Sandra Whelly

Nonpathological Extracellular Amyloid Is Present during Normal Epididymal Sperm Maturation

Amyloids are aggregated proteins characterized by a specific cross-β-sheet structure and are typically associated with neurodegenerative diseases including Alzheimers disease. Recently, however, several nonpathological amyloids have been found in intracellular organelles of normal mammalian tissues suggesting that amyloid may also carry out biological functions. We previously have shown that the epididymal cystatin CRES (cystatin-related epididymal spermatogenic), cst8, a reproductive-specific member of the cystatin superfamily of cysteine protease inhibitors, forms amyloid in vitro suggesting that CRES amyloid may also form in vivo within the epididymal lumen. Here we show that amyloid structures containing CRES are a component of the normal mouse epididymal lumen without any apparent cytotoxic effects on spermatozoa and that these structures change along the length of the tubule. These studies suggest the presence of a functional amyloid structure that may carry out roles in sperm maturation or maintenance of the luminal milieu and which itself may undergo maturational changes along the epididymis. In contrast to previous examples of functional amyloid which were intracellular, our studies now show that nonpathological/functional amyloid can also be extracellular. The presence of an extracellular and nonpathological amyloid in the epididymis suggests that similar amyloid structures may be present in other organ systems and may carry out distinctive tissue-specific functions.

Oligomerization and Transglutaminase Cross-linking of the Cystatin CRES in the Mouse Epididymal Lumen POTENTIAL MECHANISM OF EXTRACELLULAR QUALITY CONTROL

CRES (cystatin-related epididymal spermatogenic), a member of the cystatin superfamily of cysteine protease inhibitors, is expressed in the epididymis and spermatozoa, suggesting specialized roles in reproduction. Several cystatin family members oligomerize, including cystatin C that forms amyloid deposits associated with cerebral amyloid angiopathy. Our studies demonstrate that CRES also forms oligomers. Size exclusion chromatography revealed the presence of multiple forms of CRES in the epididymal luminal fluid, including SDS-sensitive and SDS-resistant high molecular mass complexes. In vitro experiments demonstrated that CRES is a substrate for transglutaminase and that an endogenous transglutaminase activity in the epididymal lumen catalyzed the formation of SDS-resistant CRES complexes. The use of a conformation-dependent antibody that recognizes only the oligomeric precursors to amyloid, negative stain electron microscopy, and Congo Red staining showed that CRES adopted similar oligomeric and fibrillar structures during its aggregation as other amyloidogenic proteins, suggesting that CRES has the potential to form amyloid in the epididymal lumen. The addition of transglutaminase, however, prevented the formation of CRES oligomers recognized by the conformation antibody by cross-linking CRES into an amorphous structure. We propose that transglutaminase activity in the epididymal lumen may function as a mechanism of extracellular quality control by diverting proteins such as CRES from the amyloidogenic pathway.

Effect of estrogen on the elongation rate and number of RNA chains being synthesized in uterine nucleoli

Administration of estradiol (E2) to ovariectomized mature rats resulted in a time-dependent increased transcriptional activity of uterine nucleoli isolated from hormone-treated animals compared to uterine nucleoli isolated from control animals. Early (4 h) E2 stimulation of uterine nucleolar transcription, resulted from an increased rate of elongation of chain growth on preinitiated nucleolar RNA with no significant effect of E2 on the number of nucleolar RNA chains being synthesized. Longer (24 h) treatment of animals with hormone resulted in both significant increased numbers of uterine nucleolar RNA chains in the act of synthesis and increased rate of elongation of nucleolar RNA chain growth. Salt extraction (150 mM NaCl) of uterine nucleoli isolated from 4 h E2-treated animals decreased transcriptional activity to the level observed in nucleoli isolated from control animals. The loss in nucleolar transcriptional activity from salt extraction was due to decreased rate of elongation of nucleolar RNA synthesis with no significant effect on the number of RNA chains being synthesized. Salt extracts from nucleoli isolated from 4 h E2-treated animals, but not control animals, contained factor(s) capable of stimulating the rate of elongation of nucleoli isolated from control animals to elongation rates observed in unextracted nucleoli isolated from 4 h E2-treated animals. Synthesis and phosphorylation of a high molecular weight uterine nucleolar protein(s) was seen after 4 h of E2 treatment with the nucleolar phosphoprotein(s) salt extractable.

