Graffeo ML et al. (2025), A novel mechanism of sperm midpiece epididymal ...

ECB-ART-54059

Cell Commun Signal 2025 Jul 01;231:319. doi: 10.1186/s12964-025-02320-x.

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A novel mechanism of sperm midpiece epididymal maturation and the role of CCDC112 in sperm midpiece formation and establishing an optimal flagella waveform.

Graffeo ML , Nguyen J , Parast FY , Dunleavy JEM , Korneev D , Yang H , Okuda H , O'Connor AE , Conrad DF , Nosrati R , Houston BJ , O'Bryan MK .

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BACKGROUND: The mitochondrial sheath is a defining feature of mammalian sperm with proposed functions in structural support and energy production for flagella movement. Recently, coiled coil domain containing (CCDC) protein 112 (CCDC112) was suggested to play a role in the regulation of ciliogenesis. CCDC112 is a poorly characterised protein and there is virtually no knowledge of its in vivo function. METHODS: Here, we define CCDC112 as crucial for male fertility using a Ccdc112 loss-of-function mouse line. To characterize and analyze male fertility, and to identify a novel process of epididymal midpiece maturation, we utilized a range of assays including fertility testing, scanning electron microscopy, high-resolution sperm motility and power output analysis, in vitro fertilization, intracytoplasmic sperm injection, mitochondria stress test assays and glycolytic flux assays. Localization of CCDC112 in cilia was assessed via the transfection of IMCD-3 cells with a CCDC112-eGFP vector and subsequent immunofluorescent staining. RESULTS: Results reveal CCDC112 as a requirement for male fertility in the mouse with an essential role in mitochondrial sheath formation. Our data reveal the critical role of CCDC112 in mitochondrial morphogenesis during midpiece formation, with the lack of CCDC112 leading to significantly reduced respiration capacity, irregular flagellar waveforms, diminished progressive motility and ultimately male sterility. In the absence of CCDC112, sperm are unable to traverse the female reproductive tract to the site of fertilization and in vitro have a poor capacity to penetrate the zonae pellucidae of oocytes or fuse with the oocyte. We further unveil a previously unrecognized process of epididymal mitochondrial sheath maturation. We show the sperm midpiece is structurally immature upon exiting the testis and maturation continues during transit from the caput to the cauda epididymis. Finally, we identify CCDC112 as a component of the distal appendages of the mother centriole in IMCD-3 cells suggestive of a facilitative role for CCDC112 in protein entry into the ciliary compartment within germ cells. CONCLUSION: Collectively, we establish CCDC112 as a key regulator of sperm midpiece assembly and function while further expanding our understanding on functional sperm production, energy generation and flagella kinematics.

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	Fig. 1. CCDC112 is required for male fertility Litter size (A), body weight (B), testis weight (C), testis daily sperm production (DSP) (D) and total epididymal sperm count (ESC) (E) in Ccdc112WT/WT mice and Ccdc112KO/KO mice (n ≥ 4/genotype). Periodic acid-Schiff stained testis sections (F-H) and hematoxylin and eosin-stained epididymis sections (J) from Ccdc112WT/WT and Ccdc112KO/KO mice. Red and blue arrowheads indicate retained spermatids in (H) and prematurely sloughed germ cells in (J), respectively. (I) Quantification of the percentage of stage IX tubules with retained spermatids in Ccdc112WT/WT and Ccdc112KO/KO mice (n ≥ 9/tubules and n = 3/genotype). Lines denote mean ± SD in A-E, I; * P < 0.05, * P < 0.001, ** P < 0.0001. Scale bars in F-G = 50 μm, H = 10 μm, J = 20 μm
