Tubulobulbar complexes are intercellular podosome-like structures that internalize intact intercellular junctions during epithelial remodeling events in the rat testis

Tubulobulbar complexes are actin-related double-membrane projections that resemble podosomes in other systems and form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat and mouse testes for junction molecules reported to be present at apical sites of attachment (ectoplasmic specializations) between Sertoli cells and spermatids. The adhesion molecules nectin 2 (PVRL2), nectin 3 (PVRL3) and alpha 6 integrin (ITGA6) are present in the elongate parts of tubulobulbar complexes and concentrated at their distal ends. Tubulobulbar complexes contain cortactin (CTTN), a key component of podosomes, and vesicles at the distal ends of tubulobulbar complexes that contain junction molecules are related to early endosome antigen (EEA1). N-cadherin (CDH2), a protein reported to be present at ectoplasmic specializations, is not localized to these unique junctions or to tubulobulbar complexes but, rather, is primarily concentrated at desmosomes in basal regions of the epithelium. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures that are responsible for internalizing intact intercellular junctions during spermatogenesis.     

Non-muscle cofilin is a component of tubulobulbar complexes in the testis

Tubulobulbar complexes are finger-like structures that form at the interface between maturing spermatids and Sertoli cells prior to sperm release and at the interface between two Sertoli cells near the base of the seminiferous epithelium. They originate in areas previously occupied by actin filament-associated intercellular adhesion plaques known as ectoplasmic specializations. Actin filaments also are associated with tubulobulbar complexes where they appear to form a network, rather than the tightly packed bundles found in ectoplasmic specializations. Cofilin, a calcium-independent actin-depolymerizing protein, previously has been identified in the testis, but has not been localized to specific structures in the seminiferous epithelium. To determine if cofilin is found in Sertoli cells and is concentrated at actin-rich structures, we reacted fixed frozen sections of rat testis, fixed fragmented tissue, and blots of seminiferous epithelium with pan-specific and non-muscle cofilin antibodies. In addition, GeneChip microarrays (Affymetrix, Santa Clara, CA) were utilized to determine the abundance of mRNA for all cofilin isoforms in Sertoli cells. Using the monoclonal pan-specific cofilin antibody, we found specific labeling exclusively at tubulobulbar complexes and not at ectoplasmic specializations. On one-dimensional (1D) Western blots this antibody reacted monospecifically with one band, and on 2D blots reacted with two dots, which we interpret as phosphorylated and nonphosphorylated forms of a single cofilin isotype. Messenger RNA for non-muscle cofilin in Sertoli cells is about 8.5-fold higher than for muscle-type cofilin. To confirm that the non-muscle isoform of cofilin is present at tubulobulbar complexes, we used antibodies specific to non-muscle cofilin for immunofluorescent localization. As with the pan-specific antibody, we found that the non-muscle cofilin antibody exclusively labeled tubulobulbar complexes. Results presented here indicate that non-muscle cofilin is concentrated at tubulobulbar complexes. Our results also indicate that cofilin is not concentrated at ectoplasmic specializations.     

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

Coxiella burnetii is an intracellular bacterial pathogen which causes Q fever, a human infection with the ability to cause chronic disease with potentially life-threatening outcomes. In humans, Coxiella infects alveolar macrophages where it replicates to high numbers in a unique, pathogen-directed lysosome-derived vacuole. This compartment, termed the Coxiella-containing vacuole (CCV), has a low internal pH and contains markers both of lysosomes and autophagosomes. The CCV membrane is also enriched with CLTC (clathrin heavy chain) and this contributes to the success of the CCV. Here, we describe a role for CLTC, a scaffolding protein of clathrin-coated vesicles, in facilitating the fusion of autophagosomes with the CCV. During gene silencing of CLTC, CCVs are unable to fuse with each other, a phenotype also seen when silencing genes involved in macroautophagy/autophagy. MAP1LC3B/LC3B, which is normally observed inside the CCV, is excluded from CCVs in the absence of CLTC. Additionally, this study demonstrates that autophagosome fusion contributes to CCV size as cell starvation and subsequent autophagy induction leads to further CCV expansion. This is CLTC dependent, as the absence of CLTC renders autophagosomes no longer able to contribute to the expansion of the CCV. This investigation provides a functional link between CLTC and autophagy in the context of Coxiella infection and highlights the CCV as an important tool to explore the interactions between these vesicular trafficking pathways.     

