A behind-the-scenes role of BDNF in the survival and differentiation of spermatogonia

Abstract

Mouse spermatogenesis entails the maintenance and self-renewal of spermatogonial stem cells (SSCs), which require a complex web-like signaling network transduced by various cytokines. Although brain-derived neurotrophic factor (BDNF) is expressed in Sertoli cells in the testis, and its receptor tropomyosin receptor kinase B (TrkB) is expressed in the spermatogonial population containing SSCs, potential functions of BDNF for spermatogenesis have not been uncovered. Here, we generate BDNF conditional knockout mice and find that BDNF is dispensable for in vivo spermatogenesis and fertility. However, in vitro , we reveal that BDNF -deficient germline stem cells (GSCs) exhibit growth potential not only in the absence of glial cell line-derived neurotrophic factor (GDNF), a master regulator for GSC proliferation, but also in the absence of other factors, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin. GSCs grown without these factors are prone to differentiation, yet they maintain expression of promyelocytic leukemia zinc finger ( Plzf ), an undifferentiated spermatogonial marker. Inhibition of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and Src pathways all interfere with the growth of BDNF-deficient GSCs. Thus, our findings suggest a role for BDNF in maintaining the undifferentiated state of spermatogonia, particularly in situations where there is a shortage of growth factors.

Figure 1

BDNF is dispensable for mouse spermatogenesis in vivo. (a) Conditional knockout strategy for the BDNF gene by Tmx injection. Small horizontal arrowheads in the top scheme indicate genotyping primer positions for the detection of floxed (F) and CreERT2-recombined (FC) alleles. (b) Confirmation of knockout by genomic PCR using testicular DNA. The numbers indicate three independent animals. (c) Size of adult mouse testes comparing BDNF^F/F and BDNF^FC/FC (n = 3 mice per genotype) at 16 weeks of age. (d) Weight of adult testis (n = 3 mice per genotype; mice aged 16 weeks). Data are presented as mean±standard deviation of 6 testis samples. Two-sided Wilcoxon rank-sum test (P = 0.22). (e) IF images of seminiferous tubules (dotted lines) showing PLZF (green) and DAPI (white) from BDNF^F/F and BDNF^FC/FC testis. Yellow arrowheads: PLZF-expressing spermatogonia. Scale bars = 100 μm. (f) Representative IF images showing spermatogonia expressing PLZF (green) and/or KIT (magenta). Yellow arrowheads: PLZF⁺/KIT⁻ spermatogonia; pink arrowheads: PLZF⁺/KIT⁺ spermatogonia; dotted lines: seminiferous tubules. Scale bars = 100 μm. (g) Box plots showing quantification of PLZF⁺/KIT⁻ and PLZF⁺/KIT⁺ spermatogonia in BDNF^F/F and BDNF^FC/FC testis. n = 166 for BDNF^F/F and n = 106 for BDNF^FC/FC seminiferous tubules. NS: not significant; BDNF: brain-derived neurotrophic factor; PCR: polymerase chain reaction; PLZF: promyelocytic leukemia zinc finger; DAPI: 4’,6-diamidino-2-phenylindole; IF: immunofluorescence; CreERT2: Cre recombinase-estrogen receptor T2.

Figure 2

BDNF^FC/FC GSCs grow without GDNF. (a) RT-PCR analysis for BDNF mRNA expression in WT MEFs and GSCs. Gapdh was used as the internal control. (b) RT-PCR confirmation for BDNF mRNA depletion in BDNF^FC/FC MEFs. Gapdh was used as the internal control. (c) PCR confirmation of the conditional deletion of BDNF in GSCs after 4-OH-Tmx addition to media. Primers indicated in Figure 1a were used. (d) Knockout confirmation in MEFs by PCR. GSCs were cultured in GSC medium containing Stem Pro-34. Growth of (e) BDNF^FC/+ and (f) BDNF^FC/FC GSCs with or without GDNF. The growth rate was calculated by cell count at indicated timings. Data are presented as the mean±standard deviation of triplicate samples. Significant P values between GDNF⁺ and GDNF⁻ are indicated (one-sided t-test). BDNF: brain-derived neurotrophic factor; PCR: polymerase chain reaction; RT-PCR: reverse transcription-PCR; mRNA: messenger RNA; GDNF: glial cell line-derived neurotrophic factor; Gapdh: glyceraldehyde-3-phosphate dehydrogenase; MEF: mouse embryonic fibroblast; GSC: germline stem cell; N: negative control; WT: wild-type; CreERT2: Cre recombinase-estrogen receptor T2.

