Granulocyte colony-stimulating factor prevents loss of spermatogenesis after sterilizing busulfan chemotherapy

Abstract

Objective To determine if granulocyte colony-stimulating factor (G-CSF) could prevent loss of spermatogenesis induced by busulfan chemotherapy via protection of undifferentiated spermatogonia, which might serve as an adjuvant approach to preserving male fertility among cancer patients. Design Laboratory animal study. Setting University. Animals Laboratory mice. Intervention(s) Five week-old mice were treated with a sterilizing busulfan dose and with 7 days of G-CSF or vehicle treatment and evaluated 10 weeks later (experiment 1) or 24 hours after treatment (experiment 2). Main Outcome Measure(s) Experiment 1 - testis weights, epididymal sperm counts, testis histology. Experiment 2 - PLZF immunofluorescent co-staining with apoptotic markers. Molecular analysis of G-CSF receptor expression in undifferentiated spermatogonia. Results Ten weeks after treatment, busulfan-treated mice that also received treatment with G-CSF exhibited significantly better recovery of spermatogenesis and epididymal sperm counts than animals receiving busulfan alone. G-CSF led to increased numbers of PLZF+ spermatogonia 24 hours after treatment that was not accompanied by changes in apoptosis. To address the cellular target of G-CSF, mRNA for the G-CSF receptor, Csf3r, was found in adult mouse testes and cultured Thy1+ (undifferentiated) spermatogonia, and cell-surface localized CSF3R was observed on 3% of cultured Thy1+ spermatogonia. Conclusion(s) These results demonstrate that G-CSF protects spermatogenesis from gonadotoxic insult (busulfan) in rodents and this may occur via direct action on CSF3R+undifferentiated spermatogonia. G-CSF treatment might be an effective adjuvant therapy to preserve male fertility in cancer patients receiving sterilizing treatments.

The regulatory repertoire of PLZF and SALL4 in undifferentiated spermatogonia

“…Testes from 6-to 8-day-old pups were used to produce testis cell suspensions that were enriched for SSCs by THY1 selection, and cultured on mitomycin C-treated SNL76/7 feeders in a defined serum-free medium containing GDNF and FGF2, as described ( Fig. S1) (Benavides-Garcia et al, 2015;Kanatsu-Shinohara et al, 2003;Nagano et al, 2003;Ogawa et al, 1997). There are no apparent differences in THY1 + spermatogonial cultures over the first 6 months [i.e.…”

Adult Human Mesenchymal Stem Cell Differentiation at the Cell Population and Single-Cell Levels Under Alternating Electric Current

“…Preamplified cDNA harvested from the C1 was then used for high-throughput qRT-PCR measurement of each amplicon using a BioMark HD system as described in literature reports, [15][16][17] with modifications. Briefly, a 2.25 mL aliquot of each amplified cDNA was mixed with 2.5 mL of 2X SsoFast EvaGreen Supermix with Low ROX (Bio-Rad) and with 0.25 mL of 20X DNA Binding Dye Sample Loading Reagent (Fluidigm).…”

Molecular Mechanisms of Cell Differentiation in Gonad Development

“…Exposure of cultures of THY1+ spermatogonia to CSF1 in the presence of GDNF enhances mouse SSC self-renewal in vitro (Oatley et al 2009). A related cytokine, granulocyte colony-stimulating factor (G-CSF or CSF3), was recently identified as a potential somatic-derived self-renewal factor based on the potential of exogenous CSF3 to protect spermatogenesis from alkylating chemotherapy (Benavides- Garcia et al 2015). Furthermore, testicular macrophages were recently shown to participate in regulation of SSC or spermatogonial fate, perhaps by secreting CSF1 or another related factor .…”

“…Interestingly, exposure of cultured mouse THY1+ spermatogonia to CSF1 alone (i.e., in the absence of GDNF) does not expand SSCs (Oatley et al 2009), suggesting a requisite cooperation between both growth factors that likely involves signal transduction cross talk. The CSF3 receptor (CSF3R) is present at the mRNA and protein level in undifferentiated spermatogonia, but the cellular source of CSF3 in normal testes is not clear (Benavides- Garcia et al 2015). A related cytokine, granulocyte colony-stimulating factor (G-CSF or CSF3), was recently identified as a potential somatic-derived self-renewal factor based on the potential of exogenous CSF3 to protect spermatogenesis from alkylating chemotherapy (Benavides- Garcia et al 2015).…”