	Fig. 2. CCDC112 is essential for sperm tail structure and function and fertilization Percentage of motile (A) and progressively motile sperm (B) and average sperm velocity (VCL, curvilinear velocity; C) of cauda epididymal sperm from Ccdc112WT/WT mice and Ccdc112KO/KO mice (n = 5 mice/genotype). (D) Cauda epididymal sperm morphology as stained by hematoxylin and eosin. Abnormalities were observed in a majority of Ccdc112KO/KO sperm, including abnormal sperm head morphology (c-f), mitochondrial sheath defects (c, d), short (e) and coiled (f) sperm tails. (E) Quantification of abnormal sperm morphology phenotypes in Ccdc112WT/WT (white triangles) and Ccdc112KO/KO males (black triangles) (n = 5/genotype). (F) Sperm midpiece length as stained by annulus marker, SEPT4; designating the junction of the putative mid- and principal pieces and as measured from the base of the sperm head to the annulus (n = 3/genotype). (G) Sperm mitochondrial sheath length as measured on hematoxylin and eosin stained sperm from the base of the sperm head to end of discernable sheath (n = 3–4/genotype). (H) Sperm tail length as measured from the sperm head base to the tail tip (n = 5/genotype). (I) Axoneme ultrastructure of cauda epididymal sperm from Ccdc112WT/WT and Ccdc112KO/KO males as assessed via transmission electron microscopy. (J) Quantification of Ccdc112WT/WT (white triangles) and Ccdc112KO/KO mouse sperm (black triangles) in the isthmus and ampulla region of the oviduct in the female reproductive tract (n ≥ 5/genotype). (K) Percentage of zonae pellucidae intact oocytes (IVF) and zonae pellucidae stripped oocytes (ZF – IVF) which developed to the two-cell stage after in vitro fertilization and percentage of oocytes which developed to the two-cell stage after intracytoplasmic sperm injection (ICSI) with Ccdc112WT/WT and Ccdc112KO/KO mouse sperm (n ≥ 3/genotype). Lines denote mean ± SD in A-C, E-H, J-K; * P < 0.05, P < 0.01, * P < 0.001, **** P < 0.0001. Scale bars in D = 10 μm, I = 100 nm
	Fig. 3. CCDC112 is critical for mitochondrial sheath formation (A) A schematic of the epididymis in mice. Once sperm are released from the testis they are transported to the epididymis for maturation, quality control, and storage. They first enter into the caput (head) epididymis before transiting through the corpus (body) towards the cauda (tail) before eventually exiting through the vas deferens upon ejaculation. Scanning electron micrography images of caput (B) and cauda (C) epididymal sperm midpieces from Ccdc112WT/WT and Ccdc112KO/KO mice. Midpieces were rated on a scale of 1 to 7, based on mitochondrial sheath abnormality severity. A score of 1 represents highly disorganized and abnormal mitochondrial sheath formation and a score of 7 represents highly organized mitochondrial alignment, i.e., normal mitochondrial sheath formation. No rating 1 sperm were seen in Ccdc112WT/WT samples. Abnormalities included irregular alignment, morphology and size of mitochondria, immature mitochondria (red arrowheads), exposed outer dense fibers (white arrowheads; ODFs) and no mitochondria (yellow arrowheads). Percentage of normal caput and cauda midpieces (D-E) in Ccdc112WT/WT (white triangles) and Ccdc112KO/KO (black triangles) males (n ≥ 10 midpieces/animal and n ≥ 4/genotype). Differing lowercase letter designations denote significant differences between groups in D-E. Percentage of caput midpieces with immature ODF and/or no mitochondria (F), the rating of caput and cauda epididymal sperm midpiece normality (G), and the relative difference between caput sperm content and cauda sperm content (H) in Ccdc112WT/WT and Ccdc112KO/KO males (n ≥ 10 midpieces/animal and n ≥ 4/genotype). Lines denote mean ± SD in D, F-H; * P < 0.05, P < 0.01, * P < 0.001, **** P < 0.0001. Scale bars in B-C = 0.5 μm
