The role of dynamin 3 in the testis

We report here that dynamin 3 in the testis is associated with structures termed tubulobulbar complexes that internalize intact intercellular junctions during sperm release and turnover of the blood-testis barrier. The protein lies adjacent to an actin-Arp2/3 network that cuffs the double plasma membrane tubular invagination at the core of each complex. To explore the possible relationship between dynamin 3 and nectin-based adhesion junctions, we transiently transfected DsRed-tagged dynamin 3 into MDCK cells stably transfected with eGFP-tagged nectin 2, one of the adhesion molecules known to be expressed in Sertoli cells at adhesion junctions. Cells transfected with the dynamin 3 construct had less uniformly distributed nectin 2 at intercellular contacts when compared to control cells expressing only nectin 2 or transfected with the DsRed plasmid alone. Significantly, tubular extensions positive for nectin 2 were visible projecting into the cells from regions of intercellular contact. Our findings are consistent with the conclusion that dynamin 3 is involved with tubulobulbar morphogenesis. Dynamin 3 also occurs in concentrated deposits around the capitulum and striated columns in the connecting piece of sperm tails suggesting that the protein in these cells may function to stabilize the base of the tail or serve as a reservoir for use during or after fertilization.     

Immunoexpression of Tyro 3 family receptors--Tyro 3, Axl, and Mer--and their ligand Gas6 in postnatal developing mouse testis.

Tyro 3 family receptors contain three members-Tyro 3, Axl, and Mer-that are essential regulators of mammalian spermatogenesis. However, their exact expression patterns in testis are unclear. In this study, we examined the localizations of Tyro 3, Axl, Mer, and their ligand Gas6 in postnatal mouse testes by immunohistochemistry. All three members and their ligand were continuously expressed in different testicular cells during postnatal development. Tyro 3 was expressed only in Sertoli cells with a varied distribution during testis development. At day 3 postnatal, Tyro 3 was distributed in overall cytoplasmic membrane and cytoplasm of Sertoli cells. From day 14 to day 35 postnatal, Tyro 3 appeared on Sertoli cell processes toward the adlumenal compartment of seminiferous tubules. A stage-dependent Tyro 3 immunoexpression in Sertoli cells was shown by adulthood testis at day 56 postnatal with higher expression at stages I-VII and lower level at stages IX-XII. Axl showed a similar expression pattern to Tyro 3, except for some immunopositive Leydig cells detected in mature testis. In contrast, immunostaining of Mer was detected mainly in primitive spermatogonia and Leydig cells, whereas a relative weak signal was found in Sertoli cells. Gas6 was strongly expressed in Leydig cells, and a relative weak staining signal was seen in primitive spermatogonia and Sertoli cells. These immunoexpression patterns of Tyro 3 family receptors and ligand in testis provide a basis to further study their functions and mechanisms in regulating mammalian spermatogenesis.     

Dynamin 2 associates with complexins and is found in the acrosomal region of mammalian sperm

Previous data showed that complexin I, a SNARE regulatory protein, is localized in and/or around the acrosome and is necessary for the acrosome reaction in sperm. To understand how complexin I regulates the acrosome reaction, we used complexin-GST pulldown assays to identify interacting proteins. We showed that both complexins I and II bound mouse sperm dynamin 2. Dynamin 2 is a 100 kDa GTPase essential to many aspects of endocytosis but its potential role in exocytosis is unknown. Dynamin 2 is expressed in rat testis and widely expressed in other tissues; however, the function of dynamin 2 in germ cells is uncertain. Dynamin 2 protein was detected in mouse testis and was most abundant in or around the developing acrosome of spermatids. In addition, dynamin 2 was co-localized with complexin I in the acrosomal region of mammalian sperm. Its co-localization and interaction with complexin I suggest that dynamin 2 may play a role during acrosome formation and/or acrosomal exocytosis.     

