The role of primary cilia in the testis

Abstract

A better understanding of the role primary cilia play in the testis is becoming increasingly important in the context of male reproductive health since defects in cilia have been associated with male infertility and reproductive disorders. Primary cilia are found on most somatic cell types within the testis, including Sertoli cells, peritubular myoid cells, and Leydig cells, especially during embryonic and neonatal stages. Primary cilia are involved in signaling pathways that are important for differentiation and function of testicular somatic cells, and cilia are also implicated in testicular morphogenesis. These observations indicate that testicular primary cilia play an integral role in regulating testis development and function.

Introduction

Primary cilia are highly conserved, ubiquitous, immotile organelles containing a 9 + 0 microtubule arrangement and anchored to the cell by the basal body, which develops from the mother centriole of the centrosome [1–4]. Primary cilia are found in most mammalian cells and are an essential organelle for many tissues, being involved in signal propagation for several pathways, such as Hedgehog (Hh), Wnt, and platelet-derived growth factor (PDGF) signaling [1,2,5–9]. The establishment of cell polarity and determination of cell fate including the coordination of embryogenesis and organogenesis are roles commonly associated with the primary cilium [1,10,11]. During the mitotic cell cycle, primary cilia are resorbed [10,12]. Disruptions of cilia structure and functions have been implicated in several human diseases and developmental defects, called ciliopathies. One example is polycystic kidney disease, which presents as kidney and liver cysts with an adult onset. The disease is characterized by defective polycistin proteins, which seem to be linked to left–right patterning and localize to the primary cilia, but the precise mechanisms involved in their function and disease development are still not fully known [1,10,11]. Therefore, pathophysiological events mediated by primary cilia have been studied in many tissues, including the male reproductive tract. However, little is known about the direct involvement of primary cilia in the regulation of testis development and function, even though male infertility is associated with certain ciliopathies and cilia-related signaling pathways are important for testis development [13–20].

Primary cilia in the testis

Primary cilia are dynamic structures, and their presence in the testis seems to vary depending on the species, developmental and maturational stage, and even within subpopulations of the same cell type. Many primary cilia-related genes are expressed in the developing testes, and primary cilia are present in somatic cells, such as Sertoli, Leydig, and peritubular myoid cells (Figure 1) [18,21–25]. Some studies also point to the presence of primary cilia in unidentified/undifferentiated interstitial cells, but it remains to be elucidated whether these cells are macrophages, mesenchymal cells, or a subgroup of Leydig cells [21–24,26]. Interestingly, there is one report of primary cilia detected in some germ cells of XY embryos during a specific time point of mouse embryogenesis [23].

Figure 1. Presence of primary cilia in 1-week-old pig testis.

Immunohistochemistry for GATA4 (Sertoli cells; orange), acetylated tubulin (cilia microtubules; white arrows), γ tubulin (basal body; green; white arrowheads), and ARL13B (primary cilia; red) reveal the presence of primary cilia on Sertoli cells (white arrows on a-b; black arrowhead on c), Leydig cells (yellow arrowheads on c), and peritubular myoid cells (green arrowheads on c). Scale bars = 10 μm. Modified with permission from Ref. [22].

Peritubular myoid cells are the testicular cells more consistently reported to present primary cilia, where they were described in most species evaluated and are present during development and in adulthood [18,21–27]. It can be hypothesized that primary cilia play a role in the complex signaling interactions of peritubular myoid cells due to their strategic position between the interstitial compartment and the seminiferous epithelium. Besides being important for regulation of spermatogenesis as a mediator of this communication, peritubular myoid cells also interact closely with the extracellular matrix of the basement membrane. Primary cilia on peritubular myoid cells could act as mechanical sensors and/or influence extracellular matrix deposition [18,24,28–30]. Moreover, the PDGF signaling pathway, commonly associated with the primary cilium, is important for peritubular myoid cell development, proliferation, and function [28,31–35]. However, no study so far has focused on the specific regulation of peritubular myoid cell function by primary cilia.

