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Quantitative analysis of androgen receptor messenger ribonucleic acid in developing Leydig cells and Sertoli cells by in situ hybridization.

Testosterone produced by Leydig cells is critical for the maintenance of spermatogenesis by Sertoli cells throughout adulthood in the rat. However, the presence of androgen receptors (AR) in Leydig cells in prepubertal rats suggests additional roles for androgen in early Leydig cell function and differentiation. In the present study, AR messenger RNA (mRNA) was directly measured by in situ hybridization in sections of rat testes at three developmental stages: on day 21 postpartum, when Leydig cells exist as mesenchymal-like progenitors; on day 35, when they are still immature, producing low amounts of testosterone; and on day 90, when they are fully functional in the sexually mature animal. Testicular AR mRNA was detected in Leydig cells, pericytes, peritubular myoid cells, and Sertoli cells. On day 90, AR mRNA levels in Sertoli cells varied with the cycle of the seminiferous epithelium, achieving peak intensity at stages VII-VIII. Measurements were made by image analysis and expressed as integrated signal intensities per unit labeled area (mean +/- SEM; n = 3 rats at each age). The results showed that levels of Leydig cell and Sertoli cell AR mRNA change significantly during development (P < 0.05). Leydig cell AR mRNA was intermediate on day 21 (at 17.3 +/- 0.7), highest on day 35 (at 26.9 +/- 1.6), and lowest on day 90 (at 11.8 +/- 1.1). The trend for isolated Leydig cells from these three ages was identical. In contrast, Sertoli cell AR mRNA was lowest on day 21 (at 19.3 +/- 1.0), intermediate on day 35 (at 24.5 +/- 1.4), and highest on day 90 (at 36.9 +/- 0.5). In Leydig cells, the highest level of AR mRNA was present during puberty, whereas the greatest amount of AR mRNA in Sertoli cells was present on day 90. This indicates that Leydig cells and Sertoli cells use different mechanisms to maintain AR levels. We infer from these data that Leydig cells are maximally sensitive to androgen during puberty, which is consistent with our hypothesis that androgens facilitate their differentiation.

Androgens↗

Vitamin A modifies the glycopeptide composition of cultured Sertoli cells.

Sertoli cells obtained from prepubertal rat testes were cultured in the presence or absence of retinol. Incorporation of monosaccharides and glycopeptide composition of the cells were studied under two experimental conditions. The results indicate that retinol increases the amount of mannose and glucosamine incorporated into cellular glycoconjugates. The labeled glycopeptides obtained from control and retinol-treated cells were separated by size and lectin affinity. Gel filtration analysis showed no size differences between the glycopeptides obtained from control and vitamin A-treated cells. Affinity chromatography on Concanavalin A and Wheat Germ Agglutinin of 3H-mannose-labeled glycopeptides showed that Sertoli cells cultured in the presence of retinol contain a higher percentage of high mannose-type glycopeptides compared with control cells. The effect of retinol on Sertoli cell glycopeptide composition is partially reversed by the administration of FSH.

Animals↗

Malignant large cell calcifying Sertoli cell tumor of the testis.

An otherwise normal 48-year-old man had malignant large cell calcifying Sertoli cell tumor of the testis. There were lymph node involvement and albugineal invasion at orchiectomy, and pulmonary metastases developed despite radiotherapy and chemotherapy. Our case and, to our knowledge, the only other reported case of malignant large cell calcifying Sertoli cell tumor had clinical and histopathological features related to aggressiveness, such as unusual patient age, large tumor size, cellular pleomorphism, high mitotic rate, necrosis and aneuploid deoxyribonucleic acid. Such characteristics are not found in benign large cell calcifying Sertoli cell tumors. Associated findings included granular change of the epididymal lining, probably due to compression by the large tumor.

Calcinosis↗

Paracrine control of human Leydig cell and Sertoli cell functions.

