Loss of SUMO-specific protease 2 causes isolated glucocorticoid deficiency by blocking adrenal cortex zonal transdifferentiation in mice.

SUMOylation is a dynamic posttranslational modification, that provides fine-tuning of protein function involved in the cellular response to stress, differentiation, and tissue development. In the adrenal cortex, an emblematic endocrine organ that mediates adaptation to physiological demands, the SUMOylation gradient is inversely correlated with the gradient of cellular differentiation raising important questions about its role in functional zonation and the response to stress. Considering that SUMO-specific protease 2 (SENP2), a deSUMOylating enzyme, is upregulated by Adrenocorticotropic Hormone (ACTH)/cAMP-dependent Protein Kinase (PKA) signalling within the zona fasciculata, we generated mice with adrenal-specific Senp2 loss to address these questions. Disruption of SENP2 activity in steroidogenic cells leads to specific hypoplasia of the zona fasciculata, a blunted reponse to ACTH and isolated glucocorticoid deficiency. Mechanistically, overSUMOylation resulting from SENP2 loss shifts the balance between ACTH/PKA and WNT/ß-catenin signalling leading to repression of PKA activity and ectopic activation of ß-catenin. At the cellular level, this blocks transdifferentiation of ß-catenin-positive zona glomerulosa cells into fasciculata cells and sensitises them to premature apoptosis. Our findings indicate that the SUMO pathway is critical for adrenal homeostasis and stress responsiveness.

Targeted Disruption of Lats1 and Lats2 in Mice Impairs Testis Development and Alters Somatic Cell Fate.

Hippo signaling plays an essential role in the development of numerous tissues. Although it was previously shown that the transcriptional effectors of Hippo signaling Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) can fine-tune the regulation of sex differentiation genes in the testes, the role of Hippo signaling in testis development remains largely unknown. To further explore the role of Hippo signaling in the testes, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in the somatic cells of the testes using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that early stages of testis somatic cell differentiation were not affected in this model but progressive testis cord dysgenesis was observed starting at gestational day e14.5. Testis cord dysgenesis was further associated with the loss of polarity of the Sertoli cells and the loss of SOX9 expression but not WT1. In parallel with testis cord dysgenesis, a loss of steroidogenic gene expression associated with the appearance of myofibroblast-like cells in the interstitial space was also observed in mutant animals. Furthermore, the loss of YAP phosphorylation, the accumulation of nuclear TAZ (and YAP) in both the Sertoli and interstitial cell populations, and an increase in their transcriptional co-regulatory activity in the testes suggest that the observed phenotype could be attributed at least in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper differentiation of testis somatic cells.

Sexually dimorphic activation of innate antitumor immunity prevents adrenocortical carcinoma development.

Unlike most cancers, adrenocortical carcinomas (ACCs) are more frequent in women than in men, but the underlying mechanisms of this sexual dimorphism remain elusive. Here, we show that inactivation of Znrf3 in the mouse adrenal cortex, recapitulating the most frequent alteration in ACC patients, is associated with sexually dimorphic tumor progression. Although female knockouts develop metastatic carcinomas at 18 months, adrenal hyperplasia regresses in male knockouts. This male-specific phenotype is associated with androgen-dependent induction of senescence, recruitment, and differentiation of highly phagocytic macrophages that clear out senescent cells. In contrast, in females, macrophage recruitment is delayed and dampened, which allows for aggressive tumor progression. Consistently, analysis of TCGA-ACC data shows that phagocytic macrophages are more prominent in men and are associated with better prognosis. Together, these data show that phagocytic macrophages are key players in the sexual dimorphism of ACC that could be previously unidentified allies in the fight against this devastating cancer.

Protein kinase A drives paracrine crisis and WNT4-dependent testis tumor in Carney complex.

Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.

Constitutive activation of CTNNB1 results in a loss of spermatogonial stem cell activity in mice.

