the testis is organized in lobes of increasing maturity throughout the cephalocaudal axis. The anuran testis is organized in tubules. Spermatogenesis occurs in cysts composed by Sertoli cells enveloping germ cells at synchronous stages. Moreover, in numerous species germ cell progression lasts a year which defines the sexual cycle. Due to the above quoted features, research on factors regulating germ cell progression in amphibians may reach greater insight as compared with mammalian animal models. In particular, studies on endocrine and paracrine/autocrine factors involved in the regulation of germ cell functions reveal that fos activation and a J protein, previously specifically found in mouse testis, exert an important role in spermatogonial proliferation and maturation of post-meiotic stages, respectively.     

Apoptosis during spermatogenesis in the spotted ray Torpedo marmorata

This article is a cytological and molecular investigation on the occurrence of apoptosis during spermatogenesis in Torpedo, a cartilaginous fish characterised by a typical cystic testis. Using DNA fragmentation and Bak gene expression, it demonstrated that germ cells undergo apoptosis only at the stages of spermatocyte and spermatid, and degeneration also involves Sertoli but not Leydig cells. In immature cysts, this cellular process probably occurs when the ratio of germ cells to the only Sertoli cell (SC) forming the spermatoblast changes. Apoptosis also takes place in mature cysts after sperm release to eliminate most of the SCs. Few of them, however, become cytoplasts and probably continue secreting androgens so as to control the final events of spermatogenesis, i.e., passage of spermatozoa through the ductus deferentes. Finally, the present investigation demonstrated that, in Torpedo testis, Bak mRNA is expressed during spermatogenesis, thus suggesting that the mitochondrial pathway might be active. This observation in one of the oldest vertebrate classes indicates that, in all vertebrates, the apoptotic process during spermatogenesis is conserved, contributing to testicular homeostasis.     

A light and electron microscopic study on the organization of the testis and the semicystic spermatogenesis of the genus Scorpaena (Teleostei,Scorpaenidae)

The testicular organization and semicystic spermatogenesis of Scorpaena porcus and Scorpaena scrofa are analyzed by means of optic and electron microscopy and immunohistochemical techniques. The testicular structure of S. porcus and S. scrofa belongs to the unrestricted spermatogonial type, but has typical features of the restricted type. Moreover, the structure presents an epithelioid arrangement of Sertoli and germ cells rather than the germinal epithelium that appears in the majority of teleosts. After the cysts open, Sertoli cells hypertrophy and remain on the basement membrane, linked by interdigitations and tight junctions and bordering the lumen of the lobule, which at this moment works as an efferent duct. Secretions of Sertoli cells usually function in the nutrition of germ cells, and they seem to contribute in it even in this kind of spermatogenesis in which the free lumen spermatids do not have any connection with Sertoli cells. In addition, Sertoli cells can divide after the cysts have broken apart and hypertrophied, suggesting that they are still important for the final maturation of spermatozoa and seminal fluid formation. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Hormonal regulation of spermatogenesis and spermiogenesis

Normal testicular function is dependent upon hormones acting through endocrine and paracrine pathways both in vivo and in vitro. Sertoli cells provide factors necessary for the successful progression of spermatogonia into spermatozoa. Sertoli cells have receptors for follicle stimulating hormone (FSH) and testosterone which are the main hormonal regulators of spermatogenesis. Hormones such as testosterone, FSH and luteinizing hormone (LH) are known to influence the germ cell fate. Their removal induces germ cell apoptosis. Proteins of the Bcl-2 family provide one signaling pathway which appears to be essential for male germ cell homeostasis. In addition to paracrine signals, germ cells also depend upon signals derived from Sertoli by direct membrane contact. Somatostatin is a regulatory peptide playing a role in the regulation of the proliferation of the male gametes. Activin A, follistatin and FSH play a role in germ cell maturation during the period when gonocytes resume mitosis to form the spermatogonial stem cells and differentiating germ cell populations. In vitro cultures systems have provided evidence that spermatogonia in advance stage of differentiation have specific regulatory mechanisms that control their fate. This review article provides an overview of the literature concerning the hormonal pathways regulating spermatogenesis.     

