Table 1.
Complete list of primers used in this study.
Fig 1.
Expression of MAFB in mouse testes.
(A) Reverse transcription-PCR (RT-PCR) of the large MAF transcription factors Mafa, Mafb, c-Maf and Nrl in various mouse tissues. Total RNA isolated from mouse testis, pancreas, spleen, kidney, and eye was used for RT-PCR analysis as described in the Materials and Methods section. RT-PCR of Hprt was carried out as a positive control. NTC was a non-template control. The RT-PCR analysis shown is representative of two independent experiments. (B) Detection of MAFB expression at Sertoli and germ cells in adult mouse testes. Testicular cells obtained from WT (upper panel) or Mafb-GFP knock-in mice (lower panel) were analyzed by FACS. Propidium iodide (PI)-negative cells (R1) were selected for flow sorting, while PI-positive dead cells were removed. The R1 fractions from WT or Mafb-GFP knock-in testicular cells were classified into two fractions, R2 representing germ cells and R3 representing Sertoli cells. GFP-positive cells are represented by the R4 fraction. The proportion of each fraction is shown above the bar.
Fig 2.
Localization of MAFB in mouse testes.
(A) Localization of MAFB in E18.5 mouse testes. Double immunostaining of MAFB with E-cadherin (ECAD), GATA4, or STAR is shown. Nuclei were counterstained with DAPI. The color of each marker is indicated above the images. G; germ cells. S; Sertoli cells. L; Leydig cells. MAFB was specifically detected in Leydig cells and Sertoli cells. (B) Localization of MAFB in adult mouse testes. Double immunostaining of MAFB with E-cadherin (ECAD), KIT, SCP3, PNA Lectin, or vimentin is shown. Nuclei were counterstained with DAPI. The color of each marker is indicated above the images. All seminiferous tubules shown represent stage VII. US; undifferentiated spermatogonia. DS; differentiated spermatogonia. P; pachytene spermatocytes. Sp; spermatids. S; Sertoli cells. L; Leydig cells. MAFB was specifically detected in Leydig cells, Sertoli cells, and pachytene spermatocytes.
Fig 3.
Testis morphogenesis of Mafb KO embryos developed normally.
(A) Histological section of WT and KO E18.5 testes stained with HE. No morphological alteration or distribution was detectable. (B and C) Counts of Leydig and Sertoli cells from E18.5 WT and KO testes. Embryonic testes (n = 3 per genotype) were sectioned, and 4 sections for each gonad were randomly selected and stained with the germ cell marker E-cadherin (green) together with either STAR (red) or GATA4 (red). Numbers of STAR-positive cells outside seminiferous tubules (Leydig cells) or GATA4-positive cells inside the tubules (Sertoli cells) were counted per unit area. The values are the mean±S.D. *P<0.05. There was no significant difference between WT and KO cell counts. (D) The expression of genes involved in testes development and function. mRNA expression of the gene markers encoding for PGCs (Oct4), Leydig cells (Cyp17a1, StAR, Insl3, Hsd3b1, and Cyp11a1), and Sertoli cells (Amh, Sox9, and WT1) was determined by qRT-PCR in E18.5 WT and Mafb KO testes. Gene expression levels were normalized to Hprt. The bars represent the mean±SEM of five individuals. *P<0.05.
Fig 4.
MAFB expression in seminiferous is stage specific and induced by RA.
(A) Seminiferous tubules at stages I-III, IV-VI, VII-VIII and IX-XII were double immunostained with MAFB (red) and vimentin (green). Nuclei were counterstained with DAPI (blue). Arrowhead indicates MAFB-negative Sertoli cells, while Arrows indicated MAFB-positive Sertoli cells. MAFB in Sertoli cells was specifically detected in stages VII to XII. (B) Seminiferous tubules at stages I-III, IV-VI, VII-VIII and IX-XII were isolated from mature mouse testis, mRNAs were extracted, and the expression levels of Mafb were then compared by qRT-PCR. The proportion of stage-dependent Mafb expression is shown as columns. Hprt was used as an internal control. Bars represent the mean±S.D. (C) Cultured primary Sertoli cells were treated with 1 μM RA for 24 hours and expression changes in Mafb were quantified by qRT-PCR (n = 3). Hprt was used as an internal control. The bars represent the mean±S.D. ***P<0.001.
Fig 5.