Observations on the inter-relationships of Sertoli cells at the level of the blood- testis barrier: evidence for formation and resorption of Sertoli-Sertoli tubulobulbar complexes during the spermatogenic cycle of the rat.

Structures termed tubulobulbar complexes are known to be formed by adjoining Sertoli cells at the level of the blood-testis barrier (Russell and Clermont, '76). Here, long (2-4 micrometer) tubular evaginations of one Sertoli cell, which end in bulbous dilations, are seen in corresponding invaginations of a neighboring Sertoli cell. In most regions of the tubular and bulbous portions of the complex, the Sertoli plasma membranes were found to be separated by a 4-5-nm intercellular space, but in some areas the membranes converged to form tight and gap junctions. The numbers, distribution and properties of tubulobulbar complexes were studied in relation to the cycle of the seminiferous epithelium. From the data obtained it was concluded that tubulobulbar complexes develop and undergo regressive changes during the spermatogenic cycle. Most complexes arise during the early stages of the cycle (Stages II-V) and develop large bulbous endings. Developing tubulobulbar complexes consist of short evaginations of one Sertoli cell which face a bristle-coated pit of the opposing Sertoli cell. At midcycle (Stages VI-VII) most show regressive changes and are eventually resorbed as a consequence of the action of nearby Sertoli lysosomes. Once resorbed, the probability of seeing a tubulobulbar complex in thin sections decreases from 4- to 8-fold. The few tubulobulbar complexes which remain past this period (Stages VII-XIV-I) usually lack bulbous endings and are fequently seen above type A spermatogonia. The data suggest that small fragments of cytoplasm and plasma membrane (including junctional surfaces) are lost from one Sertoli cell as a result of the degradative processes occurring in a neighboring Sertoli cell. Tubulobulbar resorption is discussed in relation to the impending breakdown of the blood-testis barrier above spermatocytes as these cells move upward. The possible significance of the cyclic resorption of tight and gap junctional sites between Sertoli cells is also discussed.     

Calreticulin is present in the acrosome of spermatids of rat testis.

We have already succeeded in purifying a calcium-binding protein (CalBP) from rat spermatogenic cells [Nakamura et al., Biochem. Biophys. Res. Commun., 176 (1991) 1358]. In this study, the location of this protein within rat testis was examined, using a rabbit antisera for this protein. The antigen was localized on the developing acrosomes during spermiogenesis. The NH2-terminal amino acid sequence obtained for rat CalBP was identical to that of calreticulin obtained for the skeletal muscle of mice and closely resembled that for rabbit calreticulin. On the immunoblot analysis, the purified rat CalBP reacted with an antibody raised against rabbit skeletal muscle calreticulin. The results indicate that calreticulin is present in the acrosome of spermatids of rat testes.     

Steroidogenesis activator polypeptide (SAP) in the rat ovary and testis

A steroidogenesis activator polypeptide (SAP) has previously been identified in the rat adrenal cortex (Pedersen and Brownie, Proc. natn. Acad. Sci. U.S.A. 80 (1983) 1882-1886). This factor apparently facilitates the association of mitochondrial cholesterol with the cholesterol side-chain cleavage cytochrome P-450, a reaction which is generally regarded as rate-controlling in the steroid biosynthetic pathway. The same preparative techniques have now been applied in a search for this material in other rat tissues. Among those investigated, the ovary and testis demonstrate significant concentrations of a factor which is biologically and chromatographically similar to adrenal SAP. In the immature ovary the activator becomes manifest after priming with PMSG and rises dramatically during hCG-stimulated luteinization, an increase which can be blunted with cycloheximide. In the adult rat testis it is increased acutely by treatment with hCG or dibutyryl cAMP and is diminished in response to hypophysectomy or cycloheximide. At approximately equivalent concentrations (10(-7) M), preparations of the activator from the adrenal cortex, the testis, and the superovulated ovary each enhance the activity of cholesterol side-chain cleavage in adrenocortical mitochondria by 5- to 6-fold over basal controls. We conclude that steroidogenic organs share a similar or identical intracellular modulator of cholesterol----pregnenolone conversion which is under pituitary control.     