Sperm development in the teleost Oryzias latipes

Summary In Oryzias latipes the processes of spermatogenesis and spermiogenesis occur within testicular or germinal cysts which are delimited by a single layer of lobule boundary cells. These cells, in addition to comprising the structural component of the cyst wall, ingest residual bodies cast off by developing spermatids. Therefore, they are deemed to be the homologue of mammalian Sertoli cells. The germ cells within a cyst develop synchronously owing to the presence of intercellular bridges connecting adjacent cells. Since bridges also connect spermatogonia, it seems probable that all of the germ cells within a cyst may form a single syncytium and do not exist as individual cells until the completion of spermiogenesis when the residual bodies are cast off. Significant differences between spermiogenesis in O. latipes and in the related poeciliid teleosts are discussed.     

The sertoli cell--a hormonal target and 'super' nurse for germ cells that determines testicular size

The somatic Sertoli cell plays an essential role in embryonic determination of male somatic sex and in spermatogenesis during adult life. One individual Sertoli cell supplies a clone of developing germ cells with nutrients and growth factors and it is well established that the number of Sertoli cells present is closely correlated to both testicular size and sperm output. Sertoli cells continue to proliferate and differentiate until the beginning of puberty, when they cease dividing and start nursing the germ cells. At this point in time, the future capacity of the testis for sperm production has thus been determined. Prior to puberty the Sertoli cells are immature and differ considerably with respect to morphology and biochemical activity from the mature cell. The several investigations that have focused on hormonal and paracrine regulation of the functions of the mature cell are reviewed here, but the mechanisms underlying the maturation and general maintenance of well-functioning Sertoli cells remain obscure. An alarming decline in male reproductive health has been observed in several Western countries during recent decades. Disturbance of Sertoli cell differentiation is thought to be involved in the pathogenesis of both a poor sperm count and testicular cancer. It is speculated that environmental agents that disrupt the estrogenic/androgenic balance in the testis may play a role in this connection.     

Connexin 43 a potential regulator of cell proliferation and apoptosis within the seminiferous epithelium

The gap junction proteins, connexins (Cx), are present in the testis and among them Cx43 play an essential role in spermatogenesis. By using an in vitro proliferation model of germ cells and Sertoli cells, we tempted here to clarify the role of Cx43 in the control of Sertoli and germ cell proliferation and apoptosis. Cx43 was detected in purified preparations of Sertoli cells and spermatogonia and immunolocalized in both cell types identified by vimentin and c-kit, respectively. Inhibition of gap junction coupling by the gap junction inhibitor α-GA significantly enhanced BrdU incorporation in Sertoli cells and reduced the number of activated caspase-3 positive germ cells. Similarly, inhibitory Cx43 and pan-Cx mimetic inhibitory peptides increased proliferation of Sertoli cells and stimulated survival of germ cells. Cx32 mimetic inhibitory peptide also stimulated Sertoli cell proliferation without altering germ cell proliferation and apoptosis. The present results reveal that Cx43 gap junctions between Sertoli cells participate in the control of Sertoli cell proliferation and that Cx43 gap junctions between Sertoli cells and spermatogonia are indirectly involved in germ cell number increase by controlling germ cell survival rather than germ cell proliferation.     

