Analysis Tools
reproductive system
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• relative weights of testes and cauda epididymis are not significantly reduced relative to those in wild-type males
• H&E staining showed normal seminiferous tubules morphology
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• Mito Red immunostaining of cauda epididymal sperm showed various abnormalities ranging from an abnormally thick midpiece, a slightly tapered bent thick neck, and cytoplasmic droplets with amorphous sperm heads
• ~55% of Mito Red-stained sperm show flagellar defects
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• a slightly tapered bent thick neck is observed
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• thick sperm midpieces with distal cytoplasmic droplets are observed
• however, mature sperm flagella show normal F-actin organization in the midpiece
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• plasma membrane does not cover the mitochondrial sheath uniformly
• disorganized or vacuolated mitochondria are observed
• a fibrous sheet containing more than one axoneme-mitochondrial complex is observed
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necrospermia
(
J:320651
)
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• Eosin & Nigrosine (E&N) staining revealed that the percentage of viable sperm is borderline (~40%) relative to that in heterozygous and wild-type males (60-80%)
• hypo-osmotic swelling test (where intact live sperm show a swelling/curling of the tail) revealed that the percentage of hypo-osmotic reactive sperm is also borderline, indicating that sperm membrane integrity is affected
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• cauda epididymal sperm count is significantly lower than that in heterozygous and wild-type males
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• DAPI-stained sperm cells exhibit abnormal head shape with alterations in area, circularity, length of hook, bounding width, and regularity
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• 51-56% of mature epididymal spermatozoa exhibit malformed/fragmented acrosomes
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• acrosomal biogenesis is impaired at the Golgi phase: PNA staining of adult testis sections is scattered in Golgi-phase spermatids, suggesting a less uniform acrosomal compartment; TEM analysis confirmed the presence a loosely clustered granular structure
• in the cap phase, cap structures are more unevenly distributed in round spermatids; TEM showed a bulgier and detached cap
• in the acrosomal phase, acrosomal content fails to form an arrow-like shape in elongating spermatids; TEM showed a malformed developing acrosome covering
• in the maturation phase, TEM revealed a malformed acrosomal structure
• arrested acrosome formation is due to failure of proacrosomal granule formation and fusion associated with deregulation of autophagy master regulators (Trim27, AMPK, mTOR, Atg2a, LC3B, and SQSTM1)
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• failure of proacrosomal granule formation and fusion
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• 70-80% of sperm show irregular/round head morphology
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• in the cap phase, the acrosomal vesicle fails to form a continuous cap-like structure and presents as a granule detached from the acroplaxome
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• DAPI-stained sperm cells show amorphous nuclear morphology
• actin-related protein T3 (ACTRT3, aka ARPM1) is absent in the nuclear fraction of testicular sperm
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• CASA analysis revealed a significant reduction in progressive sperm motility
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• CASA analysis revealed a significant reduction in total sperm motility
• however, curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) are not significantly reduced
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• spermiogenesis is impaired as shown by defects in acrosome biogenesis, autophagy, manchette development and mitochondrial organization
• mature sperm display a cytoplasm removal defect
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• round spermatids show abnormal manchette development
• defects in manchette development are more prominent in step 9-13 spermatids, leading to an amorphous sperm head shape
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• mating of homozygous males with wild-type females results in an average litter of 1.5 relative to 7.6 for heterozygous males and 8.2 for wild-type males
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• adult males are subfertile
• however, mating behavior is normal as vaginal plugs are clearly observed
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• in vitro, the rate of A23187-induced acrosome reaction is significantly reduced, with only 37.5% of cauda epididymal sperm undergoing acrosome exocytosis versus 60% of sperm in wild-type males
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cellular