	Fig. 4. Sperm tail beating patterns and energetics are critically dependent on CCDC112 (A) Representative sperm flagella waveform in sperm from Ccdc112WT/WT and Ccdc112KO/KO mice over the proximal most 60 μm of the sperm tail. Colored curves represent the non-dimensional time scale, where the beginning of the beat cycle is blue in the waveform and the end is in red. ‘y’ denotes the normalized amplitude and ‘x’ denotes the normalized length of the sperm tail where the y-intercept of 0 represents the sperm head. Percentage of sperm per genotype displaying each beat cycle type is denoted in the top right corner of each graph. (B) Representative shape cycles of the same sperm population in (A), as visualized by plotting the two dominant shape modes coefficients (B1 and B2) against each other over time (n = 5 animals/genotype and 8–15 sperm/animal). (C) Representative flagellum curvature plots, where the red and blue waves denote the direction of the flagellar bend above or below the sperm head hook and the intensity of the color denotes smaller radius of curvature (n = 5 animals/genotype and 8–15 sperm/animal). ‘T’ denotes time (seconds), and ‘s’ denotes arclength (µm/100) where 0 represents the sperm head. Beat frequency (D), principal piece amplitude (E), motor input (F), motor dissipation (G), internal dissipation (H), hydrodynamic dissipation (I), midpiece hydrodynamic dissipation (J) and principal piece hydrodynamic dissipation (K). Lines denote mean ± SD in D-K; P < 0.01, * P < 0.001
	Fig. 5. CCDC112 is essential for normal sperm energy production Mitochondria stress test assay on non-capacitated (A) and capacitated (B) Ccdc112WT/WT (white triangles) and Ccdc112KO/KO mouse sperm (black triangles) (n = 3 mice/genotype). Glycolytic flux Seahorse assay on non-capacitated (C) and capacitated (D) Ccdc112WT/WT and Ccdc112KO/KO mouse sperm (n = 3 mice/genotype). Note the difference in the Y axis scale between mitochondrial stress test and glycolytic assays. OCR = oxygen consumption rate; ECAR = extracellular acidification rate; O/M = oligomycin; AA + R = antimycin A and rotenone; 2-DG = 2-Deoxy-D-Glucose. Lines denote mean ± SD; * P < 0.05, P < 0.01, * P < 0.001
	Fig. 6. CCDC112 is a component of the distal appendages of the mother centriole Immunofluorescence staining of somatic IMCD-3 cells induced to produce primary cilia via serum starvation expressing e-GFP tagged CCDC112 (green). CCDC112 localized to the centrosome (as marked by γ-tubulin; red) (A). Staining of the distal and subdistal appendages as marked by ODF2 (red) refined localization to the mother centriole (B). Further staining localized CCDC112 specifically to the distal appendages (as marked by CEP170; red) of the mother centriole in interphase cells (C). Blue represents nuclei labelled by DAPI. Scale bars in A = 3 μm and in B-C = 1 μm
	Fig. 7. Summary schematic of sperm formation in the absence of CCDC112 In Ccdc112KO/KO mice, sperm commonly possessed abnormal mitochondrial sheath structure with overall irregular mitochondrial morphology and poor mitochondrial elongation, staggering and compaction during sheath formation. A subset of sperm also possessed exposed outer dense fibers (ODFs) and immature mitochondria within the sheath, abnormal head morphology and shortened tails. As a result, sperm exhibited poor energy generation, reduced tail flexibility, particularly in the midpiece, and reduced sperm power. Image modified from [73]

	Fig. 1. CCDC112 is required for male fertility Litter size (A), body weight (B), testis weight (C), testis daily sperm production (DSP) (D) and total epididymal sperm count (ESC) (E) in Ccdc112WT/WT mice and Ccdc112KO/KO mice (n ≥ 4/genotype). Periodic acid-Schiff stained testis sections (F-H) and hematoxylin and eosin-stained epididymis sections (J) from Ccdc112WT/WT and Ccdc112KO/KO mice. Red and blue arrowheads indicate retained spermatids in (H) and prematurely sloughed germ cells in (J), respectively. (I) Quantification of the percentage of stage IX tubules with retained spermatids in Ccdc112WT/WT and Ccdc112KO/KO mice (n ≥ 9/tubules and n = 3/genotype). Lines denote mean ± SD in A-E, I; * P < 0.05, * P < 0.001, ** P < 0.0001. Scale bars in F-G = 50 μm, H = 10 μm, J = 20 μm
