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. 2018 Oct 25;13(10):e0206173.
doi: 10.1371/journal.pone.0206173. eCollection 2018.

Presence of aberrant epididymal tubules revealing undifferentiated epithelial cells and absence of spermatozoa in a combined neuraminidase-3 and -4 deficient adult mouse model

Regiana Oliveira  1 , Louis Hermo  1 , Alexey V Pshezhetsky  2 , Carlos R Morales  1
Affiliations

Presence of aberrant epididymal tubules revealing undifferentiated epithelial cells and absence of spermatozoa in a combined neuraminidase-3 and -4 deficient adult mouse model

Regiana Oliveira et al. PLoS One. .

Abstract

Mammalian neuraminidases are responsible for the removal of sialic acids from glycoproteins and glycolipids and function in a variety of biological phenomena such as lysosomal catabolism and control of cell differentiation and growth. Disruption of Neu3 and Neu4 genes has led to the generation of a mouse model revealing severe neurological disorders. In this study a morphological analysis was performed on the epididymis of 3 month-old neu3-/-neu4-/- mice as compared with wild type animals. In neu3-/-neu4-/- mice the majority of tubules of the main epididymal duct were large and lined by differentiated epithelial cells, but revealing lysosomal abnormalities in principal and basally located cells. Of particular note was the presence of aberrant epididymal tubules (ATs) juxtaposed next to the main tubules. ATs were small and of different shapes. Layers of myoid cells encased ATs, which they shared with those of the main tubules, but no interstitial space existed between the two. While some ATs were a dense mass of cells, others revealed a distinct lumen devoid of spermatozoa. The latter revealed an undifferentiated epithelium consisting of cuboidal cells and basal cells, with junctional complexes evident at the luminal front. The absence of spermatozoa from the lumen of the ATs suggests that they were not in contact with the main duct, as also implied by the undifferentiated appearance of the epithelium suggesting lack of lumicrine factors. Despite the presence of ATs, the main duct contained ample spermatozoa, as the neu3-/-neu4-/- mice were fertile. Taken together the data suggest that absence of Neu3 and Neu4 leads to defects in cell adhesion and differentiation of epithelial cells resulting in aberrant tubular offshoots that fail to remain connected with the main duct. Hence Neu3 and Neu 4 play an essential role in the guidance of epithelial cells during early embryonic formation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
EM micrographs of the efferent ducts of WT (A) and neu3-/-neu4-/- (B-F) mouse model. In (A), several small to medium sized dense lysosomes (L) appear in a nonciliated cell (NC), while in (B), many large vacuoles (V) are noted. Large, long cylindrical structures (arrows) with an irregular outline are evident in neu3-/-neu4-/- mice, with a matrix revealing small to medium sized spaces often aligned in parallel rows (C, E, F, inset). Electron dense bulges (arrowheads) along the length of these cylindrical structures are noted, with an internal appearance of the dense L nearby, suggesting their fusion with the tubular structures (E, F). Halo cell (HC) exhibits large L (B-D). The basement membrane (BM) of neu3-/-neu4-/- mice is highly convoluted and contains small vesicular profiles (B, D). Ciliated cells (C) are indicated. Scale bars: A, B, C, E: 2μm; D, F: 500nm.
Fig 2
Fig 2
LM micrographs of semithin sections (0.5 μm) of the initial segment of WT (A) and initial segment (B-C), caput (D-F), and proximal (G) and distal (H) cauda regions of neu3-/-neu4-/- mice. In (A), principal cells (P) are tall columnar and the lumen (Lu) is small containing spermatozoa (Spz). In (B, C), P cells appear to be larger and contain many small dense lysosomes (L). Capillaries (Cap) of large size are abundant between the epithelium and myoid cell (My) layers. In (D-G), ATs of different shapes and sizes reside in close proximity to normal tubules. Some aberrant tubules (ATs) are a dense mass of cells without a central Lu (D, F, G), while others reveal a Lu (D-G) with absence of Spz (E, F). ATs form deep depressions in the normal tubules (asterisks) (E, F). ATs with a lumen consist of undifferentiated cells with a cuboidal appearance (E, F). The wall of some ATs is kinked which disrupts the homogeneity of their tubular appearance (E, F). ATs are enveloped by My cells, which are shared with the normal tubules (D-F). Large dense masses (arrowheads) appear in the basal region of the epithelium (D, G). In the cauda region, some ATs run parallel to the main tubules (G), but some impinge upon it (H inset). P cells of the distal cauda show large vacuolar L in the cytoplasm (H). The Lu of normal tubules of neu3-/-neu4-/- mice is filled with Spz. Scale bar: 20μm.
Fig 3
Fig 3
EM of the initial segment of epididymis of WT (A) and neu3-/-neu4-/- mice (B-H) mice. In (A), principal cells (P) cells demonstrate an elaborate Golgi (G), small dense L and large spherical nuclei (N). A narrow cell (NC) and basal cell (BC) are indicated. A thin BM underlies the epithelium, beneath which lie several layers of My cells. A Cap is sandwiched between the BM and inner My cell layer. In (B), many small dense L appear in P cells as well as parallel layers of cisternae of endoplasmic reticulum (ER) next to the G. A BC contains a large lysosomal/lipidic structure (L). Large Cap reside between the BM and underlying My cells. In (D), basal area of epithelium shows a HC near a BC maintaining its contact with the BM by thin processes (arrow). In (E, F) a BC contains a gigantic L. In (B-F), large dilated intercellular spaces (asterisks) appear between adjacent P cells and contain numerous membranous vesicular profiles and principal cell interdigitations. In (G), the BM is thickened and multilayered and takes on a convoluted and anastomotic appearance. My cells appear normal. In (H) many small immune cells (IC) appear in the intertubular space (IT). Scale bars: A-F, H = 2μm; G = 500nm.
