Analysis Tools
reproductive system
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• the number of DDX4-positive germ cells per seminiferous tubule is significantly decreased at P7, P14 and P21 whereas the number of PLZF-positive cells per tubule is normal
• the ratio of DDX4-positive to WT1-positive cell numbers per seminiferous tubule is significantly increased at P7 but reduced at P14 and P21
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• many DDX4 (DEAD box helicase 4)-positive and TUNEL-positive cells are detected in the cauda epididymis at P56
• number of TUNEL-positive cells per seminiferous tubule is significantly increased at P7, P14 and P21, indicating increased male germ cell apoptosis
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• the ratio of Ki67-positive cells to WT1-positive Sertoli cells per seminiferous tubule is significantly reduced at P3 and P5, indicating decreased Sertoli cell proliferation
• in vitro, the % of EDU-positive Sertoli cells is significantly less than in controls at P3 and P5
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• F-ACTIN is no longer tightly localized at the basal ectoplasmic specialization (ES) to support the blood-testis barrier (BTB) but appears as a truncated/branched network diffusely localized at the adluminal compartment of seminiferous tubules
• basal ES proteins (N-cadherin and beta-catenin) and tight junction proteins (ZO-2) are diffusely localized at the BTB, extending well beyond the basement membrane
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• at P60, immunostainings of beta-ACTIN and alpha-TUBULIN revealed a disorganized actin and microtubular arrangement in seminiferous tubules
• the total length of vimentin-positive Sertoli cell arms is significantly shorter than in controls
• overall, the cytoskeletal organization of Sertoli cells is disrupted, thus compromising cell adhesion between germ cells and Sertoli cells
• at P21, purified primary Sertoli cells also show a disorganized F-ACTIN cytoskeletal structure
• at P56, TEM analysis of the seminiferous epithelium showed abnormally thin cytoplasmic arms, poor adherence to neighboring germ cell gaps, fragmented nucleoli, and massive accumulation of lipids in Sertoli cells
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• the number of WT1 (WT1 transcription factor)-positive Sertoli cells per seminiferous tubule is significantly reduced from P5 to P56
• the ratio of Ki67-positive cells to WT1-positive cells per seminiferous tubule is significantly reduced at P3 and P5, indicating decreased Sertoli cell proliferation
• however, no increase in Sertoli cell apoptosis is detected by TUNEL assay and WT1 co-staining at P21 and P43
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• from P14 to P56, Sertoli cell nuclei are found in the centers of >60% of the seminiferous tubules, ranging from single nuclei to intratubular clusters of 5-10 nuclei, suggesting disrupted Sertoli cell polarity
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• seminiferous tubule diameter is significantly reduced from P7 to P56
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• adult males show marked degeneration and atrophy of the seminiferous tubules
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• mRNA levels extracellular matrix (ECM)- and cell adhesion-related genes (e.g. Timp1, Trf, Spp1, Ccbe1, Lcn2, and Ahsg), are significantly upregulated in Sertoli cells at P3 (SCs in proliferation) and at P21 (SCs in differentiation) due to their DNA demethylation status
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small testis
(
J:324178
)
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• testis size is significantly reduced at P56
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• from P7 to P56, testis weight/body weight ratio is significantly lower than that in control males
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• at P56, an in vivo biotin tracing assay showed presence of the tracer dye in the adluminal compartment of seminiferous tubules, suggesting a leaky BTB
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• spermatogenic cells are gradually lost due to apoptosis from P7 leading to arrest of spermatogenesis at the elongating/elongated spermatid stage
• a massive loss of immature germ cells from the seminiferous epithelium is observed
• however, the ratio of 1N cell fractions (spermatids), 2N fractions (G1-phase spermatogonia and Sertoli cells), and 4N fractions (primary spermatocytes and G2-phase spermatogonia) is normal at P21, indicating normal spermatogonial differentiation and meiotic processes
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• epididymis size is significantly reduced at P56
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• adult males are completely infertile
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cellular