Fertility Defects in Mice Expressing the L68Q Variant of Human Cystatin C A ROLE FOR AMYLOID IN MALE INFERTILITY

Background: The L68Q variant of cystatin C is highly amyloidogenic forming aggregates in individuals with HCCAA. Results: Spermatozoa from mice expressing human L68Q cystatin C exhibit fertility defects and increased levels of amyloid. Conclusion: L68Q epididymal fluid containing cystatin C amyloid is harmful for sperm function. Significance: Amyloid in the reproductive tract may contribute to male factor infertility. Hereditary cystatin C amyloid angiopathy is an autosomal dominant disorder in which a variant form of cystatin C (L68Q) readily forms amyloid deposits in cerebral arteries in affected individuals resulting in early death. L68Q protein deposits in human cystatin C amyloid angiopathy patients have also been found in tissues outside of the brain including the testis, suggesting possible effects on fertility. Heterozygous transgenic mice (L68Q) that express the human L68Q variant of cystatin C under the control of the mouse cystatin C promoter were unable to generate offspring, suggesting the presence of L68Q cystatin C amyloid affected sperm function. In vitro studies showed that epididymal spermatozoa from L68Q mice were unable to fertilize oocytes and exhibited poor sperm motility. Furthermore, spermatozoa from L68Q mice exhibited reduced cell viability compared with wild type (WT) spermatozoa and often were detected in large agglutinated clumps. Examination of the epididymal fluid and spermatozoa from L68Q mice showed increased levels and distinct forms of cystatin C amyloid that were not present in WT mice. The addition of epididymal fluid from L68Q mice to WT spermatozoa resulted in a recapitulation of the L68Q phenotype in that WT spermatozoa showed reduced cell viability and motility compared with WT spermatozoa incubated in epididymal fluid from WT mice. L68Q epididymal fluid that was depleted of cystatin C amyloids, however, did not impair the motility of WT spermatozoa. Taken together these studies suggest that amyloids in the epididymal fluid can be cytotoxic to the maturing spermatozoa resulting in male infertility.

Role of polyamine in the regulation of RNA synthesis in uterine nucleoli.

Administration of estradiol (E2) to ovariectomized mature rats has been shown to result in synthesis of uterine polyamines in the same temporal manner as E2 regulation of nucleolar transcription. Data is presented on the in vivo and in vitro effects of polyamines on uterine nucleolar RNA synthesis. Transcervical intrauterine administration of putrescine (100 micrograms), spermidine (100 micrograms), or spermine (100 micrograms) resulted in an increased transcriptional activity of 93 and 82% in uterine nucleoli isolated from putrescine and spermidine treated animals, respectively. Spermine administration was without effect on uterine nucleolar transcription. The polyamine-induced increase in transcription was totally accounted for by an increased rate of elongation of previously initiated RNA chains. No effect on the number of nucleolar RNA chains in the act of synthesis was observed. Preincubation of uterine nucleoli, isolated from control animals (no E2) with putrescine, spermidine, or spermine in the presence, but not in the absence of ATP, resulted in 44, 83 and 31% increased nucleolar RNA synthesis, respectively. In vitro polyamine-induced nucleolar RNA synthesis was correlated with a polyamine activated phosphorylation of nucleolar proteins of 110,000 24,000, 18,000 and 14,000 Da. Results suggest that early E2 action may result in activation of the polyamine pathway which modulates nucleolar protein kinase activity; initiating an increase in nucleolar transcription.

Cystatin-Related Epididymal Spermatogenic Aggregates in the Epididymis

Cystatin-related epididymal spermatogenic (CRES) is the defining member of a reproductive subgroup within the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. CRES is synthesized and secreted by the initial segment of the epididymis and is present in the sperm acrosome, suggesting roles in sperm maturation and fertilization. We have previously demonstrated that CRES is present within the epididymal lumen as monomeric (14 and N-glycosylated 19-kd forms) as well as sodium dodecyl sulfate-sensitive and sodium dodecyl sulfate-resistant high-molecular mass complexes. We have also shown that recombinant CRES protein will self-aggregate and form amyloid structures in vitro, raising the possibility that CRES might also form amyloid in vivo. Amyloid is a large protein aggregate with a specific cross-β sheet structure, and its presence is usually associated with disease. This review discusses protein aggregation in the epididymis and provides a brief overview of amyloid formation, including recent studies in other organ systems identifying examples of amyloid that are nonpathologic and carry out biologic functions (ie, functional amyloid). Studies that were carried out to determine if amyloid is present in the epididymal lumen and if CRES is associated with these structures are also described. The presence of CRES amyloid in the mouse epididymal lumen and the absence of pathology suggest either the presence of mechanisms to neutralize the cytotoxicity associated with pathologic amyloid or that CRES is a new example of a functional amyloid with roles in epididymal function.