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Quantification of lysosomal profile areas of principal cells. Lysosomal profile areas were estimated from digital images obtained by immunocytochemical studies for control (Con), castrated (Ct) and castrated with testosterone supplementation (Ct+T) animals. Calculations were performed on principal cells of the distal IS and proximal caput designated as the IS/Caput region and the distal corpus and proximal cauda designated as the corpus/ cauda region. Bars represent the means of lysosomal profile area per cell ± SEM from three independent experiments for IS/caput (A) and corpus/cauda (B) regions. a, b and c are significantly different from each other (p < 0.0001, obtained by Tukey's Multiple Comparisons Test). https://doi.org/10.1371/journal.pone.0250454.g002
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In the epididymis, lysosomal proteins of the epithelial cells are normally targeted from the Golgi apparatus to lysosomes for degradation, although their secretion into the epididymal lumen has been documented and associated with sperm maturation. In this study, cathepsin D (CatD) and prosaposin (PSAP) were examined in adult epididymis of control,...
Contexts in source publication
Context 1
... the difference in size was confirmed in castrated animals with significant statistical values in both of the 2 regions examined, i.e. IS/caput and corpus/cauda, as compared to control values (Fig 2A and 2B). ...
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... of testosterone to castrated animals did not rescue lysosomal size to control levels (Fig 2A and 2B), nor was the appearance of lysosomes identical to controls (Fig 1I-1L). ...
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... lysosomes were also evident in castrated animals supplemented with testosterone ( Fig 3B). Quantitatively, a statistically significant difference was noted in the size of principal cell lysosomes in the IS/caput region both in the absence (Fig 2A) and presence (Fig 2B) of testosterone as compared to controls. Thus the situation for lysosomal size is similar in the initial segment as for the other epididymal regions. ...
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... lysosomes were also evident in castrated animals supplemented with testosterone ( Fig 3B). Quantitatively, a statistically significant difference was noted in the size of principal cell lysosomes in the IS/caput region both in the absence (Fig 2A) and presence (Fig 2B) of testosterone as compared to controls. Thus the situation for lysosomal size is similar in the initial segment as for the other epididymal regions. ...
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... data from western blot immunoblotting of whole adult epididymal tissue (Fig 4A) revealed a significant increase of CatD levels observed as an intermediate form of 48 kDa in castrated animals of the three regions (Fig 4A, upper panel) correlating with the increase in size of lysosomes of principal cells caused by an enhancement of the endocytic process of these cells (Fig 2A and 2B). Administration of testosterone to castrated animals reversed the situation of CatD expression to control levels in all epididymal regions (Fig 4A, lower panel) suggesting that testosterone fine-tunes the levels of CatD expression in lysosomes of principal cells in control animals but not their size. ...
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... reactive bodies were deduced to be lysosomes as demonstrated by our previous EM cytochemical studies [13,14]. Qualitatively, lysosomes in principal cells of these regions appeared to be increased in size in castrated animals ( Figs 3E, 3F, 5E-5H and 6A-6C) compared to controls, as verified by quantification of CatD reactive lysosomes (Fig 2A and 2B). Some of these large lysosomes revealed a dense ring of reactivity enveloped by a central pale stained core (Figs 3E, 5E, 5F, 5H and 6A-6C). ...
Context 7
... the expression of PSAP in lysosomes of principal cells, as well as its secretion into the lumen, was not modified by absence of testosterone. Moreover, while a qualitatively significant increase in size of lysosomes of principal cells was noted by LM-IHC in both castrated animals with and without testosterone replacement (Fig 2A and 2B), this did not correlate with an increased expression of PSAP in these cells. It is suggested that the size of lysosomes is a result of the adverse effects of castration on the endocytic activity of principal cells, and that this is independent on the expression of PSAP. ...
