Morphological and morphometric changes and epithelial apoptosis are induced in the rat epididymis by long-term letrozole treatment

The epididymis is an organ that plays a key role in sperm maturation. The aim of this study was to examine the association between the chronic treatment of mature male rats with letrozole and morphological evaluation and morphometric values of epididymis as well as changes in the number of apoptotic cells in epididymal epithelium. Adult rats were treated with letrozole for 6 months and the epididymis weight, morphology, morphometric values and the number of apoptotic cells in the epithelium were examined. Long-term aromatase inhibition resulted in presence of intraepithelial clear vacuoles, hyperplasia of clear cells and a hyperplastic alteration in the epithelium known as a cribriform change. Moreover, changes in diameters of the epididymal duct and the epididymal lumen and changes in the epididymal epithelium height were observed. The number of apoptotic epithelial cells was increased in letrozole-treated group. It can be indicated that chronic treatment with letrozole can affect morphology, morphometric values and apoptosis in the epididymis of adult male rats. Observed changes are similar to that observed in the aging processes and may also be important for patients treated with aromatase inhibitors.

Self-renewal and differentiation of rat Epididymal basal cells using a novel in vitro organoid model

The epididymis is composed of a pseudostratified epithelium comprised of various cell types. Studies have shown that rat basal cells share common properties with adult stem cells and begin to differentiate in vitro in response to fibroblast growth factor and 5α-dihydrotestosterone. The characterization of rat basal cells is therefore necessary to fully understand the role of these cells. The objectives of this study were to assess the ability of single basal cells to develop organoids and to assess their ability to self-renew and differentiate in vitro. We isolated basal cells from the rat epididymis and established 3-dimensional cell cultures from the basal and non-basal cell fractions. Organoids were formed by single adult epididymal basal cells. Organoids were dissociated into single basal cells which were able to reform new organoids, and were maintained over 10 generations. Long-term culture of organoids revealed that these cells could differentiated into cells expressing the principal cell markers aquaporin 9 and cystic fibrosis transmembrane conductance regulator. Electron microscopy demonstrated that organoids were comprised of several polarized cell types displaying microvilli and the ability to form tight junctions. Additionally, organoids could be formed by basal cells from either the proximal or distal region of the epididymis, and are able to secrete clusterin, a protein implicated in the maturation of spermatozoa. These data indicate that rat basal cells can be used to derive epididymal organoids, and further supports that notion that these may represent a stem cell population in the epididymis.

Castration causes an increase in lysosomal size and upregulation of cathepsin D expression in principal cells along with increased secretion of procathepsin D and prosaposin oligomers in adult rat epididymis

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, and 2-day castrated rats without (Ct) and with testosterone replacement (Ct+T) to evaluate their expression and regulation within epididymal epithelial cells. By light microscope-immunocytochemistry, a quantitative increase in size of lysosomes in principal cells of Ct animals was noted from the distal initial segment to the proximal cauda. Androgen replacement did not restore the size of lysosomes to control levels. Western blot analysis revealed a significant increase in CatD expression in the epididymis of Ct animals, which suggested an upregulation of its expression in principal cells; androgens restored levels of CatD to that of controls. In contrast, PSAP expression in Ct animals was not altered from controls. Additionally, an increase in procathepsin D levels was noted from samples of the epididymal fluid of Ct compared to control animals, accompanied by an increased complex formation with PSAP. Moreover, an increased oligomerization of prosaposin was observed in the epididymal lumen of Ct rats, with changes reverted to controls in Ct+T animals. Taken together these data suggest castration causes an increased uptake of substrates that are acted upon by CatD in lysosomes of principal cells and in the lumen by procathepsin D. These substrates may be derived from apoptotic cells noted in the lumen of proximal regions and possibly by degenerating sperm in distal regions of the epididymis of Ct animals. Exploring the mechanisms by which lysosomal enzymes are synthesized and secreted by the epididymis may help resolve some of the issues originating from epididymal dysfunctions with relevance to sperm maturation.

Palmitoylated GLB1L4 protein transfers via exosomes to maintain sperm function in rat epididymis

Epididymal specific proteins play a crucial role in sperm maturation. Some of post-translational modified proteins are transported from caput to cauda of epididymis through exosomes which regulate function of sperm in cauda epididymis. Rat beta-galactosidase-1-like protein 4 (GLB1L4) expresses specifically in the caput epididymis and localizes on sperm; however, the regulatory ways which GLB1L4 protein interacts with sperm to maintain sperm function are unclear. In this study, knockdown of rat GLB1L4 could inhibit in vitro capacitation of sperm in cauda epididymis and reduce fertility of the male rats by injection of special lentivirus-shRNA into caput epididymis. Moreover, a considerable proportion of GLB1L4 proteins from rat caput epididymis were loaded on exosomes. The exosomes loaded GLB1L4 from in vitro primary rat caput epididymal epithelial cells could bind with spermatozoa in cauda epididymis. Further, the palmitoylation status of cysteine residues at 12th and 15th sites of the protein molecule could significantly affect cellular localization of GLB1L4 protein. It was identified that most of GLB1L4 was palmitoylated in the presence of exosomes from primary caput epididymal cells and the level of palmitoylated GLB1L4 in the exosomes could be inhibited by 2-Bromopalmitate (2-BP). These results suggested that the palmitoylated GLB1L4 from rat caput epididymis could be transported to the cauda epididymis to regulate the sperm function by exosomes.

Contractions transport exfoliated epithelial cells through the neonatal epididymis

Contractions of the adult epididymal duct are well known in the context of sperm transport. Some reports also describe contractions of the epididymal duct during development, but data about their character, regulation and function are sparse. In the foetal human epididymis we found luminal cells and could identify them as exfoliated epithelial cells originating from the epididymis and not from testis by using antibodies against neutral endopeptidase as an epithelial epididymal duct marker. Exfoliated cells were also found in the epididymal duct after birth. Time-lapse imaging revealed directional transport of luminal cells in the neonatal rat epididymis interrupted by pendular movement. Spontaneous contractions were discovered in the neonatal epididymis and an association between these contractions and the transport of the luminal cells could be observed. Both, transport and spontaneous contractions, were affected significantly by substances known to contract (noradrenaline) or relax (the phosphodiesterase 5 inhibitor sildenafil) smooth muscle cells. Immunohistochemistry showed staining for the proliferation marker proliferating-cell-nuclear-antigen (PCNA) in cells of the ductal lumen of the neonatal rat epididymis indicating the extrusion of cells also during proliferation. Our data showed spontaneous contractions of the immature epididymal duct associated with the transport of exfoliated luminal cells before the first occurrence of sperm cells. Results suggest an important role including both (i) a mechanical place holder function of exfoliated luminal cells (ii) together with a novel idea of organized waste disposal of these cells during development.