Background: Ageing in men is believed to be associated with fertility decline and elevated risk of congenital disorders for the offspring. The previous studies also reported reduced germ and Sertoli cell numbers in older men. However, it is not clear whether ageing in men with normal spermatogenesis affects the testis and germ cell population dynamics in a way sufficient for transmitting adverse age effects to the offspring. Objectives: We examined men with normal spermatogenesis at different ages concerning effects on persisting testicular cell types, that is the germ line and Sertoli cells, as these cell populations are prone to be exposed to age effects. Material and methods: Ageing was assessed in testicular biopsies of 32 patients assigned to three age groups: (i) 28.8 AE 2.7 years; (ii) 48.1 AE 1 years; and (iii) 70.9 AE 6.2 years, n = 8 each, with normal spermatogenesis according to the Bergmann-Kliesch score, and in a group of meiotic arrest patients (29.9 AE 3.8 years, n = 8) to decipher potential links between different germ cell types. Besides morphometry of seminiferous tubules and Sertoli cell nuclei, we investigated spermatogenic output/efficiency, and dynamics of spermatogonial populations via immunohistochemistry for MAGE A4, PCNA, CREM and quantified A-pale/A-dark spermatogonia. Results: We found a constant spermatogenic output (CREM-positive round spermatids) in all age groups studied. In men beyond their mid-40s (group 2), we detected increased nuclear and nucleolar size in Sertoli cells, indirectly indicating an elevated protein turnover. From the 7th decade (group 3) of life onwards, testes showed increased proliferation of undifferentiated spermatogonia, decreased spermatogenic efficiency and elevated numbers of proliferating A-dark spermatogonia. Discussion and conclusion: Maintaining normal sperm output seems to be an intrinsic determinant of spermatogenesis. Ageing appears to affect this output and might provoke compensatory proliferation increase in A spermatogonia which, in turn, might hamper germ cell integrity.

The Hippo signaling pathway is known to regulate cell differentiation, proliferation and apoptosis. Whereas activation of the Hippo signaling pathway leads to phosphorylation and cytoplasmic retention of the transcriptional coactivator YAP, decreased Hippo signaling results in nuclear import of YAP and subsequent transcription of pro-proliferative genes. Hence, a dynamic and precise regulation of the Hippo signaling pathway is crucial for organ size control and the prevention of tumor formation. The transcriptional activity of YAP is controlled by a growing number of upstream regulators including the family of WWC proteins. WWC1, WWC2 and WWC3 represent cytosolic scaffolding proteins involved in intracellular transport processes and different signal transduction pathways. Earlier in vitro experiments demonstrated that WWC proteins positively regulate the Hippo pathway via the activation of large tumor suppressor kinases 1/2 (LATS1/2) kinases and the subsequent cytoplasmic accumulation of phosphorylated YAP. Later, reduced WWC expression and subsequent high YAP activity were shown to correlate with the progression of human cancer in different organs. Although the function of WWC proteins as upstream regulators of Hippo signaling was confirmed in various studies, their important role as tumor modulators is often overlooked. This review has been designed to provide an update on the published data linking WWC1, WWC2 and WWC3 to cancer, with a focus on Hippo pathway-dependent mechanisms.

The WW-and-C2-domain-containing (WWC) protein family is involved in the regulation of cell differentiation, cell proliferation, and organ growth control. As upstream components of the Hippo signaling pathway, WWC proteins activate the Large tumor suppressor (LATS) kinase that in turn phosphorylates Yes-associated protein (YAP) and its paralog Transcriptional coactivator-with-PDZ-binding motif (TAZ) preventing their nuclear import and transcriptional activity. Inhibition of WWC expression leads to downregulation of the Hippo pathway, increased expression of YAP/TAZ target genes and enhanced organ growth. In mice, a ubiquitous Wwc1 knockout (KO) induces a mild neurological phenotype with no impact on embryogenesis or organ growth. In contrast, we could show here that ubiquitous deletion of Wwc2 in mice leads to early embryonic lethality. Wwc2 KO embryos display growth retardation, a disturbed placenta development, impaired vascularization, and finally embryonic death. A whole-transcriptome analysis of embryos lacking Wwc2 revealed a massive deregulation of gene expression with impact on cell fate determination, cell metabolism, and angiogenesis. Consequently, a perinatal, endothelial-specific Wwc2 KO in mice led to disturbed vessel formation and vascular hypersprouting in the retina. In summary, our data elucidate a novel role for Wwc2 as a key regulator in early embryonic development and sprouting angiogenesis in mice.

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The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.