Similar publications
The selective transport to lysosomes can be mediated by either mannose-6-phosphate receptors (CD-MPR and CI-MPR) or sortilin. In mammalian epididymis, some lysosomal proteins are secreted into the lumen through unknown mechanisms. To investigate the underlying mechanisms of lysosomal protein transport in epididymal cells we studied the expression a...
Citations
... The endoplasmic reticulum is the location of protein synthesis and assembly as well as lipid and membrane synthesis, which plays crucial roles in sperm maturation modulation [13,14]. Meanwhile, the lysosome mediates the proper biochemical and molecular modifications of sperm by endocytosis and modifying glycoproteins and lipids on the sperm plasma membrane, which induces a fertile and motile state [15,16]. Epididymal cholesterol homeostasis is vital for a proper post-testicular maturation of spermatozoa [17]. ...
Background
The epididymis is a highly regionalized tubular organ possesses vectorial functions of sperm concentration, maturation, transport, and storage. The epididymis-expressed genes and proteins are characterized by regional and developmental dependent pattern. However, a systematic and comprehensive insight into the postnatal development dependent changes in gene and protein expressions of porcine epididymis is still lacking. Here, the RNA and protein of epididymis of Duroc pigs at different postnatal development stages were extracted by using commercial RNeasy Midi kit and extraction buffer (7 M Urea, 2 M thiourea, 3% CHAPS, and 1 mM PMSF) combined with sonication, respectively, which were further subjected to transcriptomic and proteomic profiling.
Results
Transcriptome analysis indicated that 198 and 163 differentially expressed genes (DEGs) were continuously up-regulated and down-regulated along with postnatal development stage changes, respectively. Most of the up-regulated DEGs linked to functions of endoplasmic reticulum and lysosome, while the down-regulated DEGs mainly related to molecular process of extracellular matrix. Moreover, the following key genes INSIG1, PGRMC1, NPC2, GBA, MMP2, MMP14, SFRP1, ELN, WNT-2, COL3A1, and SPARC were highlighted. A total of 49 differentially expressed proteins (DEPs) corresponding to postnatal development stages changes were uncovered by the proteome analysis. Several key proteins ACSL3 and ACADM, VDAC1 and VDAC2, and KNG1, SERPINB1, C3, and TF implicated in fatty acid metabolism, voltage-gated ion channel assembly, and apoptotic and immune processes were emphasized. In the integrative network, the key genes and proteins formed different clusters and showed strong interactions. Additionally, NPC2, COL3A1, C3, and VDAC1 are located at the hub position in each cluster.
Conclusions
The identified postnatal development dependent genes and proteins in the present study will pave the way for shedding light on the molecular basis of porcine epididymis functions and are useful for further studies on the specific regulation mechanisms responsible for epididymal sperm maturation.
... After cooling to room temperature, the slides were immersed in a peroxidase blocker solution containing 0.03% hydrogen peroxide and 0.031 M sodium azide (Dako Canada Inc., Mississauga, ON). They were then incubated overnight at 4˚C with either anti-prosaposin (PSAP, RRID:AB_2792974) [79], anti-cytokeratin 5 (CK5, Covance Cat# PRB-160P-100, RRID:AB_10063444) [80] or anti-cathepsin D (CathD, Santa Cruz Biotechnology Cat# sc-6487, RRID:AB_637895) [81] antibodies in 50 mM Tris-Cl, pH 7.4 containing 1% Bovine serum albumin (BSA, Sigma A7511). ...
