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TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis.

Shambharkar PB, Bittinger M, Latario B, Xiong Z, Bandyopadhyay S, Davis V, Lin V, Yang Y, Valdez R, Labow MA - PLoS ONE (2015)

Bottom Line: TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux.Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis.Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Developmental and Molecular Pathways, 100 Technology Square, Cambridge, Massachusetts, United States of America.

ABSTRACT
Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.

No MeSH data available.


Related in: MedlinePlus

Tmem203  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  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  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  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  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).
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pone.0127480.g005: Tmem203 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 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 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 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 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).

Mentions: To determine the nature of azoospermia in Tmem203 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 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 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.


TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis.

Shambharkar PB, Bittinger M, Latario B, Xiong Z, Bandyopadhyay S, Davis V, Lin V, Yang Y, Valdez R, Labow MA - PLoS ONE (2015)

Tmem203  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  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  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  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  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).
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pone.0127480.g005: Tmem203 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 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 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 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 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).
Mentions: To determine the nature of azoospermia in Tmem203 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 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 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.

Bottom Line: TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux.Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis.Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Developmental and Molecular Pathways, 100 Technology Square, Cambridge, Massachusetts, United States of America.

ABSTRACT
Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.

No MeSH data available.


Related in: MedlinePlus