Two enriched populations of Leydig cells (hLCI and hLCII) have been characterized in human testes; it is noteworthy, that in the presence of hCG the steroid ouput is higher in hLCII when compared to hLCI; conversely, the basal production of steroids is greater in hLCI than in hLCII. The addition of increasing amounts of seminiferous tubule-conditioned medium to the purified Leydig cells leads to a dose-related enhancement of the steroid production in both Leydig cell fractions under basal and hCG-stimulated conditions. It is therefore obvious that a paracrine factor (or factors) from seminiferous tubular origin influences positively and with a high efficiency the human Leydig cell function. The human Sertoli cell synthesizes lactate, estradiol-17beta and several proteins, namely transferrin, ferritin and inhibin. In the presence of germ cells the Sertoli cell production of estradiol-17beta is decreased whereas the transferrin and inhibin outputs are enhanced. In addition the lactate dehydrogenase, gamma-glutamyl transpepetidase, alkaline phosphatase and creatine phosphokinase activities have been quantitated in various human Sertoli cell preparations. It should be kept in mind that germ cells exert an important influence on the adult Sertoli cell secretory activity through either direct contact and/or via secreted factors; moreover germ cells potentialize the stimulating effect of FSH on the Sertoli cell function and indirectly the Leydig cell output of testosterone via the Sertoli cell secretion of paracrine factor(s).

Adult↗

Evaluation on the phagocytosis of apoptotic spermatogenic cells by Sertoli cells in vitro through detecting lipid droplet formation by Oil Red O staining.

During spermatogenesis, more than half of the differentiating spermatogenic cells undergo apoptosis before they mature into spermatozoa. Ultrastructure studies showed that the formation of lipid droplets in Sertoli cells was associated with phagocytosis of residual bodies and apoptotic germ cells by Sertoli cells. Here, a relationship between the phagocytosis of apoptotic spermatogenic cells and lipid droplet formation in Sertoli cells was studied in vitro by Oil Red O (ORO) staining. The results confirmed that the formation of lipid droplets was a result of phagocytosis of apoptotic spermatogenic cells in Sertoli cells. By comparing phagocytosis of apoptotic spermatogenic cells and thymocytes by Sertoli cells to that by macrophages, we demonstrated that the lipid droplets accumulation in phagocytes depended on phagocytosed apoptotic cell type, but not phagocyte type. However, the size of lipid droplets was related to the type of phagocytes. By this approach, we found that Sertoli cells at different postnatal stages of development had a similar phagocytic ability. These results suggested that the detection of lipid droplets by ORO staining was a practical method to evaluate the phagocytic functions of Sertoli cells in vitro. This approach could also be considered as an in vitro model to study the lipid formation, metabolism, and function in Sertoli cells.

Animals↗

Heregulins or neu differentiation factors and the interactions between peritubular myoid cells and Sertoli cells.

Interactions between (mesenchymal) peritubular myoid cells and (epithelial) Sertoli cells play an important role in the control of Sertoli cell function and spermatogenesis. The factors involved, however, have only partially been identified. Heregulins or neu differentiation factors (NDFs) mediate mesenchymal-epithelial interactions in a variety of tissues, but their role in the testis has not been investigated. Here we demonstrate that recombinant human heregulin-alpha (Her-alpha) and Her-beta stimulate transferrin and androgen-binding protein production by cultured rat Sertoli cells up to 2.5-fold. These effects are more pronounced than those of previously identified growth factors active in this assay, such as insulin-like growth factor I and basic fibroblast growth factor. Combination with these factors results in additive effects and in marked morphological changes in the Sertoli cell cultures, including formation of tubule-like structures. Stimulation of androgen-binding protein secretion is paralleled by increased levels of the corresponding messenger RNA. This parallelism was less consistent for transferrin. Semiquantitative RT-PCR indicates that the expression of NDF-alpha and NDF-beta is more pronounced in peritubular cells than in Leydig or Sertoli cells. Conversely, the main receptors for heregulins/NDFs, HER-3 and HER-4, are predominantly expressed in Sertoli cells. A displacement assay confirms the presence of high-affinity binding sites for [125I]Her-beta on intact Sertoli cells and reveals parallel displacement curves for Her-beta, Her-alpha, and concentrated peritubular cell-conditioned medium (PTCM; estimated ED50 values: 1 ng/ml, 50 ng/ml, and 130 microg protein/ml, respectively), indicating that peritubular cells secrete one or more factors able to compete for heregulin receptors. It is concluded that heregulins/NDFs may play a role in mesenchymal-epithelial interactions in the testis. Estimates of the concentrations of heregulins in PTCM, however, make it unlikely that they contribute significantly to the effects observed with low concentrations of PTCM and ascribed to the putative mediator PModS (peritubular factor that modulates Sertoli cell function). Further investigations will be required to define the exact role of heregulins in the testis.