Fig 4
Fig 4
EM of the caput epididymidis of WT (A) and neu3-/-neu4-/- mice (B-F) mice. In (A), P cells, BC and NC are indicated. Spz are evident in Lu. In (B), a BC contains a large lysosomal/lipidic structure (L) and the basal area of a P cell shows a large irregularly shaped L. In (C), a small AT, formed of low cuboidal undifferentiated epithelial cells, is lodged within a large indentation of a normal tubule. The AT envelops a central Lu devoid of Spz. My cells completely enclose the AT which are shared by the normal duct. A large irregularly shaped L is seen in the base of a P cell. In (D) two adjacent ATs are evident with one impinging on a normal tubule. In (E) higher mag of the tubule seen in Fig (D), the AT is smaller than the normal tubule, is made up of undifferentiated epithelial cells and does not shows a central lumen. Images (F and the inset) show an AT made up of epithelial cells (E) that extend from the BM to the central lumen (Lu). The inset is a high power of (F) revealing that the epithelial cells form junctional complexes (JC) at the apical extremities near the lumen. These cells show a G, but are in no way are they comparable to the highly differentiated P cells of the normal tubule. The Lu is devoid of Spz but contains sectional profiles of the epithelial cell microvilli (Mv). BC border the periphery of the ATs (E and F). Scale bars: A, B, E, F = 2 μm; C, D = 10 μm.
Fig 5
Fig 5
EM of the corpus epididymidis of WT (A) and neu3-/-neu4-/- (B-F) mice. In (A), P cells reach the Lu and contain Spz. Nuclei (N) are more or less spherical and regular in appearance. A thin BM underlies the epithelium and several My layers are seen. In (B), P cells reach the Lu where Spz are plentiful. Nuclei (N) of P cells are highly irregular in form. One N encircles organelles of the cytoplasm (N2). Some P cells contain more than one nucleus (N1). A BC is filled with huge L. The base of P cells and a BC shows small thin foot-like processes (arrows) that project into a highly convoluted BM. In (C) a NC is filled with large endosomes (E) and L. In (D) a BC filled with huge L reveals thin foot-like processes (arrows) that project into a highly anastomotic BM. In (E), gigantic endosomes (E) and L appear in the cytoplasm of a P cell revealing an elaborate G. In (F), a thin continuous squamous circular epithelial cell (SE) is noted surrounding a large Lu revealing microvilli but absence of Spz. The SE contacts the BM by thin foot-like processes. The Lu of the main duct contains Spz. In (E) and (F) a large L appears in the infranuclear cytoplasm of a P cell, one of which is binucleated. Scale bars: Scale bars: A-E = 2μm; F = 10μm.
Fig 6
Fig 6
EM of the distal cauda epididymidis of WT (A) and neu3-/-neu4-/- (B) mice. In (A), P cells show large irregularly shaped N and the epithelial cells envelop a Lu containing Spz. In (B), P cells contain several huge pale stained vacuoles (V) and large L filled with a dense granular material. Images (C-H) show the proximal cauda of neu3-/-neu4-/- mice. In (C), adjacent ATs are situated parallel to normal tubules. The latter shows differentiated P cells, some with a double nucleus (N). Both ATs consist of a mass of undifferentiated cells, but the AT on the right reveals a Lu. Its encasing cells are highly attenuated with microvilli projecting into the Lu, which is devoid of Spz. The ATs are enveloped by My cells, which are shared with the normal tubule. Image (D) is a low power magnification of an AT in proximity to a normal duct. Part of an AT (AT1) impinges on a normal tubule, while the remainder of the AT (AT2) stretches beneath and parallel to the normal tubules. AT1 consists of a congealed mass of cells, while cells of AT2 at the far right form a distinct Lu containing microvilli. Details of AT1 and AT2 are shown in (E) and (F). In (E), one of the ATs (AT1) reveals a mass of cells that appear to show some differentiated organelles that in part resemble P cells of the normal tubule. No Lu is evident in AT1, but a My cell projects itself into the mass of cells seemingly to separate in part AT1 from AT2. In (F) the AT labeled AT2 in image (D) is further investigated. On the left side of the AT2 tubule, cells of different shapes and sizes, some of which appear to be squamous, border a Lu. On the right side of the field, a confluence of the Lu of 2 ATs appears to be taking place. Images (G) and (H) are a high power magnification of (F) and show flat undifferentiated epithelial cells (E) bordering a Lu and revealing JC. The Lu contains microvilli (Mv) and membranous whorls (arrowheads) but no Spz. Scale bars: A, B, E, F, G and H = 2μm; C and D = 10μm.
Fig 7
Fig 7. Immunolocalization of Prosaposin in the epididymis of neu3-/-neu4-/- mice.
Initial segment in (A) shows immunoreaction of prosaposin in wild type mice. (B) shows negative control. Immunoreaction of Prosaposin in the initial segment (C), caput (D), proximal (E) and distal (F-H) cauda regions of the epididymis of neu3-/-neu4-/- mice immunostained with an anti-prosaposin antibody. In (C), small punctate reactive lysosomes (circles) are noted in the supranuclear area of P cells and in thin elongated processes of a BC. Large highly reactive cells (arrowheads) are seen at the base of the epithelium, as also demonstrated in (C inset). In (D), a narrow cell (NC) is reactive as well as several small (arrows) and large basally located cells (arrowheads). In (E-H) several large highly reactive cells (arrowheads) are noted at the base of the epithelium. In (E-G), P cells reveal infranuclear reactive L at their base (arrows). In (G and H) large vacuoles (V) are seen in the epithelium as well as basally located cells. In (G inset), P and clear (C) cells are reactive for prosaposin. Scale bar: 20μm.
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