Discussion
We report three cases of localized amyloidosis in the epididymis, two clinically detected as a nodular mass and the other obtained together with a neighbouring pathologic process (para-testicular liposarcoma). Although localized amyloidosis has been described in several locations of the male reproductive system, which contribute to the transport, maturation and/or required fluid medium of spermatozoa [
1‐
9], to the best of our knowledge, this is the first description of localized epididymal amyloidosis. Awareness of the existence of epididymal amyloidosis and of its presentation as small nodules in the epididymis, as well as knowledge of its histopathology, is of interest in clinical and pathological differential diagnoses, including tumours. Moreover, the characteristics of the lesion support the initial development of amyloid deposits in the epididymal lumen, where a specific proteome occurs [
10‐
12], and non-pathological functional amyloids and mechanisms of protein aggregation control take place [
13‐
15]. Below, we examine these issues.
Our cases were seen in patients aged 67 and over. Although this type of amyloidosis could be regarded as a senile form, larger series are needed to confirm this possibility.
Our observations indicate an initial deposit of amyloid in the epididymal lumen, with subsequent passage to the interstitium. The findings that support this sequence are as follows: a) densely grouped deposits in some sections of the epididymis occupy both the lumen and the interstitium, after distention of the epididymal lumen and partial epithelial disruption, b) presence of epithelial folds with degenerative phenomena, surrounding portions of intraluminal amyloid deposits, which are partially incorporated in the interstitium, c) epithelial strips remain on the surface of some large interstitial amyloid masses, d) several distinct aggregates of amyloid deposits are organized in a similar convoluted path to that of the epididymis, and e) the deposits occupying both the interstitium and the lumen appear with and without reticulin networks and/or stromal cells, respectively.
Both intraluminal and interstitial deposits in the epididymis show amyloid characteristics, including positivity for Congo red with yellow-green birefringence under polarized light. In our observations, apparent negativity for amyloid A, any evidence of systemic amyloidosis, paraproteinemia or underlying plasma cell dyscrasia, and the absence of amyloid deposits involving vascular walls support an organ-limited deposition of heterogeneous amyloids, including light chains κ and λ, and transthyretin. Amyloid P was also present. However, our immunohistochemical results were obtained in paraffin-embedded tissue blocks, and amyloid deposits can contain several misfolded proteins. Therefore, a more specific characterization of the proteome and misfolded proteins requires further study (see below).
The onset of amyloid deposits in the epididymal lumen may be related to our results showing amyloid P (involved in the deposition, stabilization and persistence of amyloid) and transthyretin (a transport protein in the serum and cerebrospinal fluid) expression in the apical surface of the normal epithelial cells of the epididymis. Moreover, amyloid P showed strong immunoreactivity in the spermatozoa, which concurs with the observations of others authors [
16]. Likewise, immunoreactivity for pan CK AE1 AE3 also concurs with the presence of keratins in the sperm proteome [
17].
The comparison with amyloidosis described in other locations of the male reproductive system that contribute to the transport, maturation and/or required fluid medium of spermatozoa, mainly seminal vesicles, indicates that the deposits have a different location: initially intraluminal with subsequent passage to the interstitium in the epididymis, and predominantly sub-epithelial in the seminal vesicles [
1‐
9], as occurs in choroid plexus amyloidogenic papillomas [
18]. The mechanisms to explain these differences in the polarization of the deposits require further studies. Likewise, the positive reaction for light-chain antibodies has been reported for several cases of localized amyloidosis in the urogenital tract [
19,
20], and the expression of light chains κ and/or λ in localized amyloidosis in other regions is not exceptionally rare [
21].
The unique and distinctive location of amyloid deposits in the epididymal lumen, with subsequent passage to the interstitium, could depend on the peculiar functions of this anatomical region, in which proteomic studies show the more sequentially modified milieu of the body [
10‐
12]. Indeed, the epididymis actively participates in the maturation, protection and acquisition of motility and fertility of spermatozoa by synthesis, secretion and post-transitional modifications of important molecules, including a high concentration of several hundred proteins, most of which are actively secreted by the epididymal epithelium (for review, see [
12]). Spermatozoa are dependent on this extracellular environment, since their DNA is highly compacted, making processes of transcription and translation impossible [
22‐
24]. Epididymosomes (vesicles present in the epididymis) are involved in the acquisition of new sperm proteins during epididymal transit [
22‐
24]. Some of these proteins may form functional amyloids, and in vitro studies have shown amyloid formation in this unique milieu. Thus, this milieu comprises cystatin-related epididymal spermatogenic members (CRES), which pass from monomeric forms in the proximal caput region to an aggregated amyloid state in the distal caput region [
15,
25], and amyloidogenic prion protein is present in epididymosomes and associated with hydrophobic proteins in lipophilic complex [
26‐
28]. Likewise, concentration of luminal content occurs in the epididymis (more than 90% of the fluid is removed from the epididymis) [
29], facilitating macromolecular crowding, and protein misfolding and aggregation. However, in the epididymis, amyloids act without causing pathology, due to the mechanisms of extracellular quality control [
13,
14,
25]. This control includes: a) ubiquitin-dependent proteolysis (classically considered as an intracellular quality control system and currently as also having extracellular functionality in sperm quality control) [
30], b) chaperones (involved in the prevention of protein aggregation), since chaperone clusterin is found in soluble high molecular mass lipophilic complex present in the lumen of the epididymis during sperm maturation (around 30% of total epididymal secretion [
11]), and c) transglutaminase, which prevents the formation of amyloid-type aggregates of CRES in the epididymis by post-translational modifications (transglutaminase cross-linking of cystatin CRES - [
25]). Therefore, the onset of amyloidosis deposits in the lumen of the epididymis, with subsequent passage to the interstitium, suggests a disturbance of the mechanisms mentioned above, mainly of extracellular (intraluminal) functional amyloid control.
While intracellular, post-translational quality control systems to repair or remove misfolded proteins have been well studied, extracellular mechanisms of folding control of secreted proteins are not well described, except in the lumen of the epididymis [
13,
14]. These mechanisms in the epididymis have been considered highly significant for understanding the misfolded protein formation involved in some pathological processes, including Alzheimer’s disease, cerebral angiopathies, and type II diabetes [
13,
14]. In this way, amyloid deposits in the epididymis may provide a substrate to explore not only the alteration of the reproductive function, but also the mechanism of extracellular protein misfolding control in several diseases. Thus, new studies in epididymal amyloidosis are required to explore the functional amyloids outlined above and the molecules that act in their extracellular quality control.