Figure 3

BDNF knockout GSCs grow without crucial cytokines. (a) Viability of BDNF^FC/+ and BDNF^FC/FC GSCs grown on corresponding genotypes of MEFs in the absence of GDNF (G), EGF (E), or bFGF (F). The complete medium contains all three factors. Cell viability was determined by the MTT assay after 8 days of culture. Mean absorbance values relative to the complete medium condition from duplicate samples are shown. (b) Control experiment showing viability of BDNF^FC/+ GSC on BDNF^FC/FC MEFs and BDNF^FC/FC GSCs on WT MEFs from duplicate samples. Data are presented as mean±standard deviation. Significant P values are indicated (one-sided t-test). BDNF: brain-derived neurotrophic factor; MEF: mouse embryonic fibroblast; GSC: germline stem cell; WT: wild-type; GDNF: glial cell line-derived neurotrophic factor; EGF: epidermal growth factor; bFGF: basic fibroblast growth factor.

Figure 4

BDNF-depleted GSC growth without insulin. GSC growth with or without insulin in the absence of cytokines (GDNF, EGF, and bFGF). Data are presented as mean±standard deviation of duplicate samples. Significant P values are indicated (one-sided t-test). BDNF: brain-derived neurotrophic factor; GSC: germline stem cell; GDNF: glial cell line-derived neurotrophic factor; EGF: epidermal growth factor; bFGF: basic fibroblast growth factor.

Figure 5

Dependency of GSCs on the PI3K, MAPK/ERK, and Src pathways. (a) BDNF^FC/FC GSC viability with the addition of PI3K inhibitor LY294002 for 8 days at the concentrations indicated. (b) BDNF^FC/FC GSC viability in the presence of a MEK1/2 inhibitor PD0325901 for 8 days at the concentrations indicated. (c) Effect of another Src family kinase inhibitor SU6656 for 8 days on BDNF^FC/FC GSC viability at the concentrations indicated. Data are presented as mean±standard deviation of duplicate samples. Significant P values are indicated (one-sided t-test). BDNF: brain-derived neurotrophic factor; GSC: germline stem cell; GDNF: glial cell line-derived neurotrophic factor; PI3K: phosphoinositide 3-kinase; MAPK: mitogen-activated protein kinase; ERK: extracellular signal-regulated kinase.

Figure 6

Spermatogonial marker mRNA expression in GSCs with or without essential factors. (a) qRT-PCR analysis of BDNF^F/F and BDNF^FC/FC GSCs cultured in the complete medium for the expression of spermatogonial markers. (b) qRT-PCR results showing gene expression in GDNF⁻ medium. (c) qRT-PCR results showing gene expression in EGF⁻/bFGF⁻ medium. (d) qRT-PCR results showing gene expression in insulin⁻ medium. GSCs were cultured for 3 days after depletion of each factor. BDNF^+/+ and BDNF^FC/FC MEFs were used for control (BDNF^F/F or BDNF^FC/+) and knockout (BDNF^FC/FC) GSCs, respectively. Data are presented as mean±standard deviation of duplicate samples normalized for the expression of Gapdh and Hprt1. Significant P values are indicated (one-sided t-test). NS: not significant; qRT-PCR: quantitative reverse transcription polymerase chain reaction; BDNF: brain-derived neurotrophic factor; MEF: mouse embryonic fibroblast; GSC: germline stem cell; mRNA: messenger RNA; GDNF: glial cell line-derived neurotrophic factor; EGF: epidermal growth factor; bFGF: basic fibroblast growth factor; Gapdh: glyceraldehyde-3-phosphate dehydrogenase; Hprt1: hypoxanthine phosphoribosyltransferase 1.