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. 2023 Sep 27;18(9):e0292157.
doi: 10.1371/journal.pone.0292157. eCollection 2023.

Effects of Heparan sulfate acetyl-CoA: Alpha-glucosaminide N-acetyltransferase (HGSNAT) inactivation on the structure and function of epithelial and immune cells of the testis and epididymis and sperm parameters in adult mice

Lorena Carvelli  1   2   3 , Louis Hermo  3 , Cristian O'Flaherty  3   4   5 , Richard Oko  6 , Alexey V Pshezhetsky  3   7 , Carlos R Morales  3
Affiliations

Effects of Heparan sulfate acetyl-CoA: Alpha-glucosaminide N-acetyltransferase (HGSNAT) inactivation on the structure and function of epithelial and immune cells of the testis and epididymis and sperm parameters in adult mice

Lorena Carvelli et al. PLoS One. .

Abstract

Heparan sulfate (HS), an abundant component of the apical cell surface and basement membrane, belongs to the glycosaminoglycan family of carbohydrates covalently linked to proteins called heparan sulfate proteoglycans. After endocytosis, HS is degraded in the lysosome by several enzymes, including heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and in its absence causes Mucopolysaccharidosis III type C (Sanfilippo type C). Since endocytosis occurs in epithelial cells of the testis and epididymis, we examined the morphological effects of Hgsnat inactivation in these organs. In the testis, Hgsnat knockout (Hgsnat-Geo) mice revealed statistically significant decrease in tubule and epithelial profile area of seminiferous tubules. Electron microscopy (EM) analysis revealed cross-sectional tubule profiles with normal and moderately to severely altered appearances. Abnormalities in Sertoli cells and blood-testis barrier and the absence of germ cells in some tubules were noted along with altered morphology of sperm, sperm motility parameters and a reduction in fertilization rates in vitro. Along with quantitatively increased epithelial and tubular profile areas in the epididymis, EM demonstrated significant accumulations of electrolucent lysosomes in the caput-cauda regions that were reactive for cathepsin D and prosaposin antibodies. Lysosomes with similar storage materials were also found in basal, clear and myoid cells. In the mid/basal region of the epithelium of caput-cauda regions of KO mice, large vacuolated cells, unreactive for cytokeratin 5, a basal cell marker, were identified morphologically as epididymal mononuclear phagocytes (eMPs). The cytoplasm of the eMPs was occupied by a gigantic lysosome suggesting an active role of these cells in removing debris from the epithelium. Some eMPs were found in proximity to T-lymphocytes, a feature of dendritic cells. Taken together, our results reveal that upon Hgsnat inactivation, morphological alterations occur to the testis affecting sperm morphology and motility parameters and abnormal lysosomes in epididymal epithelial cells, indicative of a lysosomal storage disease.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
LM of seminiferous epithelium (SE) of the testis stained with H&E (A-D) and anti-prosaposin antibody (E-J) of WT (A, E) and KO (B-D, F-J) mice at 11 or 14 months of age. In (F), several KO tubules demonstrate a normal appearance (NT) as seen in WT (A), while other KO tubules appear abnormal (AT) with a disrupted epithelium (B, D, G and I); some KO tubules only reveal Sertoli cells (B, G). Sertoli cells (arrowheads) immunolabeled for prosaposin are evident in WT (E) and KO (F-J) mice, as well as in the grossly altered tubules (G, I). Lu, lumen; IS, interstitial space. Scale bars = 35 μm.
Fig 2
Fig 2
EM of seminiferous epithelium (SE) of WT (A, B) and KO (C-F) KO tubules at 11 or 14 months of age. In (A, B), tubules reveal a close approximation of germ cells with Sertoli cells (S), small dense lysosomes (arrowheads) and intact BTB (curved arrows). In (C), abnormally large and medium-sized dense granulated lysosomes (Ly) are prominent in SE, as well as large lipid droplets (Li). In (D), numerous small lysosomes (arrowheads) occupy the base of Sertoli cells, along with a large myelinated-like body (mye). In (C), Sertoli cells reveal an intact BTB (curved arrows), while in (D-F), it is disrupted. In (D-F), Sertoli cells show abnormal bloated basal and adluminal compartments resulting in dilated intercellular spaces (ICS) confluent with the cytoplasm (cyt) and containing organelles and membranous profiles. Germ cells: spermatogonia (SG), early (ES) and late (LS) spermatids; pachytene (PA) and preleptotene (PL) spermatocytes; zygotene (Z) spermatocytes; As, acrosome; BM, basement membrane; My, myoid cells; N, Sertoli cell nucleus. Scale bars = 2 μm.
