Analysis Tools
reproductive system
| N |
• female mice are fertile; males exhibit normal testicular size and testis/body weight ratio relative to wild-type controls
|
 |
• CASA analysis revealed that epididymal sperm count is significantly lower than that in wild-type controls
|
|
|
• epididymal spermatozoa exhibit a significantly increased number of head malformations
• however, sperm flagella appear normal with typical 9+2 microtubule structures
|
|
|
• at step 16, round-to-elongated spermatids exhibit abnormal sperm heads
• ~64.60% of epididymal spermatozoa show abnormal sperm heads relative to ~8.70% of wild-type cells
|
|
|
• the acrosome does not normally extend when its dorsal and ventral surface are lost
|
|
|
• TEM analysis shows that the acrosome is separated from the sperm head nucleus
|
|
|
• sperm nuclei show defects in DNA condensation and a significantly lower grey intensity ratio than wild-type sperm
• in situ Hi-C sequencing results indicate that the density of spermatid nuclei is reduced, the higher-order structure of chromatin is altered, and the interaction of distal chromatin is weakened
• P(s) curve analysis (100 kb, chr1) representing the chromatin contact probability relative to the genomic distance confirms that the interaction frequency is decreased in distal regions
• analysis of the average TAD (topologically associating domain) interaction frequency and the insulation score around TADs show weakened TAD dynamics in sperm cells, consistent with defective sperm nuclear condensation and an altered 3D chromatin organization
|
|
|
• starting at step 10 of spermatid development, elongating spermatids appear malformed; the nucleus is overextended while the structural integrity (hooked head and dorsal angle) is lost
• at steps 11-16, all elongating spermatids are globally malformed
|
|
|
• percentage of progressively (forward) motile sperm is significantly lower than that in wild-type controls
|
|
|
• percentage of motile epididymal sperm is significantly lower than that in wild-type controls
|
|
|
• elongated spermatids that should not be found at steps 9 and 10 are still found at stages IX-X in the seminiferous epithelium; in contrast, all types of germ cells are present and appear normal in the epithelium before step 9
• qRT-PCR shows that testicular transcript levels of transition proteins 1 and 2 (Tnp1/2) and protamine 1 and 2 (Prm1/2) are downregulated whereas mRNA levels of other genes marking various cell-development stages (Akap3, Tssk6, Crem, Spaca9, and Odf3) are unaffected
• Western blotting shows a significant decrease in TNP1, TNP2, PRM1, and PRM2 protein expression in testes, while immunofluorescence analysis indicates a further reduction in the intensity of testicular TNP1 and PRM2 protein
• moreover, Western blot analysis of mature cauda epididymal sperm confirms that PRM protein levels are significantly reduced, whereas levels of H2A, H2B, H3 and H4 are significantly increased in mature sperm
• chromomycin A3 (CMA3) immunofluorescence staining (used to assess protamine deficiency) shows a significantly increased CMA3 signal in late-stage spermatids and epididymal sperm, indicating that histone-to-protamine exchange is impaired
|
|
|
• histologically determined morphology of the seminiferous epithelial tissue shows delayed sperm release in the testis tubules
|
|
|
• histology of the seminiferous epithelial tissue shows a spermiation failure in stage IX
|
|
|
• all males fail to produce offspring
|
|
|
• IVF assays show that sperm are rarely able to fertilize oocytes; in vitro fertilization rate is only 7.57% versus 88.20% for wild-type controls
|
cellular
|
|
• CASA analysis revealed that epididymal sperm count is significantly lower than that in wild-type controls
|
|
|
• epididymal spermatozoa exhibit a significantly increased number of head malformations
• however, sperm flagella appear normal with typical 9+2 microtubule structures
|
|
|
• at step 16, round-to-elongated spermatids exhibit abnormal sperm heads
• ~64.60% of epididymal spermatozoa show abnormal sperm heads relative to ~8.70% of wild-type cells
|
|
|
• the acrosome does not normally extend when its dorsal and ventral surface are lost
|
|
|
• TEM analysis shows that the acrosome is separated from the sperm head nucleus
|
|
|
• sperm nuclei show defects in DNA condensation and a significantly lower grey intensity ratio than wild-type sperm
• in situ Hi-C sequencing results indicate that the density of spermatid nuclei is reduced, the higher-order structure of chromatin is altered, and the interaction of distal chromatin is weakened
• P(s) curve analysis (100 kb, chr1) representing the chromatin contact probability relative to the genomic distance confirms that the interaction frequency is decreased in distal regions
• analysis of the average TAD (topologically associating domain) interaction frequency and the insulation score around TADs show weakened TAD dynamics in sperm cells, consistent with defective sperm nuclear condensation and an altered 3D chromatin organization
|
|
|
• starting at step 10 of spermatid development, elongating spermatids appear malformed; the nucleus is overextended while the structural integrity (hooked head and dorsal angle) is lost
• at steps 11-16, all elongating spermatids are globally malformed
|
|
|
• histologically determined morphology of the seminiferous epithelial tissue shows delayed sperm release in the testis tubules
|
|
|
• histology of the seminiferous epithelial tissue shows a spermiation failure in stage IX
|
|
|
• percentage of progressively (forward) motile sperm is significantly lower than that in wild-type controls
|
|
|
• percentage of motile epididymal sperm is significantly lower than that in wild-type controls
|
|
|
• Western blotting shows that the levels of the linker histone H1.0 and the testis-specific H1 variant H1.6 are decreased in testes, whereas other histone variants, including H2A/B, H3.1, H3.3, and H4, likely remain unaffected
• ubiquitination levels of H2A/B and acetylation levels in H4 histone (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) are reduced in testes, while methylation of histone 3 at K4, K9, and K36 likely remains intact
• not only the total protamine level but also the chromatin-associated protein TNPs and PRMs levels are significantly reduced in spermatids, indicating that transition proteins fail to replace histones in the testes
|
