A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies

Abstract

Objective

To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS).

Design

Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA–sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes.

Setting

A laboratory study.

Patients

Three men with a diagnosis of obstructive azoospermia (age range, 30–40 years).

Intervention

None.

Main Outcome Measures

Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue.

Results

Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%–8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1⁻/doublesex and Mab-3 related transcription factor 1⁻/STRA8⁺ spermatogonia as well as SYCP3⁺/protamine 2⁻ spermatocytes.

Conclusion

This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and in vitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.