Different subtypes of tachykinin NK(1) receptor binding sites are present in the rat brain

(2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA), which labels "septide-sensitive" but not classic NK(1) binding sites in peripheral tissues, was used to determine whether septide-sensitive binding sites are also present in the rat brain. Binding studies were performed in the presence of SR 48968 (NK(2) antagonist) and senktide (NK(3) agonist) because [(125)I]NKA also labels peripheral NK(2) binding sites and, as shown in this study, central NK(3) binding sites. [(125)I]NKA was found to label not only septide-sensitive binding sites but also a new subtype of NK(1) binding site distinct from classic NK(1) binding sites. Both subtypes of [(125)I]NKA binding sites were sensitive to tachykinin NK(1) antagonists and agonists but also to the endogenous tachykinins NKA, neuropeptide K (NPK), and neuropeptide gamma (NPgamma). However, compounds of the septide family such as substance P(6-11) [SP(6-11)] and propionyl-[Met(O(2))(11)]SP(7-11) and some NK(1) antagonists, GR 82334, RP 67580, and CP 96345, had a much lower affinity for the new NK(1)-sensitive sites than for the septide-sensitive sites. The hypothalamus and colliculi possess only this new subtype of NK(1) site, whereas both types of [(125)I]NKA binding sites were found in the amygdala and some other brain structures. These results not only explain the central effects of septide or SP(6-11), but also those of NKA, NPK, and NPgamma, which can be selectively blocked by NK(1) receptor antagonists.     

Poly (ADP-ribose) synthetase activity in rat testis mitochondria

A quite active poly (ADP-ribose) synthetase was found in isolated rat testis mitochondria. Similar levels of activity were found in mitochondria isolated from bull and hamster testis. In contrast, mitochondria isolated from rat brain or liver, and demembraned sperm, showed negligible activity. Centrifugation of testis mitochondria through a linear sucrose gradient, showed that, poly (ADP-ribose) synthetase cosediment together with succinatecytochrome c reductase and mitochondrial proteins. Furthermore, treatment with digitonin indicated that, the enzyme is localized in the inner membrane-matrix complex. Finally, kinetic studies demonstrated that, the apparent Km for NAD⁺ of the mitochondrial enzyme, was 22 μM compared with 210 μM for the nuclear enzyme.     

The beta 1 and beta 2 subunits of the AP complexes are the clathrin coat assembly components.

The beta 1 and beta 2 subunits are the closely-related large chains of the trans-Golgi network AP-1 and the plasma membrane AP-2 clathrin-associated protein complexes, respectively. Recombinant beta 1 and beta 2 subunits have been generated in Escherichia coli. It was found that, in the absence of all the other AP subunits, beta 1 and beta 2 interact with clathrin and drive the efficient assembly of clathrin coats. In addition, beta 2 subunits and AP complexes compete for the same clathrin binding site. The appearance of the clathrin/beta coats is the same as the barrel-shaped structures formed with native AP complexes. It is proposed that the principal function of the beta subunits is to initiate coat formation, while the remaining subunits of the AP complexes have other roles in coated pit and coated vesicle function.     

Precursor and deaminated forms of both luteinizing hormone-releasing hormone (LHRH) and (hydroxyproline9)LHRH are present in the rat hypothalamus.

A naturally occurring analog of the decapeptide luteinizing hormone-releasing hormone ([Hyp9]LHRH) has been described previously in the hypothalamus of several mammals. It derives from post-translational hydroxylation of the LHRH proline9 residue. In the present work, intermediate LHRH precursors exhibiting both Pro9 or Hyp9 residues in the LHRH sequence were characterized in the rat hypothalamus. Hydroxylation of the Pro9 residue can thus be assumed to occur at an early stage of post-translational maturation. Deaminated, free acid forms of both native decapeptides were also detected. They correspond most likely to catabolites from incompletely processed precursors.     

Vif and the p55(Gag) polyprotein of human immunodeficiency virus type 1 are present in colocalizing membrane-free cytoplasmic complexes

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259-5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55(Gag) polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions.     