Spermatogenesis requires that a careful balance be maintained between the self-renewal of spermatogonial stem cells (SSCs) and their commitment to the developmental pathway through which they will differentiate into spermatozoa. Recently, a series of studies employing various in vivo and in vitro models have suggested a role of the wingless-related MMTV integration site gene family/beta-catenin (WNT/CTNNB1) pathway in determining the fate of SSCs. However, conflicting data have suggested that CTNNB1 signaling may either promote SSC self-renewal or differentiation. Here, we studied the effects of sustained CTNNB1 signaling in SSCs using the Ctnnb1tm1Mmt/+; Ddx4-CreTr/+ (ΔCtnnb1) mouse model, in which a stabilized form of CTNNB1 is expressed in all germ cells. ΔCtnnb1 mice were found to have reduced testis weights and partial germ cell loss by 4 months of age. Germ cell transplantation assays showed a 49% reduction in total functional SSC numbers in 8 month-old transgenic mice. In vitro, Thy1-positive undifferentiated spermatogonia from ΔCtnnb1 mice formed 57% fewer clusters, which was associated with decreased cell proliferation. A reduction in mRNA levels of genes associated with SSC maintenance (Bcl6b, Gfra1, Plzf) and increased levels for markers associated with progenitor and differentiating spermatogonia (Kit, Rarg, Sohlh1) were detected in these cluster cells. Furthermore, RNAseq performed on these clusters revealed a network of more than 900 genes regulated by CTNNB1, indicating that CTNNB1 is an important regulator of spermatogonial fate. Together, our data support the notion that CTNNB1 signaling promotes the transition of SSCs to undifferentiated progenitor spermatogonia at the expense of their self-renewal.

Targeted Disruption of Lats1 and Lats2 in Mice Impairs Adrenal Cortex Development and Alters Adrenocortical Cell Fate.

It has recently been shown that the loss of the Hippo signaling effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in adrenocortical steroidogenic cells impairs the postnatal maintenance of the adrenal gland. To further explore the role of Hippo signaling in mouse adrenocortical cells, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in steroidogenic cells using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that developing adrenocortical cells adopt characteristics of myofibroblasts in both male and female Lats1flox/flox;Lats2flox/flox;Nr5a1-cre mice, resulting in a loss of steroidogenic gene expression, adrenal failure and death by 2 to 3 weeks of age. A marked accumulation of YAP and TAZ in the nuclei of the myofibroblast-like cell population with an accompanying increase in the expression of their transcriptional target genes in the adrenal glands of Lats1flox/flox;Lats2flox/flox;Nr5a1-cre animals suggested that the myofibroblastic differentiation could be attributed in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper adrenocortical cell differentiation and suppresses their differentiation into myofibroblast-like cells.

Lats1 and Lats2 are required for the maintenance of multipotency in the Müllerian duct mesenchyme.

WNT signaling plays essential roles in the development and function of the female reproductive tract. Although crosstalk with the Hippo pathway is a key regulator of WNT signaling, whether Hippo itself plays a role in female reproductive biology remains largely unknown. Here, we show that conditional deletion of the key Hippo kinases Lats1 and Lats2 in mouse Müllerian duct mesenchyme cells caused them to adopt the myofibroblast cell fate, resulting in profound reproductive tract developmental defects and sterility. Myofibroblast differentiation was attributed to increased YAP and TAZ expression (but not to altered WNT signaling), leading to the direct transcriptional upregulation of Ctgf and the activation of the myofibroblast genetic program. Müllerian duct mesenchyme cells also became myofibroblasts in male mutant embryos, which impeded the development of the male reproductive tract and resulted in cryptorchidism. The inactivation of Lats1/2 in differentiated uterine stromal cells in vitro did not compromise their ability to decidualize, suggesting that Hippo is dispensable during implantation. We conclude that Hippo signaling is required to suppress the myofibroblast genetic program and maintain multipotency in Müllerian mesenchyme cells.

Yes-associated protein expression in germ cells is dispensable for spermatogenesis in mice.