Heparan sulfate (HS), an abundant component of the apical cell surface and basement membrane, belongs to the glycosaminoglycan family of carbohydrates covalently linked to proteins called heparan sulfate proteoglycans. After endocytosis, HS is degraded in the lysosome by several enzymes, including heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and in its absence causes Mucopolysaccharidosis III type C (Sanfilippo type C). Since endocytosis occurs in epithelial cells of the testis and epididymis, we examined the morphological effects of Hgsnat inactivation in these organs. In the testis, Hgsnat knockout ( Hgsnat-Geo ) mice revealed statistically significant decrease in tubule and epithelial profile area of seminiferous tubules. Electron microscopy (EM) analysis revealed cross-sectional tubule profiles with normal and moderately to severely altered appearances. Abnormalities in Sertoli cells and blood-testis barrier and the absence of germ cells in some tubules were noted along with altered morphology of sperm, sperm motility parameters and a reduction in fertilization rates in vitro . Along with quantitatively increased epithelial and tubular profile areas in the epididymis, EM demonstrated significant accumulations of electrolucent lysosomes in the caput-cauda regions that were reactive for cathepsin D and prosaposin antibodies. Lysosomes with similar storage materials were also found in basal, clear and myoid cells. In the mid/basal region of the epithelium of caput-cauda regions of KO mice, large vacuolated cells, unreactive for cytokeratin 5, a basal cell marker, were identified morphologically as epididymal mononuclear phagocytes (eMPs). The cytoplasm of the eMPs was occupied by a gigantic lysosome suggesting an active role of these cells in removing debris from the epithelium. Some eMPs were found in proximity to T-lymphocytes, a feature of dendritic cells. Taken together, our results reveal that upon Hgsnat inactivation, morphological alterations occur to the testis affecting sperm morphology and motility parameters and abnormal lysosomes in epididymal epithelial cells, indicative of a lysosomal storage disease.
... After cooling to room temperature, the slides were immersed in a peroxidase blocker solution containing 0.03% hydrogen peroxide and 0.031 M sodium azide (Dako Canada Inc., Mississauga, ON). They were then incubated overnight at 4˚C with either anti-prosaposin (PSAP, RRID:AB_2792974) [79], anti-cytokeratin 5 (CK5, Covance Cat# PRB-160P-100, RRID:AB_10063444) [80] or anti-cathepsin D (CathD, Santa Cruz Biotechnology Cat# sc-6487, RRID:AB_637895) [81] antibodies in 50 mM Tris-Cl, pH 7.4 containing 1% Bovine serum albumin (BSA, Sigma A7511). ...
Heparan sulfate (HS), an abundant component of the apical cell surface and basement membrane, belongs to the glycosaminoglycan family of carbohydrates covalently linked to proteins called heparan sulfate proteoglycans. After endocytosis, HS is degraded in the lysosome by several enzymes, including heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and in its absence causes Mucopolysaccharidosis III type C (Sanfilippo type C). Since endocytosis occurs in epithelial cells of the testis and epididymis, we examined the morphological effects of Hgsnat inactivation in these organs. In the testis, Hgsnat knockout (Hgsnat-Geo) mice revealed statistically significant decrease in tubule and epithelial profile area of seminiferous tubules. Electron microscopy (EM) analysis revealed cross-sectional tubule profiles with normal and moderately to severely altered appearances. Abnormalities in Sertoli cells and blood-testis barrier and the absence of germ cells in some tubules were noted along with altered morphology of sperm, sperm motility parameters and a reduction in fertilization rates in vitro. Along with quantitatively increased epithelial and tubular profile areas in the epididymis, EM demonstrated significant accumulations of electrolucent lyso-somes in the caput-cauda regions that were reactive for cathepsin D and prosaposin anti-bodies. Lysosomes with similar storage materials were also found in basal, clear and myoid cells. In the mid/basal region of the epithelium of caput-cauda regions of KO mice, large vac-uolated cells, unreactive for cytokeratin 5, a basal cell marker, were identified morphologically as epididymal mononuclear phagocytes (eMPs). The cytoplasm of the eMPs was occupied by a gigantic lysosome suggesting an active role of these cells in removing debris from the epithelium. Some eMPs were found in proximity to T-lymphocytes, a feature of PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.
