Clusterin (10. Seminiferous System and Spermatozoa )

Clusterin is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. Clusterin mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications.the detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. Clusterin was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins ^Ref .

Later studies confirmed thatt two forms of clusterin, the beta-chain monomer and the heterodimer, are present on the spermatozoal surface membrane and in seminal plasma ^Ref .

Transfection of ASC-17D Sertoli cells with a 20-base antisense oligonucleotide against clusterin mRNA resulted in extracellular release of LDH and DNA fragmentation. Sertoli cell death by antisense oligonucleotide of clusterin was sequence specific and dose dependent. Treatment of antisense oligonucleotide induced a marked reduction of synthesis for clusterin protein, but not for clusterin mRNA expression, suggesting the translational suppression of clusterin by antisense oligonucleotide. Further, microscopic observation showed that more noticeable cell death was induced by treating the antisense prior to plating the cells than by treating after cell attachment to the plate. From these results, we speculate that down-regulation of clusterin expression in the anchorage-dependent Sertoli cells prevents them from attaching to the plate, and therefore induces cell death ^Ref .

Clusterin mRNA levels were measured by quantitative RT-PCR in RNA extracted from testicular biopsies of 49 azoospermic patients classified according to testicular histology as having normal spermatogenesis or spermatogenic failure. When related to the percentage of Sertoli cells counted on a histological section of a neighbouring tissue sample, clusterin mRNA levels were significantly lower in the ‚spermatogenic failure‘ group compared with the control group ( P = 0.0043, respectively). These levels were also significantly lower in the cases of ‚constitutive‘ (cryptorchidism and Yq microdeletion) and ‚idiopathic‘ spermatogenic failures when compared with the control group; conversely, they were not significantly decreased in the group with ‚acquired spermatogenic failure‘ (orchitis, testicular traumatism, chemoradiotherapy and varicocele). These data further demonstrate an alteration of Sertoli cell functions in some human spermatogenic failures and suggest that a lack of Sertoli cell maturation may be involved in cases of constitutive or idiopathic spermatogenic failures ^Ref .

Western blots of cauda sperm membrane extracts of control animals and orchiectomized animals treated with testosterone had a very low level of epididymal clusterin, whereas extracts collected from orchiectomized animals revealed high levels of clusterin. It was suggested that, in the normal animal, clusterin is secreted into the lumen of the proximal epididymis where it binds to the sperm membrane. In the distal epididymis, clusterin dissociates from sperm and is processed (proteolysis/en­docytosis). I tis hypothesized that, in the absence of androgen, the processing and regulation of clusterin is disrupted ^Ref .

It was proposed that protective effects of clusterin are the core of the differential tolerance of ischemia by the various testicular cells and the morpholo gy of the postischemic testis. Suppression of ischemic damage selectively in Sertoli cells might be the consequence of the ability to produce clusterin, an endogenous inhibitor of ischemic injury. This hypothetical function of clusterin is supported by its immunosuppressive properties and its structural and functional identity to several types of human complement cytolysis inhibitors ^Ref .

In the initial segment and the caput epididymidis, neither orchidectomy nor testosterone replacement, at either dose, had any effect on clusterin mRNA concentrations. In the corpus and cauda epididymidis, bilateral orchidectomy resulted in a 3.5– and 9.4-fold increase, respectively, in clusterin mRNA concentrations, whereas testosterone replacement caused a dose-dependent decrease in clusterin mRNA concentrations. Unilateral orchidectomy was done to determine if clusterin mRNA concentrations are dependent on testicular factors released in the lumen of the epididymis. In the corpus and the cauda epididymidis, unilateral orchidectomy resulted in elevated clusterin mRNA concentrations in the IPsilateral epididymis. There were no changes in clusterin mRNA concentrations in the initial segment and caput epididymidis. These results provide complementary evidence that the message for clusterin is differentially regulated along the epididymis. During postnatal development clusterin mRNA concentrations in the caput-corpus epididymidis increased dramatically between 14 and 21 days as well as between 49 and 63 days. Interestingly, between 28 and 42 days, when serum testosterone concentrations are increasing, there was no change in the concentration of clusterin mRNA in the caput-corpus epididymidis. Similar results were observed in the cauda epididymidis with the exception that between 28 and 42 days, there was a dramatic decrease in clusterin mRNA in the cauda epididymidis. Together these experiments demonstrate that the regulation of clusterin mRNA concentrations is segment specific.in the initial segment and caput epididymidis there is no apparent regulation of clusterin by testicular factors,whereas in the corpus and cauda epididymidis testosterone can repress clusterin mRNA concentrations ^Ref .

Morphologically abnormal spermatozoa have an extensive surface coating of conventional 80 kDa native clusterin, but this form of clusterin is not detectable on normal spermatozoa. Normal spermatozoa, however, contain within the acrosomal cap a different form of clusterin, reactive with an anticlusterin alpha-chain antibody. Agglutinated spermatozoa, most of which are grossly abnormal, were intensely labelled with the antibody against conventional 80 kDa clusterin, suggesting that the ‚clustering‘ properties of this protein may play a role in the aggregation of abnormal spermatozoa. Anticlusterin monoclonal antibodies may be useful for semen analysis. Staining spermatozoa with anticlusterin monoclonal antibodies is a technically simple method which provides a visually obvious means of assessing spermatozoa morphology and acrosome status simultaneously. The current data also suggest that different functions of clusterin in the reproductive tract may be attributed to different molecular forms of the protein ^Ref .

The acrosomal form of clusterin is associated with the contents of the acrosome ^Ref .

It was suggested that the percentage of clusterin positive cells (CPS) in bull semen is potentially a better predictor of fertility than sperm motility or abnormal morphology ^Ref .

It was also suggested that the percentage of CPCs in ram semen could be a useful marker in poor-quality ejaculates ^Ref .

It was demonstrated the expression of clusterin gene in four leydig tumor cell lines, including mouse MA-10 and I-10 and rat R2C and LC-540. When the cells were incubated with 0.1 mM 8-bromo-cAMP or (Bu)2cAMP for 17 h, an unexpected, profound suppression of clusterin mRNA accumulation was observed. A 60–70% decrease in clusterin mRNA was observed in MA-10 and R2C cells, 10% in i-10 cells, and no apparent change in LC-540 cells. The inhibitory effect of cAMP was specific to the clusterin gene, since in the same cells cholesterol side-chain cleavage enzyme mRNA was drastically elevated in MA-10 and I-10 cells while alpha-tubulin mRNA levels were not changed in all four cell lines. The reduction could be detected as early as 4 h, and was evident at 17 h after cAMP administration. Removal of cAMP from culture media at 17 h prevented the decline of clusterin mRNA.the suppression of clusterin gene expression can also be demonstrated by treatment with human CG or forskolin, which were known to elevate intracellular cAMP levels22. The reduction of clusterin mRNA after exposure of MA-10 cells to cAMP is not due to a decrease in its transcriptional activity, but rather to an increase in the degradation of this mRNA through synthesis of a destabilizing protein(s) and its mRNA ^Ref .

Follicle-stimulating hormone had no effect on clusterin in rat Sertoli cells ^Ref .

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