Hippo signaling in mammalian reproduction.

The Hippo signaling pathway is a highly conserved pathway that is essential for animal development and adult homeostasis. The pathway is named for overgrowth, reminiscent of a hippopotamus, observed in fruit flies carrying Hippo pathway mutations ( Harvey et al. 2003 ). Subsequent studies using biochemical and genetic approaches have uncovered core components and mechanisms of Hippo signaling, including upstream inputs and downstream outputs. Interestingly, a large variety of signals can act upstream to initiate Hippo signaling, including extracellular ligands, steroids, stress, and mechanical cues ( Zhong et al. 2024 ). In most contexts, the major output of Hippo signaling is modulation of gene expression. The mechanisms by which Hippo transcriptional targets are specifically regulated are an active area of investigation. In general, Hippo signaling represses the transcriptional activity of the paralogous transcriptional co-factors YAP1 and WWTR1 (also called TAZ) by preventing their association with TEAD family DNA-binding factors. In the absence of Hippo signaling, YAP1 and WWTR1 associate with TEADs, and the YAP1/WWTR1/TEAD complex then induces transcriptional changes. The YAP1/WWTR1/TEAD complex is generally thought to act as a transcriptional activator. However, YAP1 and WWTR1 can also repress transcription by recruiting transcriptional repressors, for example, of the RUNX, PPAR, SMAD, or VGLL families, or the Nucleosome Remodeling and Deacetylation (NuRD) complex ( Ferrigno et al. 2002 , Hong et al. 2005 , Beyer et al. 2013 , Varelas 2014 , Kim et al. 2015 , Valencia-Sama et al. 2015 , Cotton et al. 2017 , Zhang et al. 2018 ). The clinical implications of this are that, depending on molecular context, the YAP1/WWTR1/TEAD complex could possess both tumor suppressor and oncogenic activities ( Kim et al. 2018 ). Several excellent review articles have described roles for Hippo in homeostasis, disease, development, and regeneration in model organisms and in humans ( Moya & Halder 2016 , Misra & Irvine 2018 , Davis & Tapon 2019 , Zheng & Pan 2019 ). Yet, the roles of Hippo signaling in reproduction have been less comprehensively summarized. Drawing from studies in mice and humans, we review known roles for Hippo signaling in mammalian reproduction, beginning with the early embryo and then proceeding upstream to explore paternal and maternal Hippo roles in germ cell production, pregnancy, and disease.