Generation of Mafb-cKO and efficiency evaluation.
(A) Targeting strategy to generate Mafbfl/fl CAG CreER™ (Mafb-cKO) mouse. The genomic structure of the mouse Mafb gene (top line) and illustrations of the targeting vector (second line) are shown. The Mafb single exon flanked by loxP sites (green triangles), and a neomycin (Neo) resistance cassette was inserted downstream flanked by FRT sites (orange triangles). The resultant targeted allele is shown (third line). The neomycin cassette was excised in vivo by crossing with the general FLP deleter strain, and the resultant floxed allele (fourth line) is shown. The floxed allele was crossed with the desired CAG-CreER™ (fifth line). The deleted allele after induction by tamoxifen is presented (bottom line). Restriction enzyme sites are shown in blue. PCR primers sites are indicated (blue triangles). (B) Efficiency of the Cre driver mice. The R26GRR reporter mouse, containing two cassettes EGFP flanked by loxP sites and tdsRED, was mated with the CAG-CreER™ driver mouse strain that ubiquitously expresses CreER™ in all cell types. The testes from a resultant male CAG-CreER™;R26GRR mouse were sectioned and analyzed pre- (upper panel) and post-tamoxifen injection (lower panel). The ability of the reporter mice to express tdsRED exclusively in testes was confirmed by microscopic examination of unstained sections. The mice crossing strategy is shown on the right. (C) Time-course of Mafb excision in the cKO mice. Six-week-old controls as well as cKO mice were intraperitoneally injected with tamoxifen. gDNA was extracted from mouse tails at post-tamoxifen injection days 1, 5, 10 and 15, and PCR was performed using a MafbΔ primer: 400 bp represents the deleted Mafb exon. D; Day. M; Marker. bp; base pair. (D) IHC was performed to verify MAFB deletion from cKO mouse testes after tamoxifen injection. Immunostaining for MAFB (red); nuclei were counterstained with DAPI (blue) as shown. The upper panel shows the control, while the lower panel shows cKO mouse testes. (E) Western blot confirming MAFB deletion in cKO mouse testes after tamoxifen injection. Total testis proteins were isolated, ~80 μg was loaded per well, and then the blot was probed with anti-MAFB or anti-β-actin antibody (internal control) for both the control and cKO mice, as shown. kDa; kilodaltons.
Fig 6.
Spermatogenesis development of adult Mafb-cKO mice.
Six-week-old mice were injected with tamoxifen and testes of cKO mice at 3- and 8-months of age were examined compared to those of age-matched controls (n = 4 for each group). (A) Testicular sections stained with Periodic acid-Schiff (PAS). (B) Immunostaining with various testicular cell types; undifferentiated spermatogonia (GFRA1 and PLZF), differentiated spermatogonia (KIT+ inside the tubule), spermatocytes (SCP3), spermatids (PNA-Lectin), Sertoli cells (SOX9 and Vimentin), and Leydig cells (KIT+ outside the tubule and STAR). The color of markers is indicated in the left boxes in panels. Colored arrows corresponding to the boxed markers are shown. (C) The expression level of the genes involved in testis function and germ cell development. The marker genes representing undifferentiated spermatogonia (Nanos3), differentiated spermatogonia (c-Kit), undifferentiated and differentiated spermatogonia (Sohlh1), differentiated spermatogonia and preleptotene spermatocytes (Stra8), spermatids (Prm2), Leydig cells (Hsd3b1), and Sertoli cells (Sox9) were analyzed by qRT-PCR. Each reaction was performed in duplicate for each gene. The data represent the means±SEM and are shown as relative mRNA expression after normalization to Hprt. *P<0.05. (D) The proportion of seminiferous stages: I-III, IV-VI, VII-VIII, and IX-XII from each genotype. *P<0.05. M; Month.
Fig 7.
Examination of the testosterone levels and male fertility of adult cKO mice.
Six-week-old cKO mice were injected with tamoxifen and examined at 3- and 8-months of age compared with age-matched controls (n = 4 for each group). (A) Blood plasma testosterone levels. (B) Litter size. (C) Testis weights. (D) Epididymal sperm counts. (E) Sperm motility %. (F) Progressive motility. M; Month. The data represent the mean±SEM. *P<0.05.
Table 2.
KEGG pathway analysis of the DEGs in Mafb-cKO Sertoli cells.