Immunohistochemical localization of a water channel, aquaporin 7 (AQP7), in the rat testis

Cell volume reduction is one of the most distinct morphological changes during spermiogenesis and may be largely attributable to water efflux from the cell. A strong candidate for a water efflux route, aquaporin 7 (AQP7), which is a water channel, was studied immunohistochemically in the rat testis. Immunoreactivity was restricted within the elongated spermatids, testicular spermatozoa, and residual bodies remaining in the seminiferous epithelium. Weak but distinct immunoreactivity was first observed in the cytoplasmic mass of the spermatid at step 8 of spermiogenesis. The Golgi-like apparatus became steadily immunoreactive at step 10. The plasma membrane covering the cytoplasmic mass showed strong immunoreactivity after step 16. At this step, the middle piece of the tail also showed immunoreactivity at the portion protruding into the lumen. The whole head and distal tail, where the elongated spermatid had only a limited amount of cytoplasm, showed no immunoreactivity throughout spermiogenesis. After spermiation, the immunoreactivity of AQP7 remained at the middle piece and in the cytoplasmic droplet in the testicular spermatozoon. The present observations suggest that AQP7 contributes to the volume reduction of spermatids, since this water channel protein is localized on the plasma membrane covering the condensing cytoplasmic mass of the elongated spermatid, and since the seminiferous tubule fluid is hypertonic.     

Identification of GABA(B) receptor in rat testis and sperm

gamma-Aminobutyric acid (GABA) can mimic and potentiate the action of progesterone in initiating the acrosome reaction (AR) of mammalian sperm, indicating that sperm contain receptors for GABA. This contention was validated by identifying the receptor (R) subtype, GABA(A)R, in mammalian sperm. In the present study a second subtype, GABA(B)R, was identified in rat testis and sperm. Total RNAs of rat testis and sperm were prepared and used as template to synthesize the respective cDNAs by the RT-PCR method. Two splice variants of the cDNA coding GABA(B)R1 (GABA(B)R1a and GABA(B)R1c) and GABA(B)R2 were identified. Extracts of rat testis, spermatogenic cells and sperm contained two proteins with estimated molecular sizes of 130 and 100 kDa, corresponding to GABA(B)R1a and GABA(B)R1c/lb, respectively, determined by Western blot using polyclonal anti-GABA(B)R1 antibody. By an indirect immunofluorescence technique, GABA(B)R1 was located on the head of rat sperm. The present finding is the first direct demonstration that mammalian sperm contain GABA(B)R.     

Testosterone formation in testis of the immature androgen insensitive pseudohermaphrodite male rat (Stanley-Gumbreck rat)

Testosterone formation from pregnenolone (3β-hydroxy-5-pregnen-20-one) and progesterone in testis of the Stanley-Gumbreck pseudohermaphrodite (Ps) adult rat is greatly reduced in comparison to the normal (Nl) adult rat testis. In an attempt to determine whether this defect is congenital or acquired postnatally with increasing age, minced testis of 1-month-old Ps and Nl rats were incubated with progesterone, and the labeled metabolites identified. Almost equal amounts of progesterone were metabolized by both Ps and Nl testis. In mince incubations without NADPH nearly as much testosterone and 4-androstene-3,17-dione accumulated in the Ps as in the Nl testis. Very little androsterone and 5α-androstane-3α,17β-diol were formed in these incubations. When minces were incubated with progesterone in the presence of NADPH, testosterone and 4-androstene-3,17-dione accumulation was greatly reduced, and instead 5α-androstane-3α,17β-diol was formed as the major product by Nl testis and androsterone by Ps testis. Neither heparin, a 5α-reductase inhibitor, nor glucose-6-phosphate dehydrogenase alone significantly influenced progesterone metabolism or the accumulation of testosterone or 4-androstene-3,17-dione in either Ps or Nl testis. These results indicated that the 5α-reductase activity in both the Ps and N1 testis is dependent only on NADPH. Although studies were not carried out in younger rats (2–5 days of age), our results are in agreement with previous studies of Goldstein and Wilson who demonstrated equal accumulation of testosterone in incubations of testis from normal and Tfm/y mice. However, it is apparent that differences between Nl and Ps testis may be revealed only under conditions which allow maximum rates of 17-oxo- and 5α-reductions.     

