Evidence for an androgen receptor in the seminiferous tubules of the human testis

Androgen receptors (AR) were studied in seminiferous tubule cytosol and testicular nuclear extracts prepared from testes of previously untreated elderly men undergoing orchiectomy as therapy for prostatic carcinoma. Cytosol exhibited high affinity (Kd = 0.8 nM), saturable binding of [3H]methyltrienolone; however, the synthetic progestin, promegestone was a stronger competitor for MT binding sites than were 5 alpha-dihydrotestosterone (DHT) or testosterone (T), suggesting the presence of progesterone-like binding sites. Addition of triamcinolone acetonide (TA) produced the usual relative steroid specificity for AR binding and reduced the measured AR binding capacity by 19 +/- 8% (Mean +/- SD, n = 3). The umber of MT binding sites was 30-40 fmol/mg protein, or an average of 65 fmol/g testis, and the equilibrium dissociation constant at 0 degrees C was 0.6-1.4 nM. In the presence of sodium molybdate, binding was stable for 40 h at 0 degrees C and the half-time of dissociation of the MT-AR complex was 12-16 h. The binding of salt extractable (600 mM KCl) nuclear sites to MT was saturable and was specific for androgens. The number of binding sites in nuclear extracts was 170 fmol/g testis and the apparent equilibrium dissociation constant was 4.2 nM. Thus, the binding of MT to human seminiferous tubule cytosol and testicular nuclear extract exhibits properties which are nearly identical to those of the prostate AR. Further study of this androphilic protein may provide insight into the role of androgen in normal and abnormal spermatogenesis in man.     

A smaller molecular weight retinol binding protein in rat testis seminiferous tubules

In the cytosol fraction in rat testis seminiferous tubules a lower molecular weight protein of ～4,800 daltons that binds retinol with high specificity has been isolated and purified by ammonium sulfate precipitation and on Sephadex column chromatography. The hexane extract of the component gave a characteristic retinol fluorescence spectrum. The amino acid composition was qualitatively similar to the retinol binding protein in the blood with the exception that cystine and cysteine were absent.     

Intercellular communication in rat seminiferous tubules

Intercellular electrical coupling in seminiferous tubules from prepubescent and adult Wistar rats has been studied by using conventional techniques. It is found that cells in the seminiferous epithelium are electrically coupled. Experiments performed using "Sertoli cell-enriched" seminiferous tubules indicate the existence of intercellular ionic communication between Sertoli cells. Junctional conductance is independent of the direction of electrical field and it is affected by A23187 Ca ionophore (5 microM) but not by exposure to the neurotransmitter norepinephrine (1-5 X 10(-5) M). Intracellular resistivity (including junctional resistance) is higher in mature as compared to immature germinal epithelium. These findings suggest that cell metabolites or second messenger molecules could be transferred via the low-resistance pathways between epithelium cells to coordinate cellular activity.     

Premeiotic germ cell defect in seminiferous tubules of Atm-null testis

Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16(Ink4a) activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16(Ink4a)-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16(Ink4a)-mediated senescence.     

In vitro development of seminiferous tubules from immature rat testis: ultrastructural and morphometric studies

Localization of snRNA at the ultrastructural level was studied in the nucleolus of CHO cells by EM autoradiography. In conditions where snRNA U3 is the only RNA species labelled in the nucleolus, silver grains were largely found at the periphery, over the granular ribonucleoprotein component and the perinucleolar condensed chromatin; this enrichment was quantitatively significant. Inhibition of pre-rRNA synthesis with actinomycin D did not alter the concentration or the distribution of U3 inside the nucleolus. The results are consistent with the demonstration that U3 is hydrogen-bonded to 28S pre-rRNA, and thus should be found in the granular compartment where 32S-28S pre-rRNA is assembled into 55s RNP.     

Testosterone micromilieu in staged rat seminiferous tubules

Endogenous testosterone concentrations in rat seminiferous tubules were measured in relation to different stages of the cycle of the seminiferous epithelium. For this purpose, the seminiferous tubules were mechanically separated from the interstitial tissue on a cooled (1 degree C) petri dish under a stereomicroscope without added medium. After recognition of the stages of the cycle by transillumination, the specimens were rapidly transferred by dry forceps into test tubes for testosterone radioimmunoassay. The results of the dry dissection method were compared with measurements on tubules that were kept after separation in phosphate buffered saline (PBS, pH 7.4), in order to reveal the possible leakage of testosterone from the tubules. The maximal concentration of testosterone per unit length of seminiferous tubule was found in stages VII and VIII of the cycle (288 +/- 60 fmol/cm, mean +/- SEM, n = 12), and the minimal in stages IX-XII (219 +/- 57 fmol/cm, P less than 0.01). If the levels were correlated with unit volumes of the seminiferous tubules, identical concentrations of testosterone (521-542 fmol/mm3, approx. 500 nmol/l) were found in the different stages of the cycle. Despite the similarity of testosterone concentrations in the different parts of the seminiferous tubules the local concentrations of biologically active (i.e. free) testosterone may be modulated by extracellular and intracellular androgen binding components.     