Fig 3
Fig 3
Toluidine blue stained sections of different regions of the epididymis of WT (A, C, E, G) and KO (B, D, F, H) mice. In (B), principal cells (P) of the initial segment (IS) of KO mice appear taller in appearance compared to WT mice (A), and large elongated dilated spaces (DS) occupy the base of the epithelium (B). In the caput, corpus and cauda regions of KO mice (D, F, H), a plethora of small to large-sized pale lysosomes (large arrows) occupy the cytoplasm of principal cells (P), which are not noted in WT cells (C, E, G). In addition, very large pale stained foamy eMPs (asterisks) converge at the base of the epithelium of KO mice of the caput and corpus regions (D, F), which are not noted in WT mice (C, E). The lumen (Lu) of KO mice reveals an abundance of sperm. Basal cells, small arrows; IT, intertubular space. Scale bars = 50 μm.
Fig 4
Fig 4
EM of efferent ducts of WT type (C) and KO (A, B, D, E) mice. In (C), a few small dense lysosomes (arrows) appear in nonciliated (NC) and ciliated (Ci) cells. At the same time, KO mice reveal medium to large-sized pale stained and dense lysosomes supra and infranuclearly (A, B, D, E). In (B), a small portion of the initial segment (IS) is observed. Dilated intercellular spaces (stars) containing membranous profiles are evident at the base of the epithelium of KO mice (A, B, D, E). In (D), germ cells (GC) appear in the lumen. In (D, E), halo cells (HC) are seen at the base of the epithelium. BM, basement membrane; My, myoid cells; Lu, lumen; IT, intertubular space. Scale bars = 2 μm.
Fig 5
Fig 5
EM of the initial segment of WT (A) and KO (B-D) mice. In (A), tall columnar principal cells (P) reveal a few small dense lysosomes (arrows). A halo cell (H) is adjacent to a small nondescript basal cell (BC), and a large narrow cell (NC) is noted. In KO mice, the dense lysosomes (arrows) appear to be more abundant and larger in size (B-D) as compared to WT mice (A). In (B-D), basal cells of KO mice reveal different shapes, sizes and commitment to the basement membrane and are filled with small to medium-sized pale lysosomes (arrows). Basally located dilated intercellular spaces (ICS) of KO mice contain membranous and vesicular profiles (arrowheads) (B-D). Cap, capillaries; My, myoid cells; BM, basement membrane; N, nucleus. Scale bars = 2 μm.
Fig 6
Fig 6
EM of caput (A, B), corpus (C, D) and cauda (E, F) regions of WT (A, C, E) and KO (B, D, F) mice. In WT mice (A, C, E), principal cells (P) reveal few small dense supranuclear lysosomes (arrows) and occasional irregular larger dense lysosomes (Ly) infranuclearly (C, E). In all 3 regions of KO mice, principal cells (P) exhibit a plethora of medium to large-sized pale lysosomes (Ly) in their supra-and infranuclear areas (B, D, F), Clear cells (CL) are evident in KO mice (B, D, F) and at times show numerous pale stained lysosomes (F). Basal cells (BC) of KO mice (B, D, F) show prominent small to moderate pale stained lysosomes, which are not noted in WT mice (A). Large basally located eMPs (asterisks) reside in KO mice at the base of the epithelium (B, D). My, myoid cells; Lu, lumen; N, nucleus; H, halo cell. Scale bars = 2 μm.
Fig 7
Fig 7
