Dysplastic development of seminiferous tubules and interstitial tissue in rat hypogonadic (hgn/hgn) testes

The hypogonadic rat is characterized by male sterility, reduced female fertility, and renal hypoplasia controlled by a single recessive allele (hgn) on chromosome 10. Plasma testosterone is low and levels of gonadotropins are high in adult male hgn/hgn rats, indicating that the cause of hypogonadism lies within the testis itself. We found that the postnatal growth of the seminiferous tubules was severely affected. Here we describe the details of postnatal testicular pathogenesis of the hgn/ hgn rats. In these rats, gonadal sex determination and initial differentiation of each type of testicular cell occur, but proliferation, differentiation, and maturation of these cells during postnatal testicular development is severely affected. Postnatal pathological changes include reduced proliferation and apoptotic cell death of Sertoli cells, abnormal mitosis and cell death of gonocytes, reduced deposition of extracellular matrix proteins into the basal lamina, lack of the formation of an outer basal lamina, formation of multiple layers of undifferentiated peritubular cells, and the delayed appearance and islet conformation of adult-type Leydig cells. Apoptotic cell death of Sertoli cells and disappearance of FSH receptor mRNA expression indicate that this mutant rat is a useful model for Sertoli cell dysfunction. The abnormalities listed above might be caused by defective interactions between Sertoli cells and other types of testicular cells. Because the results presented here strongly indicate that a normal allele for hgn encodes a factor playing a critical role in testicular development, the determination of the gene responsible for hgn and the analysis of early alterations of gene expression caused by mutations in this gene would provide important information on the mechanisms of testicular development.     

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.     

Up-regulation of WNT-4 signaling and dosage-sensitive sex reversal in humans

Wnt-4, a member of the Wnt family of locally acting secreted growth factors, is the first signaling molecule shown to influence the sex-determination cascade. In mice, a targeted deletion of Wnt-4 causes the masculinization of XX pups. Therefore, WNT-4, the human homologue of murine Wnt-4, is a strong candidate gene for sex-reversal phenotypes in humans. In this article, we show that, in testicular Sertoli and Leydig cells, Wnt-4 up-regulates Dax1, a gene known to antagonize the testis-determining factor, Sry. Furthermore, we elucidate a possible mechanism for human XY sex reversal associated with a 1p31-p35 duplication including WNT-4. Overexpression of WNT-4 leads to up-regulation of DAX1, which results in an XY female phenotype. Thus, WNT-4, a novel sex-determining gene, and DAX1 play a concerted role in both the control of female development and the prevention of testes formation. These observations suggest that mammalian sex determination is sensitive to dosage, at multiple steps in its pathway.     

A reassessment of Y chromosomal behaviour in germ cells and Sertoli cells of the mouse as revealed by in situ hybridisation

In situ hybridisation experiments were carried out to reappraise the state of condensation of the Y chromosome in germ cells and Sertoli cells of the mouse. Previous work had suggested that all testicular cells showed a condensed Y chromosome prior to the adult stage. We now demonstrate that, although the Y chromosome is condensed in pre-pubertal Sertoli cells, it is greatly expanded in primordial germ cells (gonocytes). An expanded Y-signal is first seen in Sertoli cell nuclei at or around day 21 of postnatal development, coinciding with the first appearance of spermatids in the germinal epithelium.     

An overview of cell renewal in the testis throughout the reproductive cycle of a seasonal breeding teleost, the gilthead seabream (Sparus aurata L)

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the testicular regression process that occurs in both seasonal testicular involution and sex change. Insofar as fish reproduction is concerned, little is known about cell renewal and elimination during the reproductive cycle of seasonal breeding teleosts with asynchronous spermatogenesis. We have previously described how acidophilic granulocytes infiltrate the testis during postspawning where, surprisingly, they produce interleukin-1beta, a known growth factor for mammalian spermatogonia, rather than being directly involved in the elimination of degenerative germ cells. In this study, we are able to discriminate between spermatogonia stem cells and primary spermatogonia according to their nuclear and cytoplasmic diameters and location in the germinal epithelium, finding that these two cell types, together with Sertoli cells, proliferate throughout the reproductive cycle with a rate that depends on the reproductive stage. Thus, during spermatogenesis the spermatogonia stem cells, the Sertoli cells, and the developing germ cells (primary spermatogonia, A and B spermatogonia, and spermatocytes) in the germinal compartment, and cells with fibroblast-shaped nuclei in the interstitial tissue proliferate. However, during spawning, the testis shows few proliferating cells. During postspawning, the resumption of proliferation, the occurrence of apoptotic spermatogonia, and the phagocytosis of nonshed spermatozoa by Sertoli cells lead to a reorganization of both the germinal compartment and the interstitial tissue. Finally, the proliferation of spermatogonia increases during resting when, unexpectedly, both oogonia and oocytes also proliferate. This proliferative pattern was correlated with the gonadosomatic index, testicular morphology, and testicular and gonad areas, suggesting that complex mechanisms operate in the regulation of gonocyte proliferation in hermaphrodite fish.     