	Fig. 2. CCDC112 is essential for sperm tail structure and function and fertilization Percentage of motile (A) and progressively motile sperm (B) and average sperm velocity (VCL, curvilinear velocity; C) of cauda epididymal sperm from Ccdc112WT/WT mice and Ccdc112KO/KO mice (n = 5 mice/genotype). (D) Cauda epididymal sperm morphology as stained by hematoxylin and eosin. Abnormalities were observed in a majority of Ccdc112KO/KO sperm, including abnormal sperm head morphology (c-f), mitochondrial sheath defects (c, d), short (e) and coiled (f) sperm tails. (E) Quantification of abnormal sperm morphology phenotypes in Ccdc112WT/WT (white triangles) and Ccdc112KO/KO males (black triangles) (n = 5/genotype). (F) Sperm midpiece length as stained by annulus marker, SEPT4; designating the junction of the putative mid- and principal pieces and as measured from the base of the sperm head to the annulus (n = 3/genotype). (G) Sperm mitochondrial sheath length as measured on hematoxylin and eosin stained sperm from the base of the sperm head to end of discernable sheath (n = 3–4/genotype). (H) Sperm tail length as measured from the sperm head base to the tail tip (n = 5/genotype). (I) Axoneme ultrastructure of cauda epididymal sperm from Ccdc112WT/WT and Ccdc112KO/KO males as assessed via transmission electron microscopy. (J) Quantification of Ccdc112WT/WT (white triangles) and Ccdc112KO/KO mouse sperm (black triangles) in the isthmus and ampulla region of the oviduct in the female reproductive tract (n ≥ 5/genotype). (K) Percentage of zonae pellucidae intact oocytes (IVF) and zonae pellucidae stripped oocytes (ZF – IVF) which developed to the two-cell stage after in vitro fertilization and percentage of oocytes which developed to the two-cell stage after intracytoplasmic sperm injection (ICSI) with Ccdc112WT/WT and Ccdc112KO/KO mouse sperm (n ≥ 3/genotype). Lines denote mean ± SD in A-C, E-H, J-K; * P < 0.05, P < 0.01, * P < 0.001, **** P < 0.0001. Scale bars in D = 10 μm, I = 100 nm
	Fig. 3. CCDC112 is critical for mitochondrial sheath formation (A) A schematic of the epididymis in mice. Once sperm are released from the testis they are transported to the epididymis for maturation, quality control, and storage. They first enter into the caput (head) epididymis before transiting through the corpus (body) towards the cauda (tail) before eventually exiting through the vas deferens upon ejaculation. Scanning electron micrography images of caput (B) and cauda (C) epididymal sperm midpieces from Ccdc112WT/WT and Ccdc112KO/KO mice. Midpieces were rated on a scale of 1 to 7, based on mitochondrial sheath abnormality severity. A score of 1 represents highly disorganized and abnormal mitochondrial sheath formation and a score of 7 represents highly organized mitochondrial alignment, i.e., normal mitochondrial sheath formation. No rating 1 sperm were seen in Ccdc112WT/WT samples. Abnormalities included irregular alignment, morphology and size of mitochondria, immature mitochondria (red arrowheads), exposed outer dense fibers (white arrowheads; ODFs) and no mitochondria (yellow arrowheads). Percentage of normal caput and cauda midpieces (D-E) in Ccdc112WT/WT (white triangles) and Ccdc112KO/KO (black triangles) males (n ≥ 10 midpieces/animal and n ≥ 4/genotype). Differing lowercase letter designations denote significant differences between groups in D-E. Percentage of caput midpieces with immature ODF and/or no mitochondria (F), the rating of caput and cauda epididymal sperm midpiece normality (G), and the relative difference between caput sperm content and cauda sperm content (H) in Ccdc112WT/WT and Ccdc112KO/KO males (n ≥ 10 midpieces/animal and n ≥ 4/genotype). Lines denote mean ± SD in D, F-H; * P < 0.05, P < 0.01, * P < 0.001, **** P < 0.0001. Scale bars in B-C = 0.5 μm