Nxf3 is expressed in Sertoli cells, but is dispensable for spermatogenesis

In eukaryotes, mRNA is actively exported to the cytoplasm by a family of nuclear RNA export factors (NXF). Four Nxf genes have been identified in the mouse: Nxf1, Nxf2, Nxf3, and Nxf7. Inactivation of Nxf2, a germ cell-specific gene, causes defects in spermatogenesis. Here we report that Nxf3 is expressed exclusively in Sertoli cells of the postnatal testis, in a developmentally regulated manner. Expression of Nxf3 coincides with the cessation of Sertoli cell proliferation and the beginning of their differentiation. Continued expression of Nxf3 in mature Sertoli cells of the adult is spermatogenesis stage-independent. Nxf3 is not essential for spermatogenesis, however, suggesting functional redundancy among Nxf family members. With its unique expression pattern in the testis, the promoter of Nxf3 can be used to drive postnatal Sertoli cell-specific expression of other proteins such as Cre recombinase.     

Androgen-Responsive MicroRNAs in Mouse Sertoli Cells

Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs - small, non-coding RNAs - are one class of androgen-regulated trans-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.     

RANKL induces NFATc1 acetylation and stability via histone acetyltransferases during osteoclast differentiation

The MTM (myotubularin)/MTMR (myotubularin-related) protein family is comprised of 15 lipid phosphatases, of which nine members are catalytically active. MTMs are known to play a fundamental role in human physiology as gene mutations can give rise to X-linked myotubular myopathy or Charcot-Marie-Tooth disease, which manifest in skeletal muscle or in peripheral neurons respectively. Interestingly, studies have shown MTMR2 and MTMR5, two MTM family members, to be highly expressed in the testis, particularly in Sertoli and germ cells, and knockout of either gene resulted in spermatogenic defects. Other studies have shown that MTMR2 functions in endocytosis and membrane trafficking. In the testis, MTMR2 interacts and co-localizes with c-Src/phospho-Src-(Tyr?1?), a non-receptor protein tyrosine kinase that regulates the phosphorylation state of proteins at the apical ES (ectoplasmic specialization), a unique type of cell junction found between Sertoli cells and elongating/elongated spermatids. In the present review, we highlight recent findings that have made a significant impact on our understanding of this protein family in normal cell function and in disease, with the emphasis on the role of MTMs and MTMRs in spermatogenesis. We also describe a working model to explain how MTMR2 interacts with other proteins such as c-Src, dynamin 2, EPS8 (growth factor receptor pathway substrate 8) and ARP2/3 (actin-related protein 2/3) at the apical ES and the apical TBC (tubulobulbar complex; tubular-like invaginations that function in the disassembly of the apical ES and in the recycling of its components) to regulate spermiation at late stage VIII of the seminiferous epithelial cycle.     

An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.

Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis-promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.     

Heterophilic binding of the adhesion molecules poliovirus receptor and immunoglobulin superfamily 4A in the interaction between mouse spermatogenic and Sertoli cells

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.     

Involvement of the D-type cyclins in germ cell proliferation and differentiation in the mouse

Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D(1) is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D(1) in spermatogonial proliferation, in particular during the G(1)/S phase transition. Cyclin D(2) is first expressed at the start of spermatogenesis when gonocytes produce A(1) spermatogonia. In the adult testis, cyclin D(2) is expressed in spermatogonia around stage VIII of the seminiferous epithelium when A(al) spermatogonia differentiate into A(1) spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D(2) during spermatogenesis, cyclin D(2) expression was studied in vitamin A-deficient testis. Cyclin D(2) was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A(1) spermatogonia by administration of retinoic acid. Overall, cyclin D(2) seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A(1) spermatogonia. Cyclin D(3) is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D(3) expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D(3) may control G(1)/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D(1) and D(3) seem to be involved in cell cycle regulation, whereas cyclin D(2) likely has a role in spermatogonial differentiation.     