Developmental delay and unstable state of the testes in the rdw rat with congenital hypothyroidism

From the present study of the rdw rat, it is clear that the thyroid hormone is essential for the development and maintenance of the testes. In previous studies, the thyroid hormone has few serious effects on the testes except during the neonatal stage when the thyroid hormone receptor is mainly present. However, there is little knowledge concerning the prolonged effect of thyroid hormone deficiency throughout the rat's life span. In the present study, a morphological analysis was performed on the testes of rdw rats with congenital hypothyroidism. The rdw testes required a longer time to develop into the normal adult structure. Moreover, the developed, normal structure began to degenerate after full maturation. Specific characteristics of the rdw testes include: (i) a prolonged proliferation of Sertoli cells during postnatal development; (ii) a developmental delay in the appearance of spermatocytes and spermatid; (iii) direct contact with each other for both spermatocytes and spermatids, without Sertoli cell cytoplasm completely intervening between adjacent germ cells; (iv) subsequent apoptosis of germ cells after maturation; (v) reduction in the height of the seminiferous epithelium; and (vi) lower testosterone levels in the rdw rats, especially during old age. Thus, we conclude that the thyroid hormone plays an important role in developing and maintaining normal function of testes.     

Histological, histochemical, enzyme histochemical and ultrastructural investigations of the testis of Padogobius martensi between annual breeding seasons

From July to March, the testis of the spring‐spawning freshwater goby Padogobius martensi is characterized by spermatogonial proliferation. A close correlation exists among type of proliferating spermatogonia, gonado‐somatic (I_G) profiles and morphological and functional variations of the Leydig cells. The I_G reach their minimal levels by the end of summer and increase progressively but modestly during autumn and winter. Declining I_G levels are associated with proliferation of primary spermatogonia only, whereas increasing I_G levels are associated with predominant proliferation of secondary spermatogonia. Minimal I_G levels are reached when the germinal epithelium is formed by a continuum of primary spermatogonia and associated Sertoli cells. The proliferation of secondary spermatogonia begins only at this time. Spermatogenesis in autumn occurs when spermatogonial cysts contain at the most 16 cells and it rarely results in the maturation of several cysts so that the amount of sperm cells produced is either negligible or scarce. A number of degenerating cells are usually present within the spermatogonial and meiotic cysts. Leydig cells are the unique cells that display features of steroidogenic cells: mitochondria with tubular cristae, extensive smooth endoplasmic reticulum (SER), 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and glucose‐6‐phosphate dehydrogenase (G6PD) activity and sudanophilia. Light and dark Leydig cell varieties are always present. During regression, Leydig cells undergo a marked decrease in SER amount, mitochondrial sizes and number of mitochondrial cristae. In parallel, the 3β‐HSD and G6PD activities and sudanophilia decrease progressively until they become undetectable by the end of regression. In autumn, mitochondria increase in size, reaching sizes similar to those observed at the end of the spawning season in the light cells, but not in the dark cells. The SER, on the contrary, undergoes a modest and irregular increase only in a part of the Leydig cells, mostly of the light type. In parallel, the 3β‐HSD and G6PD activities increase until they become moderately intense by the end of autumn. At the end of winter, the SER is extensive and regularly dilated in both Leydig cell types, whereas mitochondria still have sizes similar to those observed in December. The 3β‐HSD and G6PD activities are strong and sudanophilia is again detectable. Sertoli cells undergo changes in shape and position in relation to the proliferation of primary spermatogonia and the development of cysts. A junction modulation occurs in association with these changes. Sertoli cells also undergo changes indicative of a decrease in activity immediately after spawning (loss of mitochondrial cristae and clarification of the mitochondrial matrix) and of an increase in activity by the end of the regressing phase (darkening of the mitochondrial matrix and increase in mitochondrial cristae, rough endoplasmic reticulum (RER) and free ribosomes). In addition, they are involved in the phagocytosis of degenerating germ cells at all stages of their development. Macrophages are found in the testis interstitium only, where they are usually adjacent to Leydig cells, myoid cells and blood capillaries and do not participate in the phagocytosis of degenerating germ cells. Myoid cells do not undergo ultrastructural changes except for an increase in the amount of heterochromatin by the end of spawning. The meaning of the autumnal spermatogenic wave and the relationships between the development of the germinal epithelium and the changes of the Leydig and Sertoli cells are discussed.