Fetal Leydig cells (FLCs) differentiate, but having abnormal proliferation status in the interstitium ofWt1SC-cKOmouse testes during fetal testis development

Fetal Leydig cells (FLCs) differentiate, but having abnormal proliferation status in the interstitium ofWt1SC-cKOmouse testes during fetal testis development. of PMCs, FLCs and interstitial progenitor cells from embryo to newborn, leading to abnormal fetal testis interstitial development. Specifically, the expression of PMC marker genes-Sma, Myh11andDes, and interstitial progenitor cell marker geneVcam1were down-regulated, whereas FLC marker genesStAR, Cyp11a1, Cyp17a1andHsd3b1were up-regulated, in neonatalWt1SC-cKOtestes. The ratio of PMC: FLC were also reduced inWt1SC-cKOtestes, concomitant with a down-regulation of Notch signaling moleculesJag 1, Notch 2, Notch 3, andHes1in neonatalWt1SC-cKOtestes, illustrating changes in the differentiation status of FLC from their interstitial progenitor cells during fetal testis development. In summary, Wt1regulates the development of FLC and interstitial progenitor cell lineages through Notch signaling, and it also plays a role in PMC development. Collectively, these effects confer fetal testis compartmentalization. == Introduction == During embryogenesis, Sry (sex-determining region of the Y chromosome) expression in pre-Sertoli cells of XY individuals turns on a genetic cascade by directing the bipotential genital ridge to develop into the testis [1]. The onset of Sry expression leads to Sertoli cell aggregation, encircling germ cells to form testis cords which are then surrounded by peritubular myoid cells (PMCs) [for reviews, see [24]]. Between testis cords is the interstitium, inhabited by fetal Leydig cells (FLCs), uncharacterized interstitial progenitor cells, arterial and venous blood vasculature, lymphatic PA-824 (Pretomanid) vessels, resident macrophages and nerve cells [for reviews, see [24]]. Thus, the differentiation, proliferation and movements of different testicular cell types are tightly coordinated to support fetal testis compartmentalization. Although the genetic networks and the testis cell types responsible for testis development are known [for reviews, see [2, 3, 5]], the cellular interactions that confer fetal testis compartmentalization remain unclear. Sertoli cell is thought to be the critical cell type that drives fetal testis compartmentalization [4], yet accumulating evidence has shown that FLCs and PMCs also play active roles in fetal testis development. Studies have shown that FLCs modulate Sertoli cell proliferation, and testis cord elongation and expansion via activin A [6]. PMCs also interact with Sertoli cells to deposit extracellular matrix components to form the basement membrane that defines the testis cords and interstitium [7]. However , whether Sertoli cells regulate PMC and FLC development to drive fetal testis compartmentalization is still unclear. Wt1is a tumor suppressor and also an oncogene encoding at least 24 transcription factors involved in cell proliferation, differentiation, apoptosis and organ development [reviewed in PA-824 (Pretomanid) [8, 9]]. Global knockout ofWt1in mice led to gonad agenesis and embryonic lethality [10]. In the testis, the Sertoli cell is the major cell type expressedWt1, and Sertoli cell-specific deletion ofWt1usingAmh-Cre was earlier reported to disrupt the formation of testis cords in fetal testis [11], likely mediated by a down-regulation of collagen 1(IV) and 2(IV) expression [12], and re-programming of Sertoli cells to Leydig-like Rabbit Polyclonal to HDAC7A (phospho-Ser155) cells [13]. However , it remained unknown whether Sertoli cell-specific deletion ofWt1would modulate differentiation and proliferation of FLCs and PMCs, which in turn perturbed testis compartmentalization during fetal testis development. In this study, we usedWt1SC-cKOmice to perform a detailed analysis on the differentiation, proliferation and apoptotic status of PMCs, FLCs and their shared interstitial progenitor cells to unravel the role ofWt1in fetal testis development. == Materials and Methods == == Mouse genetics == The use of mice for experiments reported herein was approved by the Animal Care Committee of the Institute of Zoology, Chinese Academy of Sciences. All mice were maintained in a C57BL/6; 129/SvEv mixed background. Wt1+/floxmice [11] were mated with mice carrying theWt1-null allele (Wt1+/) [10] andAmh-Cre transgenic mice [14] to produceWt1-/flox; Amh-Cre (Wt1SC-cKO) offspring. All mouse lines were provided by Dr . PA-824 (Pretomanid) Fei Gao (Institute of Zoology, Chinese Academy of Sciences). DNA isolated from tail biopsies was used for genotyping by PCR to confirm Sertoli cellWt1knockout (cKO) in fetal males as earlier described [10, 11, 14]. No difference was found amongWt1+/flox, Wt1+/flox; Amh-Cre, Wt1-/floxor wild-type mice, thus the age-matched male mice (Wt1+/flox, Wt1+/flox; Amh-Cre orWt1-/flox) served as the corresponding control mice ofWt1-/flox; Amh-Cre. == Immunofluorescence (IF) and immunohistochemistry (IHC) analysis == IF was performed using both paraffin and frozen sections, and IHC using paraffin sections. Testes were obtained immediately following euthanasia by CO2asphyxiation, fixed in 4% paraformaldehyde, embedded in paraffin and obtained paraffin sections at 5 m with a microtome. Frozen sections at 8 m obtained in a cryostat at -22C were fixed in 4% PFA for 10 min. IF was performed using either the FITC or TRITC-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). For IHC, tissue sections were de-paraffinized and rehydrated, to be followed by antigen retrieval in 10 mM sodium citrate buffer for 15 min..