Three Dynamin-Encoding Genes Are Differentially Expressed in Developing Rat Brain

Abstract: Dynamin proteins are members of a recently described family of GTPases involved in receptor-mediated processes. To date, three different dynamin-encoding genes have been identified in mammalian tissues. Dynamin I is expressed only in neurons, whereas dynamin II is ubiquitously expressed. A third isoform, dynamin III, was originally isolated from a rat testis cDNA library and shown to be testis-specific. However, here we report the cloning and characterization of dynamin III from brain and lung, demonstrating a more extended pattern of expression for this isoform. In addition, we have investigated the temporal pattern of expression of these three genes during brain development. We find that both dynamin I and dynamin III mRNA levels are up-regulated during embryogenesis, whereas dynamin II mRNA levels remain unchanged. From these results, we conclude that dynamin III is not a testis-specific isoform and, furthermore, that rat brain expresses three different dynamin-encoding genes that are differentially regulated during development. Therefore, this large isoform diversity of dynamin proteins in brain predicts a significant complexity in the understanding of dynamin-based processes in this tissue.     

Overlapping role of dynamin isoforms in synaptic vesicle endocytosis

The existence of neuron-specific endocytic protein isoforms raises questions about their importance for specialized neuronal functions. Dynamin, a GTPase implicated in the fission reaction of endocytosis, is encoded by three genes, two of which, dynamin 1 and 3, are highly expressed in neurons. We show that dynamin 3, thought to play a predominantly postsynaptic role, has a major presynaptic function. Although lack of dynamin 3 does not produce an overt phenotype in mice, it worsens the dynamin 1 KO phenotype, leading to perinatal lethality and a more severe defect in activity-dependent synaptic vesicle endocytosis. Thus, dynamin 1 and 3, which together account for the overwhelming majority of brain dynamin, cooperate in supporting optimal rates of synaptic vesicle endocytosis. Persistence of synaptic transmission in their absence indicates that if dynamin plays essential functions in neurons, such functions can be achieved by the very low levels of dynamin 2.     

Expression and function of class B scavenger receptor type I on both apical and basolateral sides of the plasma membrane of polarized testicular Sertoli cells of the rat

Class B scavenger receptor type I (SR-BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR-BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR-BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle-stimulating hormone (FSH). When the expression of SR-BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first-round spermatogenesis and continued to be detectable thereafter. The level of SR-BI mRNA expression in Sertoli cells was lower at spermatogenic stages I-VI than at other stages. SR-BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR-BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR-BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR-BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.     

Role of Sertoli cell number and function on regulation of spermatogenesis

Testicular function is under the control of expression and repression of several genes and gene products, and many of these works through Sertoli cells. The capability of Sertoli cells to regulate spermatogenesis is dependent on Sertoli cell functions and Sertoli cell number. Sertoli cell number has long been thought to be stable in adults with no proliferation of Sertoli cells once adult numbers have been reached. However, adult horses do not have stable Sertoli cell numbers, and new studies indicate that adult Sertoli cells can be made to re-enter mitotic phase under certain experimental conditions. This review discusses roles of Sertoli cells in regulation of spermatogenesis and methods for estimating the number of Sertoli cells, in a testis, that overcome the problems (assumptions) associated with the indented, pear-shaped of Sertoli cell nuclei which make it difficult to estimate the volume of individual nuclei. Using several approaches to overcome the problems associated with any one method, the horse is identified as a species in which Sertoli cell number is not fixed, but it fluctuates with season. In addition to Sertoli cell numbers, the functions of Sertoli cells that are very important in signaling and controlling spermatogenesis are discussed. Recent studies have shown that "post-mitotic terminally differentiated Sertoli cells" from adult animals could, under certain conditions, re-enter the cell division cycle. Can seasonal influences be a natural set of conditions to induce the Sertoli cells of the horse testis to seasonally re-enter the cell division cycle and explain the seasonal differences in Sertoli cell number as summarized in this review? Alternatively, can seasonal differences in Sertoli cell number reflect, in the horse to a greater extent, but in adults of most species, the presence of some mitotic-capable Sertoli cells in adults? In any case, both Sertoli cell number and function are important in regulation of spermatogenesis.     