Rete testis and the adjacent seminiferous tubules during postembryonic development in mice

Testicular compartment that includes rete testis and the adjacent transitional zone (TZ) of seminiferous tubules has been examined only by light and electron microscopy until now. However, recent data suggest that adult Sertoli cells (SCs) located in this compartment are capable to commence active proliferation both in vitro and in vivo, and hence, are not completely differentiated. The present study is first to investigate mouse rete testis and TZ during the postembryonic development and is intended to determine new protein markers for cells of this compartment, the state of their differentiation, and also their proliferative activity. It was demonstrated that rete testis cells were stained for SC marker Wt1 transiently, until day 25 of postembryonic development, then the staining disappeared. Another SC marker Dmrt1 that involved in the process of SC differentiation was not expressed in the rete testis cells during the postnatal development and in the adult state. One more feature that distinguished rete testis cells from SCs was lower proliferative activity of rete testis cells in 2–6 days old mice. SCs from TZ expressed Wt1 at all ages examined. However, at earlier ages, they were heterogeneous on Dmrt1 expression, and only by day 25, Dmrt1 expression was completely disappeared from TZ SCs. It is interesting that on day 18 when SCs in seminiferous tubules complete differentiation and exit from cell cycle proliferation of TZ SCs was at significantly higher level. It is also showed that in 3D culture, Wt1+ cells isolated from rete testis and TZ of 60 days old GFP male mice were capable to form seminiferous tubules de novo in cooperation with testicular cells from 6 days old mice.     

Dehydrodolichyl diphosphate synthetase from rat seminiferous tubules

Homogenates of seminiferous tubules from rat testes catalyzed the incorporation of label from [14C]isopentenyl diphosphate into a variety of polyprenyl products. Long chain polyprenyl mono- and diphosphates were formed as major products when undesirable side reactions were minimized. The long chain polyprenyl diphosphate synthetase was measured as a sum of the mono- and diphosphate derivatives formed and was dependent on the addition of t,t-farnesyl diphosphate, isopentenyl diphosphate, and divalent cation. The highest activity was associated with the membranous fractions, whereas activity was negligible in the cytosolic fraction. The products of this prenyl transferase were labile to acid and yielded petroleum ether soluble products which indicated that the alpha-isoprene unit was unsaturated. Hydrolysis of either the polyprenyl mono-or diphosphates with a testicular phosphatase in the absence of NaF yielded C75, C80, C85, and C90 polyprenols. The chain lengths of the products of the synthetase suggest that this enzyme is responsible for the de novo biosynthesis of dehydrodolichyl diphosphates which are precursors of the dolichyl derivatives found in testes.     

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.     

Contractility and structure of adult rat seminiferous tubules in organ culture

Summary Isolated pieces of seminiferous tubules of adult rats were grown in organ culture for up to 8 weeks in Petri dishes on the surface of nutrient agar. The medium consisted of newborn calf serum, Eagle's minimum essential medium, glutamate and antibiotics. This method allowed observation of the contractions of the seminiferous tubules in the culture. Contractility, light and electron microscopic structure and histochemically demonstrable activities of alkaline phosphatase and ATPase of the tubule walls were studied at 1-week intervals. The contractility and alkaline phosphatase activity were maintained in the tubule wall for 3 weeks, and the activity of ATPase was maintained for 5 weeks. The thin filaments of the myoid cells, which are responsible for the contractility, were seen with the electron microscope in tubules cultured for 5 weeks. The organ culture method described in the present paper seems to be valuable for studies concerning the functioning of the myoid cells of the seminiferous tubules and the possibility that this is regulated by hormones.     

Identification of stage-specific proteins synthesized by rat seminiferous tubules

Experiments were conducted to determine how the cycle of the seminiferous epithelium influenced synthesis and secretion of proteins by seminiferous tubules. Tubular segments were treated with collagenase and then cultured with [35S]methionine. These myoid cell-depleted tubules isolated from different stages of the epithelial cycle exhibited, at Stages VI and XII, two distinct peaks of secretion of total radiolabeled proteins. Two-dimensional gel electrophoresis indicated that the patterns of secreted proteins from these two stages were remarkably different, while those from other stages were intermediate between those at the peaks. At least 15 proteins were secreted cyclically, many of them previously unrecognized products of the seminiferous epithelium. One product, designated Cyclic Protein-2 (CP-2), exhibited a pronounced cycle of secretion, its peak at Stage VI being 30-fold greater than at its nadir at Stages XII-XIV. Further investigation indicated that CP-2 did not appear to originate from myoid cells or dispersed germ cells but could be recovered from Sertoli cell-enriched cultures prepared from Stage VI tubules. Protein secretion by tubular segments was also characterized by immunoprecipitation with two polyspecific antisera directed against Sertoli cell products. Five secretory proteins were identified which had cycles different from one another and from CP-2. In contrast to secreted products, the synthesis of most cellular proteins by tubular segments remained relatively constant throughout the cycle. It is concluded: 1) segments of the seminiferous epithelium secrete proteins into the culture medium which are distinct from cellular proteins; 2) the synthesis of many of these proteins varies with the epithelial cycle; and 3) several of the secreted proteins are of Sertoli cell origin, including a newly identified protein, CP-2. This indicates that the morphology and the protein synthetic capacity of the seminiferous epithelium are coordinated over space and time.