Our review would not be complete without discussion of Hippo roles in spermatogenesis. In fact, the Hippo study has been extensively studied in this context. Studies in mice have demonstrated that Hippo signaling is critically involved in the regulation of spermatogenesis ( St John et al. 1999 , Hossain et al. 2007 ). Spermatogenesis takes place within the seminiferous tubules of the testes, where immature, self-renewing spermatogonial stem cells (SSCs) reside along the inner edge of the tubule. SSCs gradually mature as they move inward toward the tubule lumen. SSCs first differentiate into spermatogonia, which can divide by mitosis to produce meiotically capable spermatocytes. After completing meiosis, haploid spermatocytes differentiate further into spermatids and then undergo morphological changes to become mature sperm in the process of spermiogenesis. Finally, the mature spermatid enters the lumen of the seminiferous tubule as a fully developed spermatozoon. Spermatozoa travel from the seminiferous tubule to the epididymis, where they undergo final maturation before ejaculation. Several models, including knockouts of Lats1, Lats2 , Yap1, and Wwtr1, report decreased testis size ( St John et al. 1999 , Levasseur et al. 2017 , Abou Nader et al. 2022 ). However, although YAP1 and WWTR1 protein are expressed and localized to the nucleus in male germ cells after puberty ( Levasseur et al. 2017 ), the direct, cell-autonomous effect of Hippo signaling on spermatogenesis appears to be limited. Conditional knockout of Yap1 in mouse spermatogonial germ cells had no apparent effect on the expression of germ cell markers, SSC formation, or sperm count in vivo ( Abou Nader et al. 2019 ). Although Hippo signaling is apparently dispensable in germ cells, it is essential to maintain the cell identities of somatic cells of the testis ( Fig. 4 ). This is reminiscent of the role in ovary somatic cells described above. The somatic cells of the testis, Sertoli cells, Leydig cells, and structural interstitial cells, are essential to the process of spermatogenesis. Leydig cells function primarily to produce testosterone, which, among several other targets, is received by Sertoli cells. Sertoli cells act as ‘nurse’ cells for the developing germ cells within the seminiferous tubules, providing hormones, nutrients, structural support, and aiding in waste removal. Proper function of Sertoli and Leydig cells is essential for spermatogenesis and male fertility. Testicular function requires Hippo signaling in somatic cells. When Hippo signaling is experimentally repressed in Sertoli cells, seminiferous tubules suffer adverse effects such as tissue fibrosis and germ cell death. Interestingly, Hippo signaling appears to be most important in Sertoli and Leydig cells for spermatogenesis. Specific knockout of Lats1 and Lats2 in Sertoli and Leydig cells led to smaller and disorganized testes as early as embryonic day E14.5 ( Abou Nader et al. 2022 ). These knockouts showed decreased expression of many Sertoli cell markers, including Sox9 . On the other hand, conditional double knockout of Yap1 and Wwtr1 in Sertoli cells decreased expression of male-specific genes such as Dhh, Dmrt1, Sox9 , and Wt1 at pre-pubertal stages ( Levasseur et al. 2017 ). Notably, the loss of cell identity in Sertoli and Leydig cells did not result in sex reversal, as neither Yap1/Wwtr1 knockout nor Lats1/2 knockout resulted in upregulation of granulosa cell genes in vivo ( Levasseur et al. 2017 , Abou Nader et al. 2022 ). Ultimately, the requirement for Hippo signaling in somatic cell identity leads to an essential, non-cell-autonomous role in spermatogenesis ( Sen Sharma & Majumdar 2017 , Abou Nader et al. 2022 ). Conditional double knockout of Lats1 and Lats2 in mouse Sertoli cells results in small, disorganized testes with very few observable seminiferous tubules ( Abou Nader et al. 2022 ). In addition, germ cells in this model were mostly apoptotic by E17.5, suggesting that active Hippo signaling in Sertoli cells is specifically required for early spermatogenesis. This conclusion is consistent with the finding that knockout of Yap1 and Wwtr1 in Sertoli cells does not impair early spermatogenesis, since Lats1/2 normally prevents the transcriptional activity of YAP1 and WWTR1 ( Levasseur et al. 2017 ). Altogether, these studies show that Hippo signaling is required in male reproductive somatic cells for proper regulation of spermatogenesis and male fertility. However, the specific transcriptional targets of YAP1/WWTR1 and exact regulatory mechanisms remain to be identified. Several lines of evidence suggest that Hippo signaling is required for stabilization of cilia ( Hossain et al. 2007 , Shi et al. 2023 ). This possibility is intriguing, as patients with autosomal dominant polycystic kidney disease, which affects cilia formation, also commonly display male infertility and sperm motility defects ( Shi et al. 2023 ).

R K has been supported by NIH https://doi.org/10.13039/100000002 grants T32 HD087166 and T32 DK071212. F A is supported by NIH grant R01 HD108722 to A R.

Surveying the roles of Hippo signaling broadly across reproductive systems, an unexpected theme has emerged. That is, the dysregulation of Hippo signaling tends to result in cell fate defects in diverse contexts, including the preimplantation embryo, and both male and female germ lines. Whether this is coincidental or due to a shared downstream mechanism awaits further study. Identification of YAP1/WWTR1/TEAD transcriptional targets in each setting could help illuminate this question. Moreover, discovering mechanisms downstream of YAP1/WWTR1/TEAD will also aid in the development of therapeutic strategies for treating reproductive diseases that are caused by Hippo signaling dysregulation.

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

A R conceived the paper. R K, F A, and A R wrote the paper.