... After cooling to room temperature, the slides were immersed in a peroxidase blocker solution containing 0.03% hydrogen peroxide and 0.031 M sodium azide (Dako Canada Inc., Mississauga, ON). They were then incubated overnight at 4˚C with either anti-prosaposin (PSAP, RRID:AB_2792974) [79], anti-cytokeratin 5 (CK5, Covance Cat# PRB-160P-100, RRID:AB_10063444) [80] or anti-cathepsin D (CathD, Santa Cruz Biotechnology Cat# sc-6487, RRID:AB_637895) [81] antibodies in 50 mM Tris-Cl, pH 7.4 containing 1% Bovine serum albumin (BSA, Sigma A7511). ...
... As in other organs of the male reproductive tract, the epididymis secretory activity has been found to be hormone dependent. Previous reports have shown that androgen deprivation induces changes in the expression, intracellular distribution, and secretion, of both CatD and PSAP in rat epididymis 9,10,15,16 . However, the mechanisms that regulate the secretion of these proteins are not fully understood. ...
... Experimental evidence indicates that CatD can also be transported by the membrane receptor sortilin, through its interaction with PSAP, the natural ligand of this receptor [29][30][31] . Studying the role of sortilin in the intracellular transport of PSAP-CatD complexes 29 might be key to elucidate whether this receptor participates in the CatD secretion by the epididymal cells 16 . ...
... One of the paradigms of lysosomal protein secretion occurs in the mammalian epididymis, whose epithelium synthesizes and releases abundant lysosomal proteins into the organ lumen 9,10,16,41,43 . Among them, CatD has been detected in the rat epididymal lumen, which is secreted as the 52 kDa precursor pro-CatD 10 probably under androgen control. ...
The selective transport to lysosomes can be mediated by either mannose-6-phosphate receptors (CD-MPR and CI-MPR) or sortilin. In mammalian epididymis, some lysosomal proteins are secreted into the lumen through unknown mechanisms. To investigate the underlying mechanisms of lysosomal protein transport in epididymal cells we studied the expression and distribution of cathepsin D (CatD) and prosaposin (PSAP) in a sortilin knocked down RCE-1 epididymal cell line (RCE-1 KD) in comparison with non-transfected RCE-1 cells. In RCE-1 cells, CatD was found in the perinuclear zone and co-localize with sortilin, whereas in RCE-1 KD cells, the expression, distribution and processing of the enzyme were altered. In turn, PSAP accumulated intracellularly upon sortilin knock-down and redistributed from LAMP-1-positive compartment to a perinuclear location, remaining co-localized with CatD. Interestingly, the sortilin knock-down induced CD-MPR overexpression and a redistribution of the receptor from the perinuclear zone to a dispersed cytoplasmic location, accompanied by an increased co-localization with CatD. The increase in CD-MPR could result from a compensatory response for the proper delivery of CatD to lysosomes in epididymal cells. The intracellular pathway taken by lysosomal proteins could be an approach for addressing further studies to understand the mechanism of exocytosis and therefore the role of these proteins in the epididymis. The mammalian epididymis plays a crucial role in the development of the sperm fertilizing capacity, providing a proper environment for gamete maturation 1. The composition of the epididymal luminal fluid varies among mammalian species and organ regions (caput, corpus and cauda) 2,3. Caput lining cells secrete 70-80% of the total epididymal proteins 4,5 , which play a critical role in sperm maturation. Among these proteins, there are several enzymes that participate in the remodeling of the sperm surface as a stage in the maturation of gametes 6-8. Some of these enzymes are lysosomal proteins, which are synthesized by the epididymal epithelium and secreted into the organ lumen 9,10. The secretion mechanism of these enzymes is still poorly understood, and their participation in sperm maturation is not fully known. The lysosomal protease cathepsin D (CatD) has been proposed as a key enzyme in the remodeling of the sperm plasma membrane and improvement of its fertilizing capacity 11-13. Other authors have suggested the participation of CatD in the processing of other lysosomal proteins such as prosaposin (PSAP) 14. Secreted CatD and PSAP have been shown to be acquired by the gametes in the epididymal duct, suggesting a role of these proteins OPEN
... As in other organs of the male reproductive tract, the epididymis secretory activity has been found to be hormone dependent. Previous reports have shown that androgen deprivation induces changes in the expression, intracellular distribution, and secretion, of both CatD and PSAP in rat epididymis 9,10,15,16 . However, the mechanisms that regulate the secretion of these proteins are not fully understood. ...
