br References and recommended reading Papers of particular

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:
Introduction Male sexual maturation includes gonadal maturation, secondary sexual characteristics, and the attainment of reproductive capacity [1]. Male sexual differentiation and reproductive development are mainly controlled by the regulation of various hormones [2,3], which are closely associated with genetic program [4]. In addition, accumulating signaling cascades in male sexual maturation have been reported, such as kisspeptin [5], androgen/androgen receptor [6], Wnt/β-catenin [7] and transforming growth factor-β [8]. These genetic signaling networks activated the subset genes, which acted as orchestrated networks to regulate the biological phenotypes during sexual development. Despite these studies, the regulatory mechanism involved in male sexual maturation remains unclear, especially in small ruminants like sheep. Hippo pathway is an evolutionarily conserved signaling cascade in regulating tissue development, homeostasis and regeneration [9]. The canonical Hippo pathway comprises mammalian sterile 20-like protein kinase 1 and 2 (MST1/2), large tumor suppressor homologues 1 and 2 (LATS1/2) and its effector yes-associated protein 1 (YAP1). Hippo pathway regulates the development and homeostasis at both cellular and organic levels in response to a wide range of extracellular and intracellular signals [10]. Emerging evidence has shown the important roles of Hippo signaling cascade in pubertal development and male maturity. LATS1 deficient mice showed decreased fertility as a result of reproductive hormone defects [11]. YAP1 regulated the expressions of sex-determining genes in mouse Sertoli Plumbagin [12]. Furthermore, Hippo pathway participated in regulating the process of steroidogenesis, which played critical roles in the sexual maturation [13]. While the underlying mechanism of Hippo components involved in male sexual maturity remains to be elucidated. In this study, to clarify the physiological roles of Hippo signaling component during male sexual maturation of Hu sheep, we investigated the localization and expressions of MST1/2, LATS1/2 and YAP1 in testicular, epididymal and ductus deferens tissues of prepubertal (3-month-old) and postpubertal (9-month-old) Hu sheep [14], as well as in spermatozoa from cauda epididymis to ejaculation.
Materials and methods
Results
Discussion The trigger of the puberty cascade is very important for the livestock production, while the process of sexual maturation depends on many interacting growth and reproductive pathways, which involved complex regulatory networks. Herein, an age-related and tissue-specific expressions of the Hippo pathway components in the male reproductive tract of Hu sheep were investigated during sexual maturation. Combined with previous researches that LATS1 deficient mice showed low fertility [11] and YAP1 inactivation resulted in the decreased male sex differentiation gene expression in mouse Sertoli cells [12], we speculate that Hippo pathway components may act as a novel pathway to regulate the development potential of male reproduction in Hu sheep. The high expressions of Hippo cascade components in male reproduction system of Hu sheep were similar with other animals like mouse and Atlantic salmon [12,20], while the transcripts of these genes were absent or low in Drosophila testis [21]. These discrepancies were mainly due to species-species differences, and more evidence still needs to clarify. Nevertheless, these results may facilitate a better understanding of Hippo pathway in male reproductive system. Leydig and Sertoli cells in the testicular sections play crucial roles in male development and the maintenance of reproductive functions [22]. Leydig cells function as endocrine populations to produce testosterone and Sertoli cells support normal spermatogenesis [23]. In the present study, the enrichment of Hippo pathway proteins in Leydig and Sertoli cells of Hu sheep indicated the important roles of this cascade in these steroid hormone producing cells and “nurse cells (Sertoli cells)“. Previous researches showed that aberrant expression of Hippo pathway components may lead to the steroidogenesis dysfunction of granulosa cell tumors [13], and testosterone could in turn regulate YAP1 expression by alleviating YAP1 promoter methylation in granulosa cells [24]. These data provide evidence that Hippo signaling may act as a local modulator of the endocrine activity in the steroid hormone producing cells. Additionally, the increased expression of Hippo pathway components in testis was coincident with the elevated circulating levels of testosterone during the sexual maturation in our study (Table S1). Moreover, Sertoli cells played vital roles in self-renewal and differentiation of spermatogonial stem cells by secreting various growth factors and cytokines [25], and some of these growth factors such as bone morphogenetic protein 4 (BMP4), was a downstream target of Hippo pathway [26]. These data indicated that Hippo pathway might involve in spermatogenesis through targeting Leydig cells and Sertoli cells.