Yes-associated protein (YAP), a key effector of the Hippo signaling pathway, is expressed in the nucleus of spermatogonia in mice, suggesting a potential role in spermatogenesis. Here, we report the generation of a conditional knockout mouse model (Yapflox/flox ; Ddx4cre/+ ) that specifically inactivates Yap in the germ cells. The inactivation of Yap in spermatogonia was found to be highly efficient in this model. The loss of Yap in the germ cells had no observable effect on spermatogenesis in vivo. Histological examination of the testes showed no structural differences between mutant animals and age-matched Yapflox/flox controls, nor was any differences detected in gonadosomatic index, expression of germ cell markers or sperm counts. Cluster-forming assay using undifferentiated spermatogonia, including spermatogonial stem cells (SSCs), also showed that YAP is dispensable for SSC cluster formation in vitro. However, an increase in the expression of spermatogenesis and oogenesis basic helix-loop-helix 1 (Sohlh1) and neurogenin 3 (Ngn3) was observed in clusters derived from Yapflox/flox ; Ddx4cre/+ animals. Taken together, these results suggest that YAP fine-tunes the expression of genes associated with spermatogonial fate commitment, but that its loss is not sufficient to alter spermatogenesis in vivo.

Conditional Deletion of FOXL2 and SMAD4 in Gonadotropes of Adult Mice Causes Isolated FSH Deficiency.

The glycoprotein FSH, a product of pituitary gonadotrope cells, regulates ovarian follicle development in females and spermatogenesis in males. FSH is a heterodimer of the common α gonadotropin subunit and the hormone-specific FSHß subunit (a product of the Fshb gene). Using a conditional knockout approach (Cre-lox), we previously demonstrated that Fshb expression in mice depends on the transcription factors forkhead box L2 (FOXL2) and SMAD4. Deletion of Foxl2 or Smad4 alone led to FSH deficiency, female subfertility, and oligozoospermia in males. Simultaneous deletion of the two genes yielded a greater suppression of FSH and female sterility. The Cre-driver used previously was first active during embryonic development. Therefore, it is unclear whether FOXL2 and SMAD4 play important roles in the development or adult function of gonadotropes, or both. To address this question, we developed a tamoxifen-inducible Cre-driver line, which enabled Foxl2 and Smad4 gene deletions in gonadotropes of adult mice. After tamoxifen treatment, females with previously demonstrated fertility exhibited profound reductions in FSH levels, arrested ovarian follicle development, and sterility. FSH levels were comparably reduced in males 1 or 2 months after treatment; however, spermatogenesis was unaffected. These data indicate that (1) FOXL2 and SMAD4 are necessary to maintain FSH synthesis in gonadotrope cells of adult mice, (2) FSH is essential for female reproduction but appears to be unnecessary for the maintenance of spermatogenesis in adult male mice, and (3) the inducible Cre-driver line developed here provides a powerful tool to interrogate gene function in gonadotrope cells of adult mice.

Targeted Disruption of YAP and TAZ Impairs the Maintenance of the Adrenal Cortex.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are functionally redundant transcriptional regulators that are downstream effectors of the Hippo signaling pathway. They act as major regulators of stem cell maintenance, cell growth, and differentiation. To characterize their roles in the adrenal cortex, we generated a mouse model in which Yap and Taz were conditionally deleted in steroidogenic cells (Yapflox/flox;Tazflox/flox;Nr5a1cre/+). Male Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice were characterized by an age-dependent degeneration of the adrenal cortex associated with an increase in apoptosis and a progressive reduction in the expression levels of steroidogenic genes. Evaluation of the expression levels of stem and progenitor cell population markers in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice also showed the downregulation of sonic hedgehog (Shh), a marker of the subcapsular progenitor cell population. Gross degenerative changes were not observed in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ females, although steroidogenic capacity and Shh expression were reduced, suggesting that mechanisms of adrenocortical maintenance are sex specific. These results define a crucial role for YAP and TAZ in the maintenance of the postnatal adrenal cortex.