Chemotactic role of neurotropin 3 in the embryonic testis that facilitates male sex determination

The first morphological event after initiation of male sex determination is seminiferous cord formation in the embryonic testis. Cord formation requires migration of pre-peritubular myoid cells from the adjacent mesonephros. The embryonic Sertoli cells are the first testicular cells to differentiate and have been shown to express neurotropin-3 (NT3), which can act on high-affinity trkC receptors expressed on migrating mesonephros cells. NT3 expression is elevated in the embryonic testis during the time of seminiferous cord formation. A trkC receptor tyrophostin inhibitor, AG879, was found to inhibit seminiferous cord formation and mesonephros cell migration. Beads containing NT3 were found to directly promote mesonephros cell migration into the gonad. Beads containing other growth factors such as epidermal growth factor (EGF) did not influence cell migration. At male sex determination the SRY gene promotes testis development and the expression of downstream sex differentiation genes such as SOX-9. Inhibition of NT3 actions caused a reduction in the expression of SOX-9. Combined observations suggest that when male sex determination is initiated, the developing Sertoli cells express NT3 as a chemotactic agent for migrating mesonephros cells, which are essential to promote embryonic testis cord formation and influence downstream male sex differentiation.     

In vitro studies of gonadal organogenesis in the presence and absence of H-Y antigen.

In a very strict sense, the primary (gonadal) sex of mammals is determined not so much by the presence or absence of the Y but the expression or nonexpression of the evolutionary extremely conserved plasma membrane H-Y antigen. The central somatic blastema of embryonic indifferent gonads contains one cell lineage characterized by the possession of S-F differentiation antigen that differentiates into testicular Sertoli cells in the presence of H-Y and into ovarian follicular (granulosa) cells in its absence. This cell lineage appears to play the most critical role in gonadal differentiation. Whether or not testicular Leydig cells and ovarian theca cells are similarly derived from the common cell lineage has not been determined. Nevertheless, if given H-Y antigen, presumptive theca-cell precursors of the fetal ovary acquire hCG (LH?)-receptors-the characteristic of fetal Leydig cells.     

Ultrastructure of Sertoli-cell penetrating processes found in germ cells of the golden-mantled ground squirrel (spermophilus lateralis)

We have studied the ultrastructure of Sertoli-cell processes that extend into developing germ cells of the ground squirrel (Spermophilus lateralis). In other mammals it is speculated that these processes anchor germ cells to the seminiferous epithelium and transfer materials between Sertoli and germ cells. In the ground squirrel, Sertoli-cell projections first appear in round spermatids and consist of regions containing numerous mitochondria and intermediate filaments together with areas composed mainly of a fine filamentous matrix. Also present are what may be desmosomelike junctions with adjacent germ cells. During spermatogenesis, numerous changes in the penetrating processes and their internal composition occur. Especially significant are those occurring during the movement of residual cytoplasm basally over spermatid heads: some Sertoli-cell processes contain microtubules, mitochondria, and vesicular elements, but also present are regions that lack organelles and appear simply as thin lamellae of cytoplasm that line cavernous invaginations of the germ cell. Coated vesicles and pits are present in processes and adjacent germ-cell regions at all stages of spermatogenesis. Our observations are consistent with the suggestions that Sertoli-cell processes have an attachment function and that they also may facilitate the movement of residual cytoplasm into the epithelium. Further, they indicate that these structures might be involved with receptor-mediated edocytosis.     

