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Updated: Nov 6, 2025
A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model
Published on: February 6, 2018
Gene and histomorphology alteration analysis in spermatogenesis arrest mouse model: a probable novel approach for
Amirreza Nabighadim1, Fahimeh Jafarnezhad-Ansariha1, Masoumeh Majidi Zolbin1
1Pediatric Urology and Regenerative Medicine Research Center, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran.
Introduction:
Approximately 15% of couples in the reproductive age are struggling with infertility which, in nearly half of them, is caused by male factors.
Material And Methods:
The present study comprised of two groups of sixteen C57BL/6 mice; each mouse received either an intraperitoneal injection of 30 mg/kg of an alkylating agent or the same amount of distilled water. Testes were harvested 30 days following the injection. Morphometric analysis of hematoxylin and eosin (H&E) stained slides including mean tubular area, diameter and intratubular particles were performed. Spermatogenesis rate was assessed by spermatogonial markers including promyelocytic leukemia zinc finger protein (PLZF) and neurogenin-3 (NGN3). Moreover, the expression rate of Wilms Tumor-1 (WT-1), A-Kinase Anchoring Protein 4 (AKAP4) and adenosine deaminase domain containing 1 (ADAD1) genes were evaluated via real-time polymerase chain reaction (RT-PCR).
Results:
The body weight gradually increased in both groups after a period of 30 days, however, the increase was significantly (p-value = 0.023) lower in the chemically treated group. All the morphometric parameters were considerably decreased in the azoospermic mice. Also, promyelocytic leukemia zinc finger protein and neurogenin-3 expression dramatically declined (p-value <0.001 for both markers). In comparison with the negative control group, the expression rates of A-Kinase Anchoring Protein 4 and adenosine deaminase domain containing 1, two genes participating in the sperm structure, were remarkably reduced in the intervention group (p-value <0.001); however, our investigations demonstrated that the azoospermia model could induce a 5-fold upregulation in Wilms Tumor-1 gene expression.
Conclusions:
Development of an azoospermia model can upregulate Wilms Tumor-1 gene expression in a higher rate after 30 days; however, expression of the testis-specific genes, A-Kinase Anchoring Protein 4 and adenosine deaminase domain containing 1, decreased after the intervention. To the best of our knowledge, this upregulation could be related to spermatogenesis recovery after the follow-up period.
Insights
An alkylating agent induced azoospermia in mice, significantly decreasing sperm production and related gene expression. However, Wilms Tumor-1 gene expression increased, potentially indicating spermatogenesis recovery.
Area of Science:
- Reproductive biology
- Toxicology
- Molecular biology
Background:
- Male infertility affects approximately 15% of reproductive-aged couples, with male factors contributing to nearly half of these cases.
- Understanding the molecular mechanisms underlying chemically induced male infertility is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the effects of an alkylating agent on spermatogenesis and gene expression in a mouse model.
- To assess the potential of certain genes as biomarkers for azoospermia and recovery.
Main Methods:
- C57BL/6 mice were injected with an alkylating agent or distilled water.
- Testes were analyzed for morphometric parameters and expression of spermatogenesis markers (PLZF, NGN3) and genes (WT-1, AKAP4, ADAD1) using RT-PCR.
- Body weight and gene expression levels were compared between groups.
Main Results:
- The alkylating agent significantly reduced body weight gain and all measured morphometric parameters of the testes.
- Expression of spermatogonial markers (PLZF, NGN3) and testis-specific genes (AKAP4, ADAD1) significantly decreased (p<0.001).
- Wilms Tumor-1 (WT-1) gene expression showed a 5-fold upregulation in the azoospermia model (p<0.001).
Conclusions:
- Chemically induced azoospermia in mice leads to decreased spermatogenesis and reduced expression of key sperm-related genes.
- Upregulation of Wilms Tumor-1 (WT-1) gene expression may be associated with spermatogenesis recovery following the intervention.
- This study provides insights into the molecular changes during chemically induced male infertility and potential recovery markers.
