KDM1A/LSD1 regulates the differentiation and maintenance of spermatogonia in mice

Abstract

The proper regulation of spermatogenesis is crucial to ensure the continued production of sperm and fertility. Here, we investigated the function of the H3K4me2 demethylase KDM1A/LSD1 during spermatogenesis in developing and adult mice. Conditional deletion of Kdm1a in the testis just prior to birth leads to fewer spermatogonia and germ cell loss before 3 weeks of age. These results demonstrate that KDM1A is required for spermatogonial differentiation, as well as germ cell survival, in the developing testis. In addition, inducible deletion of Kdm1a in the adult testis results in the abnormal accumulation of meiotic spermatocytes, as well as apoptosis and progressive germ cell loss. These results demonstrate that KDM1A is also required during adult spermatogenesis. Furthermore, without KDM1A, the stem cell factor OCT4 is ectopically maintained in differentiating germ cells. This requirement for KDM1A is similar to what has been observed in other stem cell populations, suggesting a common function. Taken together, we propose that KDM1A is a key regulator of spermatogenesis and germ cell maintenance in the mouse.

Fig 1. Expression of KDM1A in the testis.

KDM1A (A,C,E,G), PLZF (I) and combined DAPI (red) and KDM1A (green) (B,D,F,H) immunofluorescence from wild-type testes. (J) Combined PLZF (green) and KDM1A (red) IF. Asterisk in magnified inset (H) indicates mature spermatozoa. White arrowheads in (I,J) indicate spermatogonia marked by PLZF. The expression of KDM1A in these same PLZF+ spermatogonia is shown in G,H (white arrowheads). In all other panels, spermatogenic cell types are labeled as described in legend (dpp = days post partum). Cell types were identified based on morphology and location within the testicular cord or seminiferous tubule. Scale bars, 25 μm.

Fig 2. Spermatogonia differentiation and maintenance defect in Kdm1a^Vasa mutants.

(A) Representative images of control and Kdm1a^Vasa mutant adult testes. Histology from control (B) and Kdm1a^Vasa (C) adult testes. Arrowheads indicate Sertoli cells (C). SOX9 immunohistochemistry (brown) counterstained with hematoxylin (blue) from control (D) and Kdm1a^Vasa (E) testes at 21days post partum (dpp). Histology from control (F,H,J,L) and Kdm1a^Vasa testes (G,I,K,M) at 1dpp (F,G), 6dpp (H,I), 8dpp (J,K) and 10dpp (L,M). Arrowheads indicate spermatogonia (F-L), and asterisk indicates spermatocytes (L) and abnormal spermatocytes (M). (N) Germ cells per testis cord quantified from histology (F,G) at 1dpp, and SOX9 immunofluorescence (S2 Fig) at 6dpp, 8dpp and 10dpp (the adult testis size was not quantified). Quantification of OCT4+ (O), PLZF+ (P) and KIT+ (Q) germ cells per cord from immunofluorescence (S1 Fig) (n = >30 testis cords counted from multiple animals, Mann-Whitney U test, p < .001). (N-Q) The control is shown in black and Kdm1a^Vasa is shown in blue. Scale bars, 25 μm.

Fig 3. Germ cell apoptosis in Kdm1a^Vasa mutants.

Quantification of Cleaved Caspase-3 (CC3) positive germ cells per testicular cord (C) from control (A) and Kdm1a^Vasa (B) testes (n = >30 testis cords counted from multiple animals), Mann-Whitney U test, p < .001). Arrows (A,B) indicate CC3 positive nuclei. Magnified inset (B) shows fragmented DNA in germ cells from a different Kdm1a^Vasa testis (DAPI: blue). TUNEL (green) from control (D) and Kdm1a^Vasa mutants (E) at 10dpp. Testicular cord boundaries are indicated by dashed lines. Scale bars, 25 μm.

Fig 4. Germ cell maintenance and meiotic defects in Kdm1a^Cagg adult testes.

Histology from control (A) and Kdm1a^Cagg testes (B,E-L), 7–9 weeks after the last tamoxifen injection to delete Kdm1a, showing mostly Sertoli cells remaining (arrowheads in B), spematogonia-like cells near or in the lumen (arrows in E), seminiferous tubule lacking a lumen (E,L), abnormally clumped germ cells (asterisk in F,J), abnormal spacing around cells (arrow in G,H) with vacuoles interspersed (arrowheads in G,H), germ cells with crescent shaped apoptotic morphology (arrowheads in F,I), giant spermatocyte-like apoptotic cells (arrowhead in J), chromosomal abnormalities (arrowheads in K), and multi-nucleated germ cells (arrowheads in L). Quantification of SOX9+ Sertoli cells (C) and germ cells per seminiferous tubule (D) in control and Kdm1a^Cagg testis (n = 3, controls and n = 4, Kdm1a^Cagg mutants, >25 seminiferous tubules per animal, Mann-Whitney U test, p < .05). Apoptosis marker CLEAVED CASPASE-3 (CC3)(green) merged with DAPI (red) (M,N) and meiotic marker SYCP-1 (yellow) (P,Q) and from control (M,P) and Kdm1a^Cagg mutants (N,Q). Quantification of the CC3 (O) and SYCP-1 (R) immunofluorescence (n = >30 seminiferous tubules counted from multiple animals, Mann-Whitney U test, p < .05). Scale bars, 25 μm.

Fig 5. KDM1A and H3K4me2 chromatin immunoprecipitation at Oct4.

(A) Average fold enrichment (KDM1A/no Ab) from chromatin immunoprecipitation (ChIP) at Oct4 in wild-type adult testes calculated from S4A Fig (n = 2, Unpaired t-test, p < .05). (B) Average fold enrichment (H3K4me2/no Ab) from ChIP at Oct4 in Kdm1a^Vasa (black) and control (white) testes calculated from S4C Fig (n = 3 animals, Unpaired t-test, p < .05). The location of the Oct4 promoter (prom) primers and the Oct4 distal (DE) and proximal enhancer (PE) primers are shown below panel A.

Fig 6. Ectopic expression of spermatogonia genes in Kdm1a^Cagg testes.

DAPI (A,D), OCT4 (B,E), and combined (C,F) immunofluorescence from wild-type (A-C) and Kdm1a^Cagg testes (D-F). Arrowheads in (B) indicate spermatogonia marked by OCT4, whereas arrows in (E) indicate OCT4 protein in spermatocyte-like cells. Asterisk (E) indicates OCT4 protein in post-meiotic spermatid-like cells. Quantification of the OCT4 (G) ectopic protein phenotype (n = >50 tubules counted from multiple animals, Mann-Whitney U test, p < .001). Scale bars, 25 μm.