Yes-associated protein and WW-containing transcription regulator 1 regulate the expression of sex-determining genes in Sertoli cells, but their inactivation does not cause sex reversal.

Yes-associated protein (YAP) and WW-containing transcription regulator 1 (WWTR1) are two functionally redundant transcriptional regulators that are downstream effectors of the Hippo signaling pathway, and that act as major regulators of cell growth and differentiation. To elucidate their role in Sertoli cells, primary Sertoli cell culture from Yapflox/flox; Wwtr1flox/flox animals were infected with a Cre recombinase-expressing adenovirus. Concomitant inactivation of Yap and Wwtr1 resulted in a decrease in the mRNA levels of the male sex differentiation genes Dhh, Dmrt1, Sox9, and Wt1, whereas those of genes involved in female differentiation (Wnt4, Rspo1, and Foxl2) were induced. SOX9, FOXL2, and WNT4 proteins were regulated in the same manner as their mRNAs in response to loss of YAP and WWTR1. To further characterize the role of YAP and WWTR1 in Sertoli cells, we generated a mouse model (Yapflox/flox; Wwtr1flox/flox; Amhcre/+) in which Yap and Wwtr1 were conditionally deleted in Sertoli cells. An increase in the number of apoptotic cells was observed in the seminiferous tubules of 4 dpp mutant mice, leading to a reduction in testis weights and a decrease in the number of Sertoli cells in adult animals. Gene expression analyses of testes from 4 dpp Yapflox/flox; Wwtr1flox/flox; Amhcre/+ mice showed that Sertoli cell differentiation is initially altered, as Dhh, Dmrt1, and Sox9 mRNA levels were downregulated, whereas Wnt4 mRNA levels were increased. However, expression of these genes was not changed in older animals. Together, these results suggest a novel role of the Hippo signaling pathway in the mechanisms of sex differentiation.

mTOR Regulates Gap Junction Alpha-1 Protein Trafficking in Sertoli Cells and Is Required for the Maintenance of Spermatogenesis in Mice.

The mammalian target of rapamycin (Mtor) gene encodes a serine/threonine kinase that acts as a master regulator of processes as diverse as cell growth, protein synthesis, cytoskeleton reorganization, and cell survival. In the testis, physiological roles for Mtor have been proposed in perinatal Sertoli cell proliferation and blood-testis barrier (BTB) remodeling during spermatogenesis, but no in vivo studies of Mtor function have been reported. Here, we used a conditional knockout approach to target Mtor in Sertoli cells. The resulting Mtor(flox/flox); Amhr2(cre/+) mice were characterized by progressive, adult-onset testicular atrophy associated with disorganization of the seminiferous epithelium, loss of Sertoli cell polarity, increased germ cell apoptosis, premature release of germ cells, decreased epididymal sperm counts, increased sperm abnormalities, and infertility. Histopathologic analysis and quantification of the expression of stage-specific markers showed a specific loss of pachytene spermatocytes and spermatids. Although the BTB and the ectoplasmic specializations did not appear to be altered in Mtor(flox/flox);Amhr2(cre/+) mice, a dramatic redistribution of gap junction alpha-1 (GJA1) was detected in their Sertoli cells. Phosphorylation of GJA1 at Ser373, which is associated with its internalization, was increased in the testes of Mtor(flox/flox); Amhr2(cre/+) mice, as was the expression and phosphorylation of AKT, which phosphorylates GJA1 at this site. Together, these results indicate that Mtor expression in Sertoli cells is required for the maintenance of spermatogenesis and the progression of germ cell development through the pachytene spermatocyte stage. One mechanism of mTOR action may be to regulate gap junction dynamics by inhibiting AKT, thereby decreasing GJA1 phosphorylation and internalization. mTOR regulates gap junction alpha-1 protein distribution in Sertoli cells and is necessary for progression through the pachytene spermatocyte stage.