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

Abstract

Objective: To determine whether 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.

Animal(s): 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 costaining with apoptotic markers. Molecular analysis of G-CSF receptor expression in undifferentiated spermatogonia.

Result(s): 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.

Keywords: Spermatogonial stem cells; cancer therapy; cytokines; fertility preservation; infertility.

Figure 1. G-CSF prevents loss of spermatogenesis after busulfan treatment in mice

Animals from Experiment 1 were evaluated 10 weeks after the final G-CSF/vehicle treatment (see Supplemental Figure 1). (A) Testis weight, (B) Epididymal sperm counts. Labels above bars signify statistically-significant differences between groups as determined by student’s t-test. Tiled brightfield images of H&E-stained sections of testes from (C) Control group, (D) Busulfan Only group, and (E) Busulfan + G-CSF group. Scale bars = 500µm. Enlarged images of the dashed boxes in C–E are shown in (F–H), respectively. Scale bars = 50µm. Filled arrowheads = seminiferous tubules with spermatogenesis. Open arrowheads = no spermatogenesis. Further enlarged images of dashed boxes in F–H are shown in (I–K), respectively. Scale bars = 50µm. (L) Stacked bars show the percentage of all seminiferous tubule cross-sections counted from all animals in each group which exhibit differing degrees of spermatogenesis: complete spermatogenesis (complete), up to round spermatid spermatids, up to 1° spermatocytes, or containing no spermatogenesis (empty or Sertoli cell-only). A, B, and C categorical notations above bars denote statistically significant differences between groups (p<0.05) as determined by Tukey-Kramer ANOVA. The number of animals in each experimental group (n) is indicated at the base of each bar. The number of seminiferous tubule cross-sections evaluated per animal is shown in Supplemental Table 3. Details of experimental groups are in Supplemental Figure 1.

Figure 2. G-CSF treatment improves PLZF+ spermatogonial numbers after busulfan treatment without changing apoptosis

Testes from mice in Experiment 2 were evaluated on day 8 (24 hours after the last G-CSF/vehicle treatment, see Supplemental Figure 1). (A–D) PLZF+ spermatogonia were quantifed per round seminiferous tubule cross-section from testes of mice obtained from each group. Sections co-stained for (E–H) PLZF and activated Caspase3 or (I–L) PLZF and TUNEL were used to determine the percentage of PLZF+ spermatogonia that were positive for activated Caspase 3 and TUNEL, respectively. The A, B, and C categorical notations above bars denote statistically significant differences between groups (p<0.05) as determined by Student’s T-test. Scale bars = 50µm. The number of round seminiferous tubule cross-sections and cells counted per animal is in Supplemental Table 5.

Figure 3. Undifferentiated spermatogonia express CSF3R (G-CSF receptor)

(A) RT-PCR detected mRNAs for (top) Csf3r and (bottom) Gapdh in adult mouse testis, cultured Thy1+ spermatogonia and SNL76/7 STO feeders (STO). Template cDNA samples were from reactions with and without reverse transcriptase (RT: + or −). (B) Western blot detection of CSF3R in liver (+ control), adult testis and cultured Thy1+ spermatogonia. (C) Single-cell qRT-PCR measurement of the steady-state mRNA levels for the noted genes in 75 individual Thy1+ spermatogonia. Data are presented as violin plots of the log2-transformed Ct values from all 75 cells analyzed (curve height = mRNA levels, width = relative cell number). In addition to Csf3r (black), genes were separated into three groups: undifferentiated spermatogonia genes in blue (Dazl, Ddx4, Foxo1, Gfra1, Sall4, Sohlh1, Sohlh2, and Zbtb16), somatic cell genes in red (Gdnf Hsd3b1), and “housekeeping” control genes in white (Actb, Gapdh, Rpl7). Immunofluorescent staining for CSF3R protein in mouse Thy1+ spermatogonia cultures using antibodies that recognize (D) CSF3R and (E) SALL4, a marker of undifferentiated spermatogonia and (F) merged of CSF3R and SALL4 with Hoechst 33342 counterstain (blue, DNA). Staining was compared to omission of primary antibodies (data not shown). Flow cytometry dot plots show Thy1+ spermatogonia cultures stained with (G) isotype control antibodies or (H) CSF3R antibodies, both on the X-axis. Y-axis depicts autofluorescence in the AlexaFluor488 channel. Quadrant statistics are shown in the upper-right of each dot plot and depict the percentage of propidium iodide (PI) negative (viable) cells that fall within the noted quadrant.