Sex differentiation in mammals and tempo of growth: probabilities vs. switches

In the conventional model of sex differentiation in placental mammals, a switch is envisaged to steer the indifferent gonad into the path of either testicular or ovarian development. The immediate cause of the switch is thought to be the presence or absence of Sertoli cells, which in turn is controlled by the presence or absence of the testis-determining factor on the Y chromosome (TDF in humans, Tdy in mice). Quantitative investigations indicate, however, that the rate of growth of XY gonads is faster than that of XX gonads before the formation of Sertoli cells, and furthermore, that XY embryos develop faster than XX embryos long before the formation of gonadal ridges. Since the genetic constitution of the sex chromosomes appears to manifest itself from the earliest embryonic stages onwards, the concept of indifferent gonads being switched into alternate pathways becomes inappropriate. A model is proposed in which gonadal differentiation depends on developmental thresholds: the formation of Sertoli cells needs to occur by a particular stage in time in a sufficiently developed gonad, failing which the gonad will enter the ovarian pathway. While TDF is the principal factor enhancing the rate of gonadal growth, other factors which influence development rates can modulate the probability of a gonad becoming either a testis or an ovary.     

Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX----XY chimaeric mouse testes

The distribution of XX and XY cells in XX----XY chimaeric mouse testes was analysed by enzyme marker analysis of separated testicular tissues and by in situ DNA marker analysis of air-dried testicular cells and testis sections. XX cells contributed to the Leydig cells, the peritubular cells and the vascularized connective tissue of the tunica albuginea. The Sertoli cells, on the other hand, appeared to be exclusively XY. These results indicate that during the development of the testis, Sertoli cell differentiation is triggered by cell-autonomous activity of the Y chromosomal testis-determining gene Tdy. Subsequent steps in testis differentiation may be a consequence of Sertoli cell activity.     

Toll-like receptor 3 activation induces antiviral immune responses in mouse sertoli cells

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and elicit antimicrobial immune responses. In the testis, viruses can induce pathological conditions, such as orchitis, and may participate in the etiology of testicular cancer; however, the molecular mechanisms involved remain under investigation. It has been suggested that because they constitutively express interferon (IFN)-inducible antiviral proteins, Sertoli cells participate in the testicular antiviral defense system. Previously, we demonstrated a key function of mouse Sertoli cells in the bactericidal testicular defense mechanism mediated by a panel of TLRs. To better characterize the potential role of Sertoli cells in the response against testicular viral infections, we investigated the TLR3 expression and function in these cells. Sertoli cells express TLR3, and under stimulation with the synthetic double-stranded RNA analogue poly (I:C), they produce the proinflammatory molecule ICAM1 and secrete functionally active CCL2 chemokine. Using both pharmacological and genetic approaches, we found that these effects are TLR3-dependent. Moreover, using ELISA, we found that IFNA is constitutively produced and not further inducible, whereas IFNB1 is absent and dramatically induced only by transfected poly (I:C), indicating different control mechanisms underlying IFNA and IFNB1 production. To conclude, poly (I:C) elicits both inflammatory and antiviral responses in Sertoli cells.     

Effects of androgen receptor mutation on testicular histopathology of patient having complete androgen insensitivity

Androgens are required for normal male sex differentiation and development of male secondary sexual characteristics. Mutations in AR gene are known to cause defects in male sexual differentiation. In current study, we enrolled a 46,XY phenotypically female patient bearing testes in inguinal canal. DNA sequencing of the AR gene detected a missense mutation C.1715A?>?G (p. Y572C) in exon 2 which is already known to cause complete androgen insensitivity syndrome (CAIS). We focused on the effects of this mutation on the testicular histopathology of the patient. Surface spreading of testicular tissues showed an absence of spermatocytes while H&E staining showed that seminiferous tubules predominantly have only Sertoli cells. This meiotic failure is likely due to the effect of the AR mutation which ultimately leads to Sertoli cell only syndrome. Tubules were stained with SOX9 and AMH which revealed Sertoli cells maturation arrest. Western blot and realtime PCR data showed that patient had higher levels of AMH, SOX9 and inhibin-B in the testis. Therefore, we suggest that the dysfunctioning of AR by mutation enhances AMH expression which ultimately leads to the failure in maturation of Sertoli cells.     