Androgen-Binding Protein↗

Mouse Sertoli cells secrete mannose 6-phosphate containing glycoproteins that are endocytosed by spermatogenic cells.

Sertoli cells were isolated from prepubertal mice and cultured in serum-free medium to determine whether they secrete glycoproteins containing mannose 6-phosphate (M6P). Assays of the conditioned medium for lysosomal enzyme precursors, which typically bear the M6P recognition marker, indicated that Sertoli cells selectively secreted beta-N-acetylhexosaminidase and alpha-mannosidase, but not beta-glucuronidase or beta-galactosidase. Sertoli cells were labeled metabolically with [35S]methionine and the conditioned medium was fractionated on a cation-independent M6P receptor affinity column. Most of the secreted proteins did not bind to the column (peak A); however, approximately 10% of the radioactivity eluted as a low-affinity fraction (peak B), and 5-11% of the recovered cpm bound to the column and were eluted with 2.5 mM M6P (peak C). The radiolabeled proteins in each fraction were analyzed by one- and two-dimensional electrophoresis and fluorography. Two protein bands with molecular weights of 30,000 and 35,000 were present in peak B. Peak C contained at least ten M6P-containing glycoproteins with molecular weights between 30,000 and 135,000 and isoelectric points < 6.5. The 35,000-molecular-weight constituent prominent both in peaks B and C was identified as procathepsin L by immunoprecipitation with a specific antibody. When pachytene spermatocytes and round spermatids were cultured overnight in the presence of peak C glycoproteins radiolabeled with 125I, both germ cell types accumulated these Sertoli M6P-glycoproteins by a receptor-mediated process that was specifically inhibited by M6P. The Sertoli M6P-glycoproteins taken up by germ cells were processed to lower molecular weight forms. These results provide evidence that M6P receptors on the surface of spermatogenic cells endocytose secrete glycoproteins that are likely to be present in the seminiferous epithelium.

Animals↗

Condensation behaviour of the human X chromosome in male germ cells and Sertoli cells examined by fluorescence in situ hybridization.

The chromatin condensation behaviour of the human X chromosome has been studied by fluorescence in situ hybridization (FISH) analysis in germ cells and Sertoli cells of the adult testis, and comparisons are made with previous findings for the human Y chromosome and for chromosome 7. In meiotic prophase, the X chromosome can be seen to extend greatly at zygotene and to contract through pachytene into the sex vesicle. Such extension, which has also been noted for the human Y chromosome at this stage of meiosis, could be a prerequisite for XY pairing and crossing-over. By in situ hybridization analysis, the sex chromosomes of patients with 'Sertoli-cell-only' syndrome appear extremely contracted compared with the normally extended state of those in adult Sertoli cells of fertile men. By contrast, the state of expansion for chromosome 7 in Sertoli cells appears identical for sterile and fertile testes. This could suggest an association between gene-controlled germ cell losses and failure of expansion of the sex chromosome axes. The variable patterns of extension and contraction for the X and Y chromosome axes in germ cells and Sertoli cells might provide underlying clues to patterns of expression noted for sex-linked genes in the human testis.

Adult↗

Regulatory influence of germ cells on sertoli cell function in the pre-pubertal rat after acute irradiation of the testis.