... Experimental evidence indicates that CatD can also be transported by the membrane receptor sortilin, through its interaction with PSAP, the natural ligand of this receptor [29][30][31] . Studying the role of sortilin in the intracellular transport of PSAP-CatD complexes 29 might be key to elucidate whether this receptor participates in the CatD secretion by the epididymal cells 16 . ...
... One of the paradigms of lysosomal protein secretion occurs in the mammalian epididymis, whose epithelium synthesizes and releases abundant lysosomal proteins into the organ lumen 9,10,16,41,43 . Among them, CatD has been detected in the rat epididymal lumen, which is secreted as the 52 kDa precursor pro-CatD 10 probably under androgen control. ...
The selective transport to lysosomes can be mediated by either mannose-6-phosphate receptors (CD-MPR and CI-MPR) or sortilin. In mammalian epididymis, some lysosomal proteins are secreted into the lumen through unknown mechanisms. To investigate the underlying mechanisms of lysosomal protein transport in epididymal cells we studied the expression and distribution of cathepsin D (CatD) and prosaposin (PSAP) in a sortilin knocked down RCE-1 epididymal cell line (RCE-1 KD) in comparison with non-transfected RCE-1 cells. In RCE-1 cells, CatD was found in the perinuclear zone and co-localize with sortilin, whereas in RCE-1 KD cells, the expression, distribution and processing of the enzyme were altered. In turn, PSAP accumulated intracellularly upon sortilin knock-down and redistributed from LAMP-1-positive compartment to a perinuclear location, remaining co-localized with CatD. Interestingly, the sortilin knock-down induced CD-MPR overexpression and a redistribution of the receptor from the perinuclear zone to a dispersed cytoplasmic location, accompanied by an increased co-localization with CatD. The increase in CD-MPR could result from a compensatory response for the proper delivery of CatD to lysosomes in epididymal cells. The intracellular pathway taken by lysosomal proteins could be an approach for addressing further studies to understand the mechanism of exocytosis and therefore the role of these proteins in the epididymis.
... Previous studies show that of the four primary epididymal epithelial cell types enriched based on DEGs, principal cells are particularly sensitive to the elimination of circulating androgens, while basal cells, narrow cells and clear cells are less likely to be affected [20]. One of the most obvious morphological changes after castration is the decline in the number of apical microvilli on principal cells. ...
Background: Prostate cancer (PCa) is one of the most diagnosed cancers in the world. PCa inevitably progresses to castration-resistant prostate cancer (CRPC) after androgen deprivation therapy treatment, and castration-resistant state means a shorter survival time than other causes. Here we aimed to define castration-dependent and -independent diver genes and molecular pathways in CRPC which are responsible for such lethal metastatic events.
Methods: By employing digital gene expression (DGE) profiling , the alterations of the epididymal gene expression profile in the mature and bilateral castrated rat were explored. Then we detect and characterize the castration-dependent and -independent genes and pathways with two data set of CPRC-associated gene expression profiles publicly available on the NCBI.
Results: We identified 1,632 up-regulated and 816 down-regulated genes in rat’s epididymis after bilateral castration. Differential expression analysis of CRPC samples compared with the primary PCa samples was also done. In contrast to castration, we identified 97 up-regulated genes and 128 down-regulated genes that changed in both GEO dataset and DGE profile, and 120 up-regulated genes and 136 down-regulated genes changed only in CRPC, considered as CRPC-specific genes independent of castration. CRPC-specific DEGs were mainly enriched in cell proliferation, while CRPC-castration genes were associated with prostate gland development. NUSAP1 and NCAPG were identified as key genes, which might be promising biomarkers of the diagnosis and prognosis of CRPC.