The role of tumor necrosis factor-alpha and interleukin-1 in the mammalian testis and their involvement in testicular torsion and autoimmune orchitis

This review will focus the roles of TNF-alpha, IL-1 alpha, and IL-1 beta in the mammalian testis and in two testicular pathologies, testicular torsion and orchitis. TNF alpha in the testis is produced by round spermatids, pachytene spermatocytes, and testicular macrophages. The type 1 TNF receptor has been found on Sertoli and Leydig cells and numerous studies suggest a paracrine mode of action for TNF alpha in the normal testis. IL-1 alpha has been reported to be produced by Sertoli cells, testicular macrophages, and possibly postmeiotic germ cells. IL-1 receptors have been reported on Sertoli cells, Leydig cells, testicular macrophages, and germ cells suggesting both autocrine and paracrine functions. While these proinflammatory cytokines have important roles in normal testicular homeostasis, an elevation of their expression can lead to testicular dysfunctions. Testicular torsion is a clinical pathology with results in testicular ischemia and surgical intervention is often required for reperfusion. A pivotal role for IL-1beta in the pathology of testicular torsion has been recently described whereby an increase in IL-1beta production after reperfusion of the testis is correlated with the activation of the stress-related kinase, c-jun N-terminal kinase, and ultimately resulting in neutrophil recruitment to the testis and germ cell apoptosis. In autoimmune orchitis, on the other hand, TNF alpha produced by T-lymphocytes and macrophages of the testis has been implicated in the development and progression of the disease. Thus, both proinflammatory cytokines, TNF alpha and IL-1, have significant roles in normal testicular functions as well as in certain testicular pathologies.     

Aromatase in the human testis

Low levels of testicular estrogen synthesis have been reported in a number of species, but the cellular localization has not been unequivocally established. To study aromatase in the human testis, we have combined immunocytochemistry with direct measurement of enzyme activity in the testicular 6μm cryosections. Thus, the functionality of the immunoreaction and its sensitivity can be assessed in quantitative terms. Testes were obtained from immediate autopsy from men aged 18–53 years, from surgery from two patients with prostatic cancer (67 and 74 years) and from two normal children aged 8 months and 3 years at autopsy. Benign testicular sex cord tumors were also examined from two unrelated patients aged 5 and 8 years with gynecomastia and diagnosed with Peutz-Jeghers syndrome. Our results consistently showed low to moderate staining intensity of immunoreactive aromatase in comparison to that of normal human placental cryosections. Immunoreactive aromatase was only present in the interstitial Leydig cells and absent from the Sertoli cells of all normal adult testes showing spermatogenesis. Aromatase activity correlated well with the intensity of the immunostain. However, there was no obvious relationship between the level of aromatase activity and increasing age. Generally higher levels were present in testes of young men (18–22 years). No immunostain in any cell type was detected in one 33-year-old patient with testicular cancer. In the testes of the two normal prepubertal boys, no immunostaining was observed. However, intensely stained Sertoli cells as well as high aromatase activity were observed in the testicular tumors of the patients with Peutz-Jeghers syndrome. Our results suggest that Leydig cells are the source of aromatase in normal men but that Sertoli cells may express this enzyme under abnormal conditions. The combined methods for measuring enzyme activity and immunoreactive aromatase are suitable for application to tissues expressing low levels of aromatase.     

Regulation of human SRY subcellular distribution by its acetylation/deacetylation

SRY, a Y chromosome-encoded DNA-binding protein, is required for testis organogenesis in mammals. Expression of the SRY gene in the genital ridge is followed by diverse early cell events leading to Sertoli cell determination/differentiation and subsequent sex cord formation. Little is known about SRY regulation and its mode of action during testis development, and direct gene targets for SRY are still lacking. In this study, we demonstrate that interaction of the human SRY with histone acetyltransferase p300 induces the acetylation of SRY both in vitro and in vivo at a single conserved lysine residue. We show that acetylation participates in the nuclear localisation of SRY by increasing SRY interaction with importin beta, while specific deacetylation by HDAC3 induces a cytoplasmic delocalisation of SRY. Finally, by analysing p300 and HDAC3 expression profiles during both human or mouse gonadal development, we suggest that acetylation and deacetylation of SRY may be important mechanisms for regulating SRY activity during mammalian sex determination.