Triiodothyronine receptors (TR) are present in breast cancer tissues

INTRODUCTION: Possible relationships between breast cancer and thyroid hormones have been suggested for many years. The aim of this study was qualitative examination of triiodothyronine receptors (TR) in breast cancer tissues and in non cancerous breast tissue taken from the opposite side to the localization of the tumor. MATERIAL AND METHODS: The material consisted of 15 breast cancer tissues of grades G1 to G3 and the same number of control tissues obtained during radical mastectomy or local tumor resection. Tissues were homogenized. Protein fraction was isolated. Protein for TR was assessed in Western Blot reaction. RESULTS: Protein fraction for TR was present in all cancer tissues and 6 healthy controls. CONCLUSIONS: Obtained data may suggest so far unknown role of thyroid hormones and their nuclear receptors in the generation and development of breast cancer.     

Effects of cryptorchidism and orchidopexy on deoxyribonucleoside-activated nucleotidase (DAN) in the rat testis

1. Unilateral cryptorchidism was associated with an increase in specific DAN activity in the abdominal testis of adult rats. 2. In the scrotal, immature testis, there was an age-dependent decrease in specific DAN activity similar to that normally seen in rats. This decrease was less pronounced in the abdominal testis of unilaterally cryptorchid rats. 3. Total DAN activity in the abdominal testis showed a decrease in both immature and adult rats, when compared to that of the scrotal testis. 4. Orchidopexy following cryptorchidism in adult rats, resulted in complete restoration of DAN activity in spite of poor recovery of spermatogenesis.     

Role of neurotropins in rat embryonic testis morphogenesis (cord formation)

The process of seminiferous cord formation is the first morphological event that differentiates a testis from an ovary and indicates male sex determination. Cord formation occurs by embryonic Day 14 (Day 0 = plug date; E14) in the rat. A series of experiments were conducted to determine if neurotropins and their receptors are important for the process of rat embryonic cord formation. The expression of low affinity neurotropin receptor (p75/LNGFR) was determined by immunohistochemistry on sections of both testis and ovary from E13 through birth (Day 0, P0) with an antibody to p75/LNGFR. The staining for p75/LNGFR was present in the mesonephros of E13 gonads and in a sex-specific manner appeared around developing cords at E14 in the embryonic testis. At birth, staining for p75/LNGFR was localized to a single layer of cells (i.e., peritubular cells) that surrounded the seminiferous cords. The genes for both neurotropin 3 (NT3) and for corresponding high affinity neurotropin trkC receptor were found to be expressed in the E14 rat testis, as well as other neurotropins and receptors. Immunocytochemical analysis of E14 rat testis demonstrated that NT3 was localized to the Sertoli cells and trkC was present in individual cells of the interstitium at E16 and in selected preperitubular cells at E18. Previously, the peritubular cells adjacent to the cords were demonstrated to be derived from migrating mesonephros cells around the time of cord formation. To determine if neurotropins were involved in cord formation, the actions of neurotropins were inhibited. A high affinity neurotropin receptor (trk)-specific kinase inhibitor, K252a, was used to treat organ cultures of testes from E13 rats prior to cord formation. Treatment of E13 testis organ cultures with K252a completely inhibited cord formation. K252a-treated organ cultures of E14 testis that contained cords did not alter cord morphology. A second experiment to inhibit neurotropin actions utilized a specific antagonist trk-IgG chimeric fusion protein and E13 testis organ cultures. The trk-IgG molecules dimerize with endogenous trk receptors and inhibit receptor signaling and activation of ligand function. Forty percent of E13 testis organ cultures treated with trkC-IgG had significantly reduced cord formation. TrkA-IgG had no effect on initiation of cords; however, in fifty percent of the treated organs, a "swollen" appearance of the cord structures was observed. Experiments using trkB-IgG chimeric protein on E13 organ cultures had no effect on cord formation or cord morphology. The testes from trkC and NT3 knockout mice were examined to determine if there were any morphological differences in the testis. NT3 knockouts appeared to have normal cord morphology in E15 and E17 testis. TrkC knockout mice also had normal cord morphology in E14 and P0 testis. Both NT3 and trkC knockout-mice testis had less interstitial area than wild-type controls. In addition, the trkC knockout mice have an increased number of cells expressing p75LNGFR within the cords when compared to controls or NT3 knockout mice. Combined observations suggest compensation between the different neurotropin ligands, receptors, and/or possibly different growth factors for this critical biological process. In summary, results suggest a novel nonneuronal role for neurotropins in the process of cord formation during embryonic rat testis development. The hypothesis developed is that neurotropins are involved in the progression of male sex differentiation and are critical for the induction of embryonic testis cord formation.