JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):100
Poster Presentation
29th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263687
P-88. Evaluation of the proliferative capacity of spermatogonial stem cells
Laura Malta Lima Fernandes Dantas1, Alice Eduarda Valadares1, Bárbara Maria Campos Cardoso1, Laura Cecilia Borba1, Reginaldo Martello2, Augusto Barbosa Reis1, Deila Jordão Franco Sábato1, Andre Lucas Caldeira-Brant1
1 Pontifícia Universidade Católica de Minas Gerais - Contagem - MG – Brasil
2 Universidade Federal de Minas Gerais - Belo Horizonte - MG – Brasil
Objective: The main objective of this research is to describe the proliferative capacity of spermatogonial stem cells (SSCs) in human and non-human primates, and to validate the marmoset as an experimental model for male fertility studies. Histological assessment of SSC behavior is essential for developing stem cell therapies to preserve fertility.
Methods: Testes from untreated prostate cancer patients were collected by total bilateral orchiectomy, while samples from adult marmosets (Callithrix penicillata) were also obtained. The respective ethic committee approved sample collection. For spermatogonial counting, a portion of the samples was fixed in Karnovsky's, embedded in glycol methacrylate resin, sectioned at 3µm, and stained with toluidine blue. Spermatogonial subtypes were identified and counted, with numbers normalized per 100 Sertoli cell nucleoli (SCN) in humans and per seminiferous tubule (ST) in marmosets. The remaining human samples were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned at 5µm for immunohistochemistry. These sections were used with the proliferation marker MCM7 to analyze the spermatogonial mitotic index.
Results: Spermatogonial counting from three human revealed that the AdVac had the lowest average count (36±12,5 SPG/SCN) among all cell types analyzed (p<0.05). Its counterpart, the Adnovac, occupied an intermediate position, with an average of 84.5±9.9 SPG/SCN. The Apale was the most abundant, with an average of 142±19.6 SPG/SCN (p<0.05). When analyzing the dynamics of SPG across the six stages of the seminiferous epithelium cycle (SEC), AdVac numbers remained low (6.1±0.6 SPG/SCN) and relatively stable throughout the stages. The AdNovac population also varied, with a peak of 18.4±3.2 SPG/SCN in stage VI. The Apale showed greater variation, reaching its peak in stage V, with an average of 142±19.6 SSC/SCN. Immunohistochemical analysis using the MCM7 proliferation marker revealed significant differences in proliferative rates among the cell types. The AdVac population exhibited the lowest proliferative rate of 8.6±2.4 SSC/ SCN, which was significantly lower than the others (p<0.05). The AdNovac and Apale spermatogonia showed intermediate values of 47.6±3.5 SPG/SCN, whereas the differentiated type B cells displayed the highest index of 68±5.8 SPG/SCN. The spermatogonial count in the four marmoset revealed that the AdVac showed the lowest mean (8.4±1.0 SPG/ST) among all cell types analyzed (p<0.05). The AdNovac had a mean of 12.7±0.7 SPG/ST, occupying an intermediate position relative to the other SPG and remaining relatively stable throughout the nine stages of the seminiferous epithelium cycle (SEC). The Apale was the most abundant (p<0.05), with a mean of 23.5±2.9 SPG/ST. The differentiated spermatogonia B1 and B2 showed means of 12.2±2.8 SPG/ST and 11.6±2.4 SPG/ST, respectively. In the analysis of the dynamics of these populations over the nine stages of the SEC, the AdVac remained low and relatively stable in all stages. The AdNovac exhibited variation, peaking at stage IX. The Apale also varied, reaching its peak in stage IV. Finally, it was observed in both humans and monkeys that the reduction in the number of Apale was accompanied by an increase in type B SPG, a pattern that was also seen in the B to preleptotente spermatocyte (Pl) transitions.
Conclusion: Preliminary data suggest that AdVac and AdNovac spermatogonial stem cells (SSCs) are quiescent cells. In contrast, Apale cells behave as more mitotically active spermatogonia, showing greater population variation. As expected, the growth and decline pattern of differentiated spermatogonia reflects the successive renewal of these germ cells. Further immunohistochemical analysis is needed to better understand SSC behavior.