Tmem203 is essential for spermiogenesis
Tmem203 deficient mice lacked mature spermatozoa (complete azoospermia) as detected by a computer assisted sperm analyzer (CASA: Fig 4A). Histologic evaluation of the epididymides harvested from either 38-week-old or 48-week-old male Tmem203 deficient mice also showed a complete lack of mature spermatozoa in the epididymides (azoospermia) compared to wild type mice (Fig 4B). No abnormalities were seen in Tmem203 heterozygous mice (data not shown).
10.1371/journal.pone.0127480.g004 Fig 4  Tmem203 null mice completely lack mature spermatozoa in epididymis.
(A) Computer assisted sperm analyzer based analysis of epididymis preparations from wild type and Tmem203 null mice showed complete absence (*) of mature spermatozoa in Tmem203 null mice. Data is representative of two independent experiments. (B) Representative photomicrographs illustrating hematoxylin and eosin (H&E)-stained sections of proximal epididymis (caput; upper left and right panels) and distal epididymis (cauda; lower left and right panels) from a 48-week-old wild type mouse (upper and lower left panels) and from a 48-week-old Tmem203 null mice (upper and lower right panels). Note the complete absence of mature spermatozoa in the epididymis of the Tmem203 null mice compared to the wild type mice in which numerous mature spermatozoa are observed; tubular lumina of the epididymis from Tmem203 null mice contains eosinophilic proteinaceous material mixed with cellular debris. Scale bars = 50 μm. To determine the nature of azoospermia in Tmem203 null mice, spermatogenesis was examined in detail. We first evaluated the distribution of propidium iodide stained testicular cells from 35 day old or 30 week old Tmem203 null and wild type mice. DNA staining during the first wave of spermiogenesis at day 35 was indistinguishable from WT mice (Fig 5A). In contrast, at 30 weeks old, there was a large reduction of the PI stained population of Tmem203 deficient spermatocytes corresponding to the post-meiotic condensed haploid population (1n-C) (Fig 5B). Peaks representing the other developmental stages of spermatogenic cells and progression of spermatogenesis till elongated spermatids were comparable in Tmem203 null and wild type samples. Thus FACs analysis suggests that meiosis appears to be normal but Tmem203 deficient spermiogenesis fails beginning by the stage of nuclear condensation.
10.1371/journal.pone.0127480.g005 Fig 5  Tmem203 null mice exhibit a disruption of spermiogenesis.
(A-B) Propidium iodide based DNA flow cytometry analysis of testicular cell suspensions from wild-type (red tracer) and Tmem203 null mice (green tracer) at 35 day (A) or 30 week (B) (n = 2 or 3 for each genotype). Arrows highlight the differences between the wild-type and Tmem203 null samples. Abbreviations: haploid-condensed (1n-C)-elongated spermatids; haploid (1n) round spermatids; diploid (2n)—Sertoli cells, spermatogonia; S-ph, spermatogonia synthesizing DNA and the tetraploid (4n)—pachytene spermatocytes and G2 spermatogonia (C) Representative photomicrographs illustrating hematoxylin and eosin (H&E) stained sections of Stage VII seminiferous tubules from a 48-week-old wild type mouse (left panels) and from a 48-week-old cMAC knockout mouse (right panels). Compared to the seminiferous tubule from the wild type mouse (left panels) the predominant morphological changes observed in the seminiferous tubule of the cMAC knockout mouse (right panels) are characterized by an overall subtle, relative reduction in numbers of late stage post-meiotic spermatids (steps 9–16), degenerative intracytoplasmic vacuolar changes most prominent in step 16 spermatids and complete lack of spermiation (disengagement of step 16 spermatozoa from the Sertoli cell and release into the tubular lumen). Lower panels illustrate higher magnification of areas enclosed by square boxes in the upper left and right panels. Scale bars = 50 μm (upper panels) and 25 μm (lower panels). (D-E) Representative transmission electron micrographs of Stage VII seminiferous tubules from a 32-week-old wild type mouse (left panels) and from a 32-week-old Tmem203 null mouse (right panels). Labeled are step 7 spermatocytes (7), residual bodies (rb), endoplasmic reticulum (er) and degenerate, misshapen spermatid heads (asterisks) mitochondrial sheath (ms) surrounding the outer dense fibers, axoneme and axoneme complex of microtubules. Phagocytosis by Sertoli cells of degenerate spermatids is illustrated in both the top and bottom panel on the right for the Tmem203 null mouse. Residual bodies (rb) contain dense aggregations of RNA, lipid, clear vesicles, multivesicular bodies and other organelles. Scale bars = 5.0 μm (for D),2.0 μm (for E). Microscopic evaluation of the testes harvested from either 38-week-old or 48-week-old male mice showed prominent defective spermiogenesis in Tmem203 deficient mice, despite a relatively normal overall structural appearance and organization of the seminiferous tubular epithelium (Fig 5C). The predominant morphological changes observed in the seminiferous tubules of Tmem203 null mice were characterized by: a) an overall but subtle reduction in numbers of late stage post-meiotic spermatids (steps 9–16) in the stages of the cycle of spermatogenesis in which progressive elongation and condensation of the nucleus occurs [30,31], b) degenerative intra-cytoplasmic vacuolar changes most prominent in step 16 spermatids, c) complete lack of spermiation (disengagement of step 16 spermatozoa from the Sertoli cell and release into the tubular lumen), and d) retention of step 16 spermatids in stage VIII seminiferous tubules. Morphological abnormalities were not observed microscopically in Tmem203 null mice in non-spermatogenic cells including Sertoli cells and interstitial Leydig cells. In Tmem203 null mice, atrophy of individual seminiferous tubules was occasionally observed multifocally scattered randomly throughout testes (data not shown).
Transmission electron microscopy performed on testes harvested from 32-week-old Tmem203 null mice and age-matched wild type mice confirmed changes observed by bright field light microscopy including degenerative intra-cytoplasmic vacuolar changes as well defective spermiation. In addition, ultra-structural evaluation of the testes from Tmem203 null mice revealed abnormalities associated with the shape of the head of individual elongated spermatids (teratozoospermia) despite apparent normal condensation of chromatin and morphology of the acrosome (Fig 5D and 5E). Numerous, large, ovoid phagocytic structures which contained degenerate and fragmented elongated spermatids as well as remnants of elongated spermatid tail fragments were often observed throughout all levels of the seminiferous tubule; in Stage VII tubules these structures were most commonly observed near the lumina of seminiferous tubules (Fig 5D and 5E). Abundant amounts of intra-cytoplasmic material which ultra-structurally resembled endoplasmic reticulum were often observed in association with ovoid phagocytic structures. Despite degenerative changes associated with the head of elongated spermatids in Tmem203 null mice, the cytoskeletal components of the connecting, middle, principle and end pieces of the flagellum of individual mature elongated spermatids appeared to be ultra-structurally normal including the axoneme, axoneme complex of microtubules (two central microtubules surrounded by nine microtubule doublets) and fibrous sheath. Individual mitochondria helically wrapped around the outer dense fibers in the middle piece of the sperm tail (mitochondrial sheath) associated with step 15 spermatids in Stage VI seminiferous tubules and step 16 spermatids in Stage VII seminiferous tubules also appeared to be ultra-structurally normal (Fig 5D and 5E). To summarize, spermatogenesis defects appeared post-meiotically in Tmem203 deficient mice resulting in a severe reduction of late stage spermatids, appearance of only abnormal condensed haploid spermatocytes that fail to undergo spermiation.