While germ cell regulation of Sertoli cells has been extensively explored in adult rats in vivo, in contrast, very little is known about germ cell influence on Sertoli cell function at the time when spermatogenesis begins and develops. In the present study various Sertoli cell parameters (number, testicular androgen binding protein (ABP) and testin, serum inhibin-B and, indirectly, follicle-stimulating hormone (FSH)) were investigated after the exposure of 19-day-old rats to a low dose of 3 Grays of gamma-rays. Differentiated spermatogonia were the primary testicular targets of the gamma-rays, which resulted in progressive maturation depletion, sequentially and reversibly affecting all germ cell classes. Testicular weight declined to a nadir when pachytene spermatocytes and spermatids were depleted from the seminiferous epithelium and complete or near complete recovery of spermatogenesis and testicular weight was observed at the end of the experiment. Blood levels of FSH and ABP were normal during the first 11 days after irradiation, when spermatogonia and early spermatocytes were depleted. While the number of Sertoli cells was not significantly affected by the irradiation, from days 11-66 after gamma-irradiation, ABP production declined and FSH levels increased when pachytene spermatocytes and spermatids were depleted and the recovery of these parameters was only observed when spermatogenesis was fully restored. Comparison of the pattern of change in serum levels of inhibin-B and testicular levels of testin and of germ cell numbers strongly suggest a relationship between the disappearance of spermatocytes and spermatids from the seminiferous epithelium and the decrease in levels of inhibin-B and increase in levels of testin from 7 to 36 days post-irradiation. Levels of testin and inhibin-B were restored before spermatogenesis had totally returned to normal. In conclusion, this in vivo study shows that pre-pubertal Sertoli cell function is under the complex control of various germ cell classes. This control presents clear differences when compared with that previously observed in adult animals and depends on the Sertoli cell parameter of interest, as well as on the germ cell type.

Androgen-Binding Protein↗

Lamellar germ cell processes: structures for possible interaction between germs cells and Sertoli cells during spermatogonial differentiation and early meiotic prophase.

The recent finding of a stage-dependent topographical relationship of basally located germ cells to Sertoli-Sertoli interspaces in the rat testis ( Ulvik 1983a ) has suggested a focal interaction between Sertoli cells and early germ cells at the sites where 2 Sertoli cells meet over a basal germ cell. Focal, stage-dependent interaction between Sertoli cells and germ cells may require specializations of the germ cell membrane. Therefore, in the present work the surface of spermatogonia and spermatocytes has been studied under the electron microscope throughout the cycle of the seminiferous epithelium. Lamellar processes, 1-3 micron long, were found to emerge from germ cells and protrude into the spaces between the overlying Sertoli cells. The processes appeared on differentiating type A spermatogonia after stage VI, on intermediate and type B spermatogonia, and on leptotene, zygotene, and early pachytene spermatocytes; however, they were not found on type A spermatogonia prior to stage VII or on preleptotene spermatocytes at stage VII. The lamellar processes are thus stage-specific and may be sites of focal interaction with the Sertoli cells. Since the cell processes always protrude into the Sertoli-Sertoli interspaces this finding supports the hypothesis of focal, stage-dependent interaction between early germ cells and Sertoli cells via the narrow Sertoli-Sertoli interspaces.

Animals↗

Influence of germ cells upon Sertoli cells during continuous low-dose rate gamma-irradiation of adult rats.