Conclusions: Our study will provide insights into gene regulation of CRPC dependent or independent of castration and will improve understandings of CRPC development and progression.
The epididymal function and gene expression in mammals are under the control of the testis. Sex steroids are secreted from the testis and act on the epididymis in an endocrine manner. There is another, non-sex steroidal secreted signaling, named lumicrine signaling, in which testis-derived secreted proteins go through the male reproductive tract and act on the epididymis. The effects of such multiple regulations on the epididymis by the testis have been investigated for many genes. The recent development of high-throughput next-generation sequencing now enables us a further comparative survey of endocrine and lumicrine action-dependent gene expression. In the present study, testis-derived endocrine and lumicrine actions on epididymal gene expression were comparatively investigated by RNA-seq transcriptomic analyses. This investigation utilized experimental animal models in which testis-derived endocrine and/or lumicrine actions were interfered with, such as unilateral or bilateral orchidectomy. By bilateral orchidectomy, which interferes with both endocrine and lumicrine actions, 431 genes were downregulated. By unilateral orchidectomy, which also interferes with endocrine and lumicrine actions by the unilateral testis, but the endocrine action was compensated by the contralateral testis, 283 genes were downregulated. The content of such genes downregulated by unilateral orchidectomy was like those of lumicrine action-interfered efferent duct-ligation, W/Wv, and Nell2−/− mice. When genes affected by unilateral and bilateral orchidectomy were compared, 154 genes were commonly downregulated, whereas 217 genes were specifically downregulated only by bilateral orchidectomy, indicating the distinction between endocrine and lumicrine actions on the proximal epididymal transcriptome. Comparative transcriptome analyses also showed that the expressions of genes emerging since Amniota were notably impacted by bilateral orchidectomy, unilateral orchidectomy, and lumicrine action-interfering treatments; the degree of influence from these treatments varied based on the evolutionary stage beyond Amniota. These findings unveil an evolutional transition of regulated gene expression in the proximal epididymis by two different testis-derived signaling mechanisms.
The physiological functions of the mammalian epididymis are typically regulated by the testes. In addition to sex steroids secreted by testicular Leydig cells, which act on the epididymis in an endocrine manner, there is a non-sex-steroidal signaling pathway known as the lumicrine pathway. This lumicrine signaling pathway involves ligand proteins secreted from germ cells within the testicular seminiferous tubules traversing the male reproductive tract, which induce epithelial differentiation in the epididymis. These findings prompted an inquiry into whether treatments influencing testis physiology can disrupt epididymal function by interfering with testis-epididymis communication. Busulfan, an alkylating agent commonly used to deplete testicular germ cells in reproductive biology, has not been sufficiently explored because of its effects on the epididymis. This study investigated the effects of busulfan administration on the proximal epididymis using histological and transcriptomic analyses. Notably, busulfan, as opposed to the vehicle dimethyl sulfoxide (DMSO), altered the morphology of the initial segment of the epididymis, leading to a reduction in the cell height of the luminal epithelium. RNA sequencing identified 185 significantly downregulated genes in the proximal epididymis of busulfan-administered mice compared to DMSO-administered mice. Comparative transcriptome analyses revealed similarities between the epididymal transcriptome of busulfan-administered mice and lumicrine-deficient mice, such as efferent-duct-ligated W/Wv and Nell2-/- mice. However, this differed from that of bilaterally orchidectomized mice, in which both the endocrine and lumicrine signaling pathways were simultaneously ablated. Collectively, these results suggested that the harmful effects of busulfan on the proximal epididymis are secondary consequences of the ablation of testis-epididymis lumicrine signaling.
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