Dynamin2 and cortactin regulate actin assembly and filament organization

The GTPase dynamin is required for endocytic vesicle formation. Dynamin has also been implicated in regulating the actin cytoskeleton, but the mechanism by which it does so is unclear. Through interactions via its proline-rich domain (PRD), dynamin binds several proteins, including cortactin, profilin, syndapin, and murine Abp1, that regulate the actin cytoskeleton. We investigated the interaction of dynamin2 and cortactin in regulating actin assembly in vivo and in vitro. When expressed in cultured cells, a dynamin2 mutant with decreased affinity for GTP decreased actin dynamics within the cortical actin network. Expressed mutants of cortactin that have decreased binding of Arp2/3 complex or dynamin2 also decreased actin dynamics. Dynamin2 influenced actin nucleation by purified Arp2/3 complex and cortactin in vitro in a biphasic manner. Low concentrations of dynamin2 enhanced actin nucleation by Arp2/3 complex and cortactin, and high concentrations were inhibitory. Dynamin2 promoted the association of actin filaments nucleated by Arp2/3 complex and cortactin with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing lipid vesicles. GTP hydrolysis altered the organization of the filaments and the lipid vesicles. We conclude that dynamin2, through an interaction with cortactin, regulates actin assembly and actin filament organization at membranes.     

Asynchronous expression of the homeodomain protein CUX1 in Sertoli cells and spermatids during spermatogenesis in mice

The homeodomain CUX1 protein exists as multiple isoforms that arise from proteolytic processing of a 200-kDa protein or an alternate splicing or from the use of an alternate promoter. The 200-kDa CUX1 protein is highly expressed in the developing kidney, where it functions to regulate cell proliferation. Transgenic mice ectopically expressing the 200-kDa CUX1 protein develop renal hyperplasia associated with reduced expression of the cyclin kinase inhibitor p27. A 55-kDa CUX1 isoform is expressed exclusively in the testes. We determined the pattern and timing of CUX1 protein expression in developing testes. CUX1 expression was continuous in Sertoli cells from prepubertal testes but became cyclic when spermatids appeared. In testes from mature mice, CUX1 was highly expressed only in round spermatids at stages IV-V of spermatogenesis, in both spermatids and Sertoli cells at stages VI-X of spermatogenesis, and only in Sertoli cells at stage XI of spermatogenesis. While most of the seminiferous tubules in wild-type mice were between stages VI and X of spermatogenesis, there was a significant reduction in the percentage of seminiferous tubules between stages VI and X in Cux1 transgenic mice and a significant increase in the percentage of seminiferous tubules in stages IV-V and XI. Moreover, CUX1 was not expressed in proliferating cells in testes from either wild-type or transgenic mice. Thus, unlike the somatic form of CUX1, which has a role in cell proliferation, the testis-specific form of CUX1 is not involved in cell division and appears to play a role in signaling between Sertoli cells and spermatids.     

Zinc-regulating proteins, ZnT-1, and metallothionein I/II are present in different cell populations in the mouse testis.

Zinc ions play an important role in testis development and spermatogenesis. Thus, nutritional zinc deficiency leads to aberrant testicular development, reduced spermatogenesis, and male sterility. The precise actions of zinc in mediating these functions and the mechanisms by which zinc is itself regulated in the testis, however, have not been adequately elucidated. We have assessed the distribution of the zinc-regulating proteins ZnT-1 and metallothionein I/II (MT I/II) in the mouse seminiferous tubule. Co-labeling for ZnT-1 and MT I/II demonstrated unique patterns of distribution for these proteins, with ZnT-1 present in Sertoli cells in addition to luminal spermatozoa and MT I/II restricted to spermatocytes. These findings were confirmed by dual-label immunofluorescence for ZnT-1 and the Sertoli cell marker, vimentin, and by immunoelectron microscopy. The differential expression patterns of ZnT-1 and MTs support the hypothesis that ZnT-1 and MTs play different roles in the regulation of intracellular zinc in this organ. The specific expression of ZnT-1 in the Sertoli cells, moreover, is consistent with their role in maintaining a nurturing, closely regulated environment for spermatogenesis.