MAFB is dispensable for the fetal testis morphogenesis and the maintenance of spermatogenesis in adult mice

The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using Mafb-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not. MAFB was localized in Leydig and Sertoli cells at embryonic day (E) 18.5 but in Leydig cells, Sertoli cells, and pachytene spermatocytes in adults. Mafb-deficient testes at E18.5 showed fully formed seminiferous tubules with no abnormal structure or differences in testicular somatic cell numbers compared with those of control wild-type mice. Additionally, the expression levels of genes related to development and function of testicular cells were unchanged between genotypes. In adults, the expression of MAFB in Sertoli cells was shown to be stage specific and induced by RA. By generating Mafbfl/fl CAG-CreER™ (Mafb-cKO) mice, in which Cre recombinase was activated upon tamoxifen treatment, we found that the neonatal cKO mice died shortly upon Mafb deletion, but adult cKO mice were alive upon deletion. Adult cKO mice were fertile, and spermatogenesis maintenance was normal, as indicated by histological analysis, hormone levels, and germ cell stage-specific markers. Moreover, there were no differences in the proportion of seminiferous stages between cKO mice and controls. However, RNA-Seq analysis of cKO Sertoli cells revealed that the down-regulated genes were related to immune function and phagocytosis activity but not spermatogenesis. In conclusion, we found that MAFB is dispensable for fetal testis morphogenesis and spermatogenesis maintenance in adult mice, despite the significant gene expression in different cell types, but MAFB might be critical for phagocytosis activity of Sertoli cells

Dynamics of notch pathway expression during mouse testis post-natal development and along the spermatogenic cycle

Articles in International JournalsThe transcription and expression patterns of Notch pathway components (Notch 1–3, Delta1 and 4, Jagged1) and effectors (Hes1, Hes2, Hes5 and Nrarp) were evaluated (through RT-PCR and IHC) in the mouse testis at key moments of post-natal development, and along the adult spermatogenic cycle. Notch pathway components and effectors are transcribed in the testis and expressed in germ, Sertoli and Leydig cells, and each Notch component shows a specific cell-type and timewindow expression pattern. This expression at key testis developmental events prompt for a role of Notch signaling in prepubertal spermatogonia quiescence, onset of spermatogenesis, and regulation of the spermatogenic cycle

MAFB is dispensable for the fetal testis morphogenesis and the maintenance of spermatogenesis in adult mice

<div><p>The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using <i>Mafb</i>-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not. MAFB was localized in Leydig and Sertoli cells at embryonic day (E) 18.5 but in Leydig cells, Sertoli cells, and pachytene spermatocytes in adults. <i>Mafb</i>-deficient testes at E18.5 showed fully formed seminiferous tubules with no abnormal structure or differences in testicular somatic cell numbers compared with those of control wild-type mice. Additionally, the expression levels of genes related to development and function of testicular cells were unchanged between genotypes. In adults, the expression of MAFB in Sertoli cells was shown to be stage specific and induced by RA. By generating <i>Mafb</i>^fl/fl CAG-CreER^™ (<i>Mafb</i>-cKO) mice, in which Cre recombinase was activated upon tamoxifen treatment, we found that the neonatal cKO mice died shortly upon <i>Mafb</i> deletion, but adult cKO mice were alive upon deletion. Adult cKO mice were fertile, and spermatogenesis maintenance was normal, as indicated by histological analysis, hormone levels, and germ cell stage-specific markers. Moreover, there were no differences in the proportion of seminiferous stages between cKO mice and controls. However, RNA-Seq analysis of cKO Sertoli cells revealed that the down-regulated genes were related to immune function and phagocytosis activity but not spermatogenesis. In conclusion, we found that MAFB is dispensable for fetal testis morphogenesis and spermatogenesis maintenance in adult mice, despite the significant gene expression in different cell types, but MAFB might be critical for phagocytosis activity of Sertoli cells.</p></div

Testis morphogenesis of <i>Mafb</i> KO embryos developed normally.

<p>(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 (<i>n</i> = 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. *<i>P<0</i>.<i>05</i>. 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 (<i>Oct4</i>), Leydig cells (<i>Cyp17a1</i>, <i>StAR</i>, <i>Insl3</i>, <i>Hsd3b1</i>, and <i>Cyp11a1</i>), and Sertoli cells (<i>Amh</i>, <i>Sox9</i>, and <i>WT1</i>) was determined by qRT-PCR in E18.5 WT and <i>Mafb</i> KO testes. Gene expression levels were normalized to <i>Hprt</i>. The bars represent the mean±SEM of five individuals. *<i>P</i><0.05.</p

KEGG pathway analysis of the DEGs in <i>Mafb</i>-cKO Sertoli cells.

<p>KEGG pathway analysis of the DEGs in <i>Mafb</i>-cKO Sertoli cells.</p

Expression of MAFB in mouse testes.

<p>(A) Reverse transcription-PCR (RT-PCR) of the large MAF transcription factors <i>Mafa</i>, <i>Mafb</i>, <i>c-Maf</i> and <i>Nrl</i> 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 <i>Hprt</i> 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 <i>Mafb</i>-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 <i>Mafb</i>-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.</p