	Fig. 4. Sperm tail beating patterns and energetics are critically dependent on CCDC112 (A) Representative sperm flagella waveform in sperm from Ccdc112WT/WT and Ccdc112KO/KO mice over the proximal most 60 μm of the sperm tail. Colored curves represent the non-dimensional time scale, where the beginning of the beat cycle is blue in the waveform and the end is in red. ‘y’ denotes the normalized amplitude and ‘x’ denotes the normalized length of the sperm tail where the y-intercept of 0 represents the sperm head. Percentage of sperm per genotype displaying each beat cycle type is denoted in the top right corner of each graph. (B) Representative shape cycles of the same sperm population in (A), as visualized by plotting the two dominant shape modes coefficients (B1 and B2) against each other over time (n = 5 animals/genotype and 8–15 sperm/animal). (C) Representative flagellum curvature plots, where the red and blue waves denote the direction of the flagellar bend above or below the sperm head hook and the intensity of the color denotes smaller radius of curvature (n = 5 animals/genotype and 8–15 sperm/animal). ‘T’ denotes time (seconds), and ‘s’ denotes arclength (µm/100) where 0 represents the sperm head. Beat frequency (D), principal piece amplitude (E), motor input (F), motor dissipation (G), internal dissipation (H), hydrodynamic dissipation (I), midpiece hydrodynamic dissipation (J) and principal piece hydrodynamic dissipation (K). Lines denote mean ± SD in D-K; P < 0.01, * P < 0.001
	Fig. 5. CCDC112 is essential for normal sperm energy production Mitochondria stress test assay on non-capacitated (A) and capacitated (B) Ccdc112WT/WT (white triangles) and Ccdc112KO/KO mouse sperm (black triangles) (n = 3 mice/genotype). Glycolytic flux Seahorse assay on non-capacitated (C) and capacitated (D) Ccdc112WT/WT and Ccdc112KO/KO mouse sperm (n = 3 mice/genotype). Note the difference in the Y axis scale between mitochondrial stress test and glycolytic assays. OCR = oxygen consumption rate; ECAR = extracellular acidification rate; O/M = oligomycin; AA + R = antimycin A and rotenone; 2-DG = 2-Deoxy-D-Glucose. Lines denote mean ± SD; * P < 0.05, P < 0.01, * P < 0.001
	Fig. 6. CCDC112 is a component of the distal appendages of the mother centriole Immunofluorescence staining of somatic IMCD-3 cells induced to produce primary cilia via serum starvation expressing e-GFP tagged CCDC112 (green). CCDC112 localized to the centrosome (as marked by γ-tubulin; red) (A). Staining of the distal and subdistal appendages as marked by ODF2 (red) refined localization to the mother centriole (B). Further staining localized CCDC112 specifically to the distal appendages (as marked by CEP170; red) of the mother centriole in interphase cells (C). Blue represents nuclei labelled by DAPI. Scale bars in A = 3 μm and in B-C = 1 μm
	Fig. 7. Summary schematic of sperm formation in the absence of CCDC112 In Ccdc112KO/KO mice, sperm commonly possessed abnormal mitochondrial sheath structure with overall irregular mitochondrial morphology and poor mitochondrial elongation, staggering and compaction during sheath formation. A subset of sperm also possessed exposed outer dense fibers (ODFs) and immature mitochondria within the sheath, abnormal head morphology and shortened tails. As a result, sperm exhibited poor energy generation, reduced tail flexibility, particularly in the midpiece, and reduced sperm power. Image modified from [73]