The effects of continuous gamma-irradiation of adult rats at two low-dose rates (7 cGy and 12 cGy/day; up to a total dose of 9.1 Gy and 10.69 Gy 60Co gamma-ray, respectively) were investigated. Over a period of 3-131 days of irradiation, groups of experimental and control animals were killed. Body weight, testis, epididymis, prostate and seminal vesicle weights, the number of germ cells and Sertoli cells, tubular ultrastructure, epididymal and testicular levels of biologically active androgen-binding protein (ABP), and the plasma concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone were monitored. Irradiation had no effect on body weight, whereas testicular and epididymal weight began to decrease following 35 and 50 days of irradiation at 7 and 12 cGy, respectively. At 7 cGy the target cells of the gamma-rays were essentially A spermatogonia, whereas at 12 cGy A spermatogonia and preleptotene spermatocytes were primarily affected. This resulted in a progressive and sequential dose-related reduction in the number of pachytene spermatocytes, round spermatids and late spermatids (LS). Under both irradiation procedures the Sertoli cell number remained unchanged whereas partial (7 cGy) or no change (12 cGy) was seen at the Leydig cell level. Whatever the irradiation protocol, from the time LS numbers decreased, vacuolisation of the Sertoli cell cytoplasm progressively occurred, followed by thickening and folding of the peritubular tissue. Moreover, in parallel to the drop in the number of these germ cell types, ABP production fell whereas FSH levels rose. A highly significant positive correlation was found between LS numbers and these Sertoli cell parameters. This study supports our previous concept of a control of certain important aspects of Sertoli cell function by late spermatids in the adult rat.

Androgen-Binding Protein↗

Effects of basic fibroblast growth factor and nerve growth factor on lactate production, gamma-glutamyl transpeptidase and aromatase activities in cultured Sertoli cells.

Sertoli cells are under the control of FSH and androgens and also respond to polypeptidic factors locally produced. Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) have been proposed to belong to the large set of intratesticular regulators. The aim of the present investigation was to analyze the effects of bFGF and NGF on lactate production, gamma-glutamyl transpeptidase (gamma-GTP) and aromatase activities. Cultured Sertoli cells dose-dependently responded to bFGF by increasing lactate production and gamma-GTP activity under basal conditions. In FSH-stimulated cultures, a synergistic effect of FSH with bFGF for lactate production was observed. NGF did not produce changes in lactate production or gamma-GTP activity at any dose tested. Both peptides decreased FSH-stimulated aromatase activity. These results provide additional evidence for the participation of bFGF and NGF in the complex network of intratesticular regulators. bFGF has pleiotropic effects on Sertoli cell function while the actions of NGF seem to be more limited.

Animals↗

A Novel voltage-dependent chloride current activated by extracellular acidic pH in cultured rat Sertoli cells.

Sertoli cells from mammalian testis are key cells involved in development and maintenance of stem cell spermatogonia as well as secretion of a chloride- and potassium-rich fluid into the lumen of seminiferous tubules. Using whole-cell patch clamp experiments, a novel chloride current was identified. It is activated only in the presence of an extracellular acidic pH, with an estimated half-maximal activation at pH 5.5. The current is strongly outwardly rectifying, activated with a fast time-dependent onset of activation but a slow time-dependent kinetic at depolarization pulses. The pH-activated chloride current was not detected at physiological or basic pH and is not sensitive to intracellular or extracellular Ca2+ variation. Diphenylamine-2-carboxylic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid blocked the induced currents, and its anionic selectivity sequence was Cl- > Br- > I-> gluconate. We have performed a reverse transcription-PCR analysis to search for voltage-dependent chloride rClC channels in cultured rat Sertoli cells. Among the nine members of the family only rClC-2, rClC-3, rClC-6, and rClC-7 have been identified. The inwardly rectifying rClC-2 chloride current was activated by hyperpolarization but not by pH variation. A different depolarization-activated outwardly rectifying chloride current was activated only by hypotonic challenge and may correspond either to rClC-3 or rClC-6. Immunolocalization experiments demonstrate that rClC-7 resides in the intracellular compartment of Sertoli cells. This study provides the first functional identification of a native acid-activated chloride current. Based on our molecular analysis of rClC proteins, this new chloride current does not correspond to rClC-2, rClC-3, rClC-6, or rClC-7 channels. The potential physiological role of this native current in an epithelial cell from the reproductive system is discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Two plasma membrane antigens of testicular sertoli cells and H-2-restricted versus unrestricted lysis by female T cells.