EM of KO (A-C) and WT (D) mice. In (A-C), large eMPs straddle the basement membrane and reveal a cytoplasm dominated by a single gigantic pale stained lysosome (Gly) containing numerous membranous profiles and finely dense granular material. Invaginations of the plasma membrane of Gly appear as thin needle-like projections (curved arrows) that protrude deep into the Gly and, at times, branch but do not penetrate its interior. During the formation of the Gly by fusion of smaller lysosomes with each other, these projections trap the cytoplasmic matrix (A-C) as the Gly greatly increases in size and excludes organelles leaving only a thin layer of deeply stained homogeneous material between the entrapped invaginated plasma membrane of the Gly. Principal cells (P) demonstrate numerous pale stained lysosomes (Ly) of different shapes and sizes and contain membranous profiles. Notably, their large lysosomes also reveal thin invaginations (curved arrows) of their plasma membrane, which extend deep into its interior and likewise trap the cytoplasmic matrix excluding organelles. In WT mice (D), a dendritic cell (DC) reveals a thin process (arrowheads) which appears to wrap itself around a halo cell identified as a T lymphocyte (TL). In (B), a small spherical T-lymphocyte (TL) is adjacent to an eMP cell, suggested to be a dendritic cell. BM, basement membrane; N, nucleus; My, myoid cells. Scale bars = A and B = 2 μm, and C and D = 500 nm.
Fig 8
Fig 8
LM immune of the epididymis of WT (A-C) and KO (D-L) mice with anti-CK5 antibody. Basal cells (large arrows) are intensely reactive. However, the eMPs are unreactive (asterisks). Some basal cells contact the lumen (small arrows). Isolated processes of basal cells hug the basement membrane (small thin arrows). Lu, lumen; IT, intertubular space; P, principal cells. Scale bars = 20 μm.
Fig 9
Fig 9
LM immune reaction of the epididymis of WT (A, C, E) and KO (B, D, F) mice with anti-PSAP. Intense reactions appear over large, dilated eMPs (asterisks) at the base of the epithelium of KO mice which are not prominent in WT mice. Basal cells are reactive (arrows), as well as principal cells (P) and clear cells (arrowheads). Lu, lumen; IT, intertubular space. Scale bars = 20 μm.
Fig 10
Fig 10
LM immunoreaction of the epididymis of WT (A, D, G) and KO (B, C, E, F, H, I) mice with anti-CathD antibody. Note reactions over principal (P) and basal (arrows). Some clear cells (arrowheads) are reactive. eMPs of KO mice (asterisks) (B, C, E, F, H, I) show intense reactivity. Lu, lumen; IT, intertubular space. Scale bars = 20 μm.
Fig 11
Fig 11
TEM (A-D) and SEM (E-H) of sperm in the epididymal lumen of KO mice. (A-D) Abnormal shapes of sperm heads (large arrows) are noted. Some sperm tails (curved arrows) house more than one cross-sectional profile of the midpiece. Coiling of the sperm tail is noted for some sperm, with few being extreme (E-H). Scale bars A-D = 500 nm; E, F = 5 μm; G = 2 μm; H = 3 μm.
Fig 12
Fig 12. Motility parameters of sperm from cauda epididymis of WT and KO mice were quantified by computer-assisted sperm analysis (CASA).
Bars represent the means of VAP (velocity average path, μm/s; P = 0.022); VSL (velocity straight-line, μm/s; P = 0.063); DAP (distance average path, μm; P = 0.032); ALH (amplitude of lateral sperm head displacement, μm; P = 0.046); AOC (average orientation change of the head, μm; P = 0.014) and WOB (wobble coefficient: VAP/VCL; P = 0.014). Error bars indicate the standard error of means. The asterisk (*) indicates a significant decreased pattern change, and # indicates an increased one.
Fig 13
Fig 13. In vitro fertilization (IVF) of KO (Hgsnat−/−) and WT (Hgsnat+/+) mice.
IVF rates of WT values were adjusted to 100% for comparative purposes, and experimental values obtained were adjusted accordingly. The block graphs depict the mean percentage of oocytes that were fertilized after incubation with sperm of Hgsnat KO animals (n = 33) in IVF medium, as compared to wild type (n = 31). A significant difference at * P = 0,0442 (Fisher exact test, two-tailed, 2x2) was observed between both groups and error bars indicate the standard error of the mean.
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Grants and funding

This investigation was supported by an NSERC grant 03896-2017 to Dr. Carlos R. Morales, and a Canadian Institutes of Health Research (CIHR, https://cihr-irsc.gc.ca/e/193.html) grant PJT-156345 and Canadian Glycomics Network (https://canadianglycomics.ca) grant ND-1 to Dr. Alexey V. Pshezhetsky. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.