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• Mito Red immunostaining of cauda epididymal sperm showed various abnormalities ranging from an abnormally thick midpiece, a slightly tapered bent thick neck, and cytoplasmic droplets with amorphous sperm heads
• ~55% of Mito Red-stained sperm show flagellar defects
|
|
|
• a slightly tapered bent thick neck is observed
|
|
|
• thick sperm midpieces with distal cytoplasmic droplets are observed
• however, mature sperm flagella show normal F-actin organization in the midpiece
|
|
|
• plasma membrane does not cover the mitochondrial sheath uniformly
• disorganized or vacuolated mitochondria are observed
• a fibrous sheet containing more than one axoneme-mitochondrial complex is observed
|
necrospermia
(
J:320651
)
|
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• Eosin & Nigrosine (E&N) staining revealed that the percentage of viable sperm is borderline (~40%) relative to that in heterozygous and wild-type males (60-80%)
• hypo-osmotic swelling test (where intact live sperm show a swelling/curling of the tail) revealed that the percentage of hypo-osmotic reactive sperm is also borderline, indicating that sperm membrane integrity is affected
|
|
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• cauda epididymal sperm count is significantly lower than that in heterozygous and wild-type males
|
|
|
• DAPI-stained sperm cells exhibit abnormal head shape with alterations in area, circularity, length of hook, bounding width, and regularity
|
|
|
• 51-56% of mature epididymal spermatozoa exhibit malformed/fragmented acrosomes
|
|
|
• acrosomal biogenesis is impaired at the Golgi phase: PNA staining of adult testis sections is scattered in Golgi-phase spermatids, suggesting a less uniform acrosomal compartment; TEM analysis confirmed the presence a loosely clustered granular structure
• in the cap phase, cap structures are more unevenly distributed in round spermatids; TEM showed a bulgier and detached cap
• in the acrosomal phase, acrosomal content fails to form an arrow-like shape in elongating spermatids; TEM showed a malformed developing acrosome covering
• in the maturation phase, TEM revealed a malformed acrosomal structure
• arrested acrosome formation is due to failure of proacrosomal granule formation and fusion associated with deregulation of autophagy master regulators (Trim27, AMPK, mTOR, Atg2a, LC3B, and SQSTM1)
|
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• failure of proacrosomal granule formation and fusion
|
|
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• 70-80% of sperm show irregular/round head morphology
|
|
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• in the cap phase, the acrosomal vesicle fails to form a continuous cap-like structure and presents as a granule detached from the acroplaxome
|
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• DAPI-stained sperm cells show amorphous nuclear morphology
• actin-related protein T3 (ACTRT3, aka ARPM1) is absent in the nuclear fraction of testicular sperm
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• round spermatids show abnormal manchette development
• defects in manchette development are more prominent in step 9-13 spermatids, leading to an amorphous sperm head shape
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• spermatids exhibit defects and disorganization in cis- and trans-Golgi network
• disruption of the Golgi sub-domains causes abnormal formation of Golgi-derived proacrosomal vesicles leading to acrosome malformation
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• mTOR signaling is activated while AMPK is suppressed, leading to accumulation of LC3B and SQSTM1 proteins in the testes, indicating disruption of the autophagic flux
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• CASA analysis revealed a significant reduction in progressive sperm motility
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• CASA analysis revealed a significant reduction in total sperm motility
• however, curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) are not significantly reduced
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• acrosome development is impaired due to defective transport of vesicles released from Golgi complexes
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homeostasis/metabolism
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• mTOR signaling is activated while AMPK is suppressed, leading to accumulation of LC3B and SQSTM1 proteins in the testes, indicating disruption of the autophagic flux
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• LC3B, P62/SQSTM1, mTOR and p-mTOR protein levels are increased in testes, indicating inhibition in autophagy
• actin-related protein T3 (ACTRT3, aka ARPM1) is absent in the nuclear fraction of testes and sperm, suggesting that destabilization of the PFN3-ARPM1 complex results in the degradation of ARPM1
• testes show increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and destrin (DSTN, aka actin depolymerizing factor or ADF)
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