Sertoli cell-only seminiferous tubules of sterile XX,Sxrl-male mice served as an excellent source of pure Sertoli cells. When H-2-compatible female mice were immunized 3 times with these Sertoli cells, resulting antibodies recognized two antigens on the plasma membrane of testicular Sertoli cells. They were male-specific, but ubiquitously expressed H-Y antigen and the cell lineage-specific antigen which Sertoli cells shared with ovarian follicular cells. Doubly primed (2 or 3 times in vivo, and once in vitro) cytotoxic T cells from these females lysed target Sertoli cells in both H-2-restricted and nonrestricted manners. While H-2-restricted killings were attributable to H-Y antigen, further work is needed to identify the Sertoli follicular cell lineage-specific antigen as the cause of H-2-nonrestricted killings.

Animals↗

Changes in the arrangement of actin filaments in myoid cells and Sertoli cells of rat testes during postnatal development and after experimental cryptorchidism.

BACKGROUND: Abundant actin filaments are present in myoid cells and Sertoli cells in the testis. In the adult rat, the filaments form a lattice arrangement within the myoid cell, and show a hexagonal pattern in the basal junctional regions of Sertoli cells. METHODS: Isolated seminiferous tubules and frozen sections were prepared from juvenile to adult Wistar rat testes, stained with FITC-conjugated phalloidin, and observed by confocal microscopy. Unilateral cryptorchidism was induced in adult rats, and seven days later, their testes were also examined. RESULTS: In the myoid cell, parallel actin filaments running circularly around the seminiferous tubules were observed at 15 and 20 days of age. Then, at 30 days, actin filaments arranged longitudinally along the tubular long axis appeared in addition to the circular bundles. A lattice arrangement of actin-filament bundles in myoid cells became obvious at 40 days, when elongated spermatids are found in the tubule. Actin filaments in the basal junctional regions of Sertoli cells did not acquire the hexagonal pattern seen in the adult testis until 30 days of age. In the cryptorchid testes, the arrangement of actin filaments in the both cells showed a remarkable change compared to the control testis; the filaments became thinner and disrupted. CONCLUSIONS: A lattice arrangement of the actin filaments in the myoid cell appear at around 30 days, before the completion of spermatogenesis. A hexagonal pattern of the filaments in the junctional regions of Sertoli cells has already developed at this age. Cryptorchidism affects the actin filaments of the both cells.

Actins↗

Bilateral testicular large-cell calcifying sertoli cell tumor and recurrent cardiac myxoma in a patient with Carney's complex.

Large-cell calcifying Sertoli cell tumors are exceedingly rare testicular tumors which can be distinguished from pure Sertoli cell tumors by distinctive microscopic features and multifocal and bilateral calcifications [1]. We describe the characteristic sonographic appearance of the tumor with pathologic correlation. This tumor also constitutes one of the conditions of Carney's complex [2], which includes cardiac and skin myxomas, myxoid mammary fibroadenomas, spotty skin pigmentation, primary pigmented nodular adrenocortical disease, pituitary adenoma and unusual testicular tumors, especially large-cell calcifying Sertoli cell tumor. We report the case of a 10-year-old boy with four of the above conditions. Recognition of this complex is important as cardiac myxomas can lead to lethal complications. Because of a tendency for familial occurrence, family members should be screened carefully [3].

Calcinosis↗

Large cell, calcifying, Sertoli cell tumour: a case report.

Large cell, calcifying, Sertoli cell tumour, because of its distinct pathological and clinical presentations, has recently been recognized as a variant of Sertoli cell tumour. A 26-year-old man with this tumour is described, with a 3-year follow-up. Large cell, calcifying, Sertoli cell tumours occur mostly in young people. At least 6 of the 14 cases reported in the literature were associated with endocrine disorders or cardiac myxoma, or both. The patient described in this report had no endocrine or other disorders.

Adult↗

Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells.

The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. A cellular proteoglycan associated with the peritubular cells is described which has properties similar to those of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. SC III has properties similar to those of a major Sertoli cell-secreted protein previously defined as a dimeric acidic glycoprotein. The stimulation by follicle-stimulating hormone of the incorporation of [35S]SO2(-4) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III (i.e. dimeric acidic glycoprotein), and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.

Animals↗