Leydig cells, the steroidogenic cells of the testis, are also commonly shown to possess primary cilia, although this varies according to the cell’s maturational status and/or specific subpopulations, such as fetal or adult Leydig cells. Primary cilia have been reported in Leydig cells of adult rabbits [36], mouse embryos, the TM3 immature Leydig cell line [23,37,38], mature and estrogenized rats [26], neonatal pigs [22,25], and humans [21,24,39]. Furthermore, recent studies suggest that adult Leydig cells may lose their primary cilia as they differentiate and become mature. This could be, at least partially, regulated by Desert hedgehog (Dhh) and PDGF signaling in the cilia since activation of these pathways is involved in Leydig cell specification, development, and function [24,37,40–44]. The primary cilia of mouse progenitor TM3 Leydig cell line are responsive to Hh signaling and participate in the PDGF signaling that regulates Leydig cell function [24,44]. PDGF-AA treatment promoted the growth of TM3 Leydig cells via AKT/ERK signaling, whereas the depletion of ciliary genes IFT88 and CEP164 resulted in decreased Leydig cell growth, migration, and invasion [44].

The presence of primary cilia in Sertoli cells seems to vary according to their maturational status, subpopulations, and species. There are reports of immature Sertoli cells possessing primary cilia in most species, such as in mice, rats, pigs, and humans [18,21–27,30]. Interestingly, in adult humans, Sertoli cells with primary cilia were observed in Klinefelter syndrome testis, where these cells are mostly immature or dedifferentiated, and in transformed Sertoli cells of an ovarian Sertoli-cell tumor [24,45]. Similarly, in pigs, the frequency of ciliated Sertoli cells decreases with development and Sertoli cell maturation [22]. These findings suggest an important role for Sertoli-cell primary cilia in neonatal testicular morphogenesis and germ-cell niche formation, but a less critical function during maturation. Additionally, Nygaard et al. [24] reported an interesting observation on their Klinefelter-syndrome human samples: Anti-Müllerian hormone (AMH)-negative (mature) Sertoli cells seem to possess cilia foci oriented toward the basement membrane, whereas AMH-positive (immature) Sertoli-cell cilia mostly orient toward the lumen. Since seminiferous tubules in these patients are devoid of germ cells, it is difficult to interpret this observation without further studies. Similarly, in rats, Sertoli cells from the transition region (the junction between the seminiferous tubule and rete testis) exhibit varying degrees of differentiation, potentially representing different subpopulations with specific functions, and it may be interesting to investigate the presence of primary cilia in these cells and, if present, what role they might be playing [46–49].

Primary cilia and testicular morphogenesis

Although there are no direct studies focused on the role of primary cilia in sex determination and sexual differentiation, some studies have investigated the role of primary cilia in testicular morphogenesis. The temporal expression pattern of primary cilia in the testis suggests a functional role in morphogenesis and development since the organelle is mostly present in immature and not fully differentiated Leydig and Sertoli cells [22,24].

The number of Sertoli cells carrying a primary cilium in the testis cords decreases significantly when comparing testes from 2-week-old (approximately 5 per 10 Sertoli cells), 4-week-old (approximately 2.3 cells with a primary cilium per 10 Sertoli cells), and 8-week-old (approximately 1.8 cells with a primary cilium per 10 Sertoli cells) pigs, and the same pattern could be recapitulated in de-novo-formed testicular tissue after xenografting of isolated porcine testicular cells to mice [22]. Furthermore, primary cilia appear to be essential for seminiferous tubule morphogenesis in vitro. The reduction of primary cilia on testicular cells isolated from 1-week-old pig testes via treatment with ciliobrevin D (a selective ATPase motor cytoplasmic dynein inhibitor) impaired the ability of cells to rearrange into three-dimensional tubules. Cilia-ablated testicular cells formed tubules that were significantly shorter and narrower than the ones formed by untreated cells [25].