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• the number of DDX4-positive germ cells per seminiferous tubule is significantly decreased at P7, P14 and P21 whereas the number of PLZF-positive cells per tubule is normal
• the ratio of DDX4-positive to WT1-positive cell numbers per seminiferous tubule is significantly increased at P7 but reduced at P14 and P21
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• the ratio of P27-positive Sertoli cells to total Sertoli cells is significantly lower than in controls at P14, suggesting a delay in initiating cell cycle arrest
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• many DDX4 (DEAD box helicase 4)-positive and TUNEL-positive cells are detected in the cauda epididymis at P56
• number of TUNEL-positive cells per seminiferous tubule is significantly increased at P7, P14 and P21, indicating increased male germ cell apoptosis
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• the ratio of Ki67-positive cells to WT1-positive Sertoli cells per seminiferous tubule is significantly reduced at P3 and P5, indicating decreased Sertoli cell proliferation
• in vitro, the % of EDU-positive Sertoli cells is significantly less than in controls at P3 and P5
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• methylation levels in promoter regions of extracellular matrix (ECM)- and cell adhesion-related genes Timp1, Trf, and Spp1 are reduced in Sertoli cells at P3 and P21 relative to those in control Sertoli cells
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endocrine/exocrine glands
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• the ratio of Ki67-positive cells to WT1-positive Sertoli cells per seminiferous tubule is significantly reduced at P3 and P5, indicating decreased Sertoli cell proliferation
• in vitro, the % of EDU-positive Sertoli cells is significantly less than in controls at P3 and P5
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|
• F-ACTIN is no longer tightly localized at the basal ectoplasmic specialization (ES) to support the blood-testis barrier (BTB) but appears as a truncated/branched network diffusely localized at the adluminal compartment of seminiferous tubules
• basal ES proteins (N-cadherin and beta-catenin) and tight junction proteins (ZO-2) are diffusely localized at the BTB, extending well beyond the basement membrane
|
|
|
• at P60, immunostainings of beta-ACTIN and alpha-TUBULIN revealed a disorganized actin and microtubular arrangement in seminiferous tubules
• the total length of vimentin-positive Sertoli cell arms is significantly shorter than in controls
• overall, the cytoskeletal organization of Sertoli cells is disrupted, thus compromising cell adhesion between germ cells and Sertoli cells
• at P21, purified primary Sertoli cells also show a disorganized F-ACTIN cytoskeletal structure
• at P56, TEM analysis of the seminiferous epithelium showed abnormally thin cytoplasmic arms, poor adherence to neighboring germ cell gaps, fragmented nucleoli, and massive accumulation of lipids in Sertoli cells
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|
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• the number of WT1 (WT1 transcription factor)-positive Sertoli cells per seminiferous tubule is significantly reduced from P5 to P56
• the ratio of Ki67-positive cells to WT1-positive cells per seminiferous tubule is significantly reduced at P3 and P5, indicating decreased Sertoli cell proliferation
• however, no increase in Sertoli cell apoptosis is detected by TUNEL assay and WT1 co-staining at P21 and P43
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• from P14 to P56, Sertoli cell nuclei are found in the centers of >60% of the seminiferous tubules, ranging from single nuclei to intratubular clusters of 5-10 nuclei, suggesting disrupted Sertoli cell polarity
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|
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• seminiferous tubule diameter is significantly reduced from P7 to P56
|
|
|
• adult males show marked degeneration and atrophy of the seminiferous tubules
|
|
|
• mRNA levels extracellular matrix (ECM)- and cell adhesion-related genes (e.g. Timp1, Trf, Spp1, Ccbe1, Lcn2, and Ahsg), are significantly upregulated in Sertoli cells at P3 (SCs in proliferation) and at P21 (SCs in differentiation) due to their DNA demethylation status
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small testis
(
J:324178
)
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• testis size is significantly reduced at P56
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• from P7 to P56, testis weight/body weight ratio is significantly lower than that in control males
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• at P56, an in vivo biotin tracing assay showed presence of the tracer dye in the adluminal compartment of seminiferous tubules, suggesting a leaky BTB
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