The Hh signaling pathway is exclusively transduced through primary cilia in vertebrates and is suggested to be involved in testicular morphogenesis [7,8,50]. The inhibition of primary cilia formation on neonatal porcine testicular somatic cells through ciliobrevin-D treatment affected elements of the Hh signaling pathway, as shown by a decrease of protein expression for transcription factor Gli-2 and transmembrane protein Smoothened [25]. Ciliobrevin D, however, does not cause permanent primary ciliary inhibition. Therefore, the genetic disruption of structural and functional cilia components to inhibit cilia assembly have been tested [30]. CRISPR-Cas9-mediated gene editing of ODF2 and siRNA-mediated gene silencing of IFT88 in 1-week-old porcine Sertoli cells resulted in reduction in the number and length of primary cilia [30]. Additionally, cilia-disrupted cells formed more disorganized seminiferous tubules of varying size/shape in vitro [30], similar to effects of ciliobrevin D treatment [25]. However, the disruption of Hh signaling via cyclopamine, a Hedgehog antagonist, alone without directly targeting the primary cilia caused an attenuation in Gli-1 expression but did not affect in vitro seminiferous tubule formation [30]. Hence, primary cilia are of importance for tubule morphogenesis in vitro, but Hh signaling does not seem to be the responsible cilia-mediated pathway [30].

Similar results were observed when in vitro testicular morphogenesis was studied in testicular organoids, which form via self-organization of testicular cells and present a similar cytoarchitecture to testis in vivo [30,51]. Cyclopamine-treated porcine testicular cells were able to form organoids with a distinct basement membrane, yet smaller in size [30], while the ablation of porcine testicular cells’ primary cilia with ciliobrevin D inhibited organoid formation [51]. Testicular organoids that formed after knockdown of ODF2 and IFT88 in porcine cells did not show a well-defined basement membrane and the characteristic testicular cytoarchitecture [30].

Summary and future directions

Primary cilia are present on testicular somatic cells and appear to play a role in tubular morphogenesis in vitro. However, the current knowledge about this unique organelle in the testes remains very limited, especially considering the increasing evidence that relates primary cilia dysfunctions to impaired male reproductive system development and male fertility problems [18,19,52].

A link between Hh signaling and primary cilia in both Leydig and Sertoli cells has been established, and Hh signaling is strongly regulated by primary cilia. The loss of primary cilia is consistently observed in malignancies, including endocrine-related cancers such as breast, ovarian, and prostate cancer, and ciliary disfunction is believed to result in tumorigenesis [53,54]. Similarly, recent studies suggest a paracrine role for the cilia-related Hh signaling during cancer development, although the direct relationship between primary cilia and the Hh pathway affecting tumorigenesis still needs more evidence [53,54]. As primary cilia take part in coordinating several signaling pathways that work together to preserve the cell equilibrium necessary for normal tissue homeostasis, it is imperative to explore other cilia-mediated signaling pathways such as IGF, PDGF, TGFβ-related families, and mTOR signaling in the testes. This will provide a better understanding of the sensory function of primary cilia in response to environmental changes and uncover the mechanisms by which these pathways, including hormone-mediated signaling pathways, mediate ciliogenesis.

The importance of primary cilia in the testes is underscored by the observation that a mutation in IFT88 can have detrimental effects on spermatogonial stem cell (SSC) niche dynamics. SSCs play a critical role in maintaining normal spermatogenesis and sustaining fertility through balancing self-renewal and differentiation, and any dysfunction of SSCs leads to male infertility [55]. IFT88 loss of function established the regulatory role of primary cilia in regulating both mitochondrial biogenesis and lipophagy to produce free fatty acid, and a shift towards glycolytic metabolism was specifically dependent on the loss of primary cilia [56]. These findings indicate a role that primary cilia might play in the metabolic transitions that accompany the development and maturation of SSCs, from oxidative phosphorylation to anaerobic metabolism [57]. Therefore, dissecting the primary cilia-mediated regulatory network between somatic and germ cells is important to better understand spermatogenesis and pathogenesis of male infertility.

Given its complexity, the testis represents a promising model for studying the dynamic role of primary cilia in cell–cell communications during development and spermatogenesis. A better understanding of primary cilia function in the testis and the deleterious effects that malfunctioning primary cilia can have on the male reproductive system will help identify markers for diagnostic or prognostic assessment for infertility and potentially develop strategies for treatment.

Data availability

Since this is a review paper, there are no original data to share.