Bioactive fragments of laminin and collagen chains: lesson from the testis

in Reproduction
Authors:
Huitao LiThe Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University
The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, New York, USA

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Shiwen LiuThe Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University
The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, New York, USA

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Siwen WuThe Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University
The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, New York, USA

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Linxi LiThe Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University
The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, New York, USA

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Renshan GeThe Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University

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C Yan ChengThe Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University
The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, New York, USA

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Correspondence should be addressed to C Y Cheng; Email: y-cheng@popcbr.rockefeller.edu or ccheng@rockefeller.edu

*(H Li and S Liu contributed equally to this work)

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Recent studies have shown that the testis is producing several biologically active peptides, namely the F5- and the NC1-peptides from laminin-γ3 and collagen α3 (IV) chain, respectively, that promotes blood–testis barrier (BTB) remodeling and also elongated spermatid release at spermiation. Also the LG3/4/5 peptide from laminin-α2 chain promotes BTB integrity which is likely being used for the assembly of a ‘new’ BTB behind preleptotene spermatocytes under transport at the immunological barrier. These findings thus provide a new opportunity for investigators to better understand the biology of spermatogenesis. Herein, we briefly summarize the recent findings and provide a critical update. We also present a hypothetical model which could serve as the framework for studies in the years to come.

Abstract

Recent studies have shown that the testis is producing several biologically active peptides, namely the F5- and the NC1-peptides from laminin-γ3 and collagen α3 (IV) chain, respectively, that promotes blood–testis barrier (BTB) remodeling and also elongated spermatid release at spermiation. Also the LG3/4/5 peptide from laminin-α2 chain promotes BTB integrity which is likely being used for the assembly of a ‘new’ BTB behind preleptotene spermatocytes under transport at the immunological barrier. These findings thus provide a new opportunity for investigators to better understand the biology of spermatogenesis. Herein, we briefly summarize the recent findings and provide a critical update. We also present a hypothetical model which could serve as the framework for studies in the years to come.

Introduction

Throughout the epithelial cycle of spermatogenesis which has a duration of about 12.8 (stages I-XIV), 8.6 (stages I-XII) and 16 (stages I-XII) days in the testis of rats, mice and humans respectively (Heller and Clermont 1963, Clermont 1972, de Kretser and Kerr 1988, Hess and de Franca 2008, Muciaccia et al. 2013), dynamic remodeling of cell junction takes place continuously at the Sertoli cell–cell and Sertoli–germ cell interface to support the various events of spermatogonial stem cell self renewal through mitotic proliferation, meiosis, spermiogenesis and the release of sperm at spermiation (Hermo et al. 2010a ,c , O’Donnell et al. 2011, Cheng and Mruk 2015). This is necessary to facilitate timely communications between testicular cells in the seminiferous epithelium to support spermatogenesis. It is noted that the detailed morphological events pertinent to cell junction remodeling have been delineated, and some of the involving ultrastructures and biomolecules that modulate these events have been identified in recent years (Cheng and Mruk 2002, 2010, Hermo et al. 2010b ,c , Vogl et al. 2013, 2014, Berruti and Paiardi 2014). Nonetheless, the precise molecular mechanism that regulates these cellular events locally across the seminiferous epithelium in the testis to support the epithelial cycle remains relatively unexplored. Herein, we seek to summarize some recent findings in this area of research, highlighting the latest advances and to identify specific areas that deserve future investigation. We also seek to understand the advances made in this area of research in light of findings based on studies in other epithelia. This should provide a better framework for functional studies in future investigations.

ES (ectoplasmic specialization) – apical ES and basal ES

The ES is a testis-specific, actin-rich adherens junction, found at the Sertoli cell–cell interface near the basement membrane designated basal ES, which coexists with the actin-based tight junction (TJ) and gap junction (GJ) (Vogl et al. 2008, Wong et al. 2008, Berruti and Paiardi 2014). These actin-based junctions, together with the intermediate filament-based desmosome, in turn, constitute the blood–testis barrier (BTB), which is considered to be one of the tightest blood–tissue barriers in the mammalian body (Setchell 2008, Mital et al. 2011, Pelletier 2011, Cheng and Mruk 2012, Franca et al. 2012, Stanton 2016). The ES is also found at the Sertoli cell–spermatid interface (steps 8–19 or 8–16 in the rat and mouse testis, respectively) called apical ES (Vogl et al. 2008, Wong et al. 2008, Berruti and Paiardi 2014). But unlike the basal ES that coexists with TJ and GJ, once the apical ES appears between Sertoli cells and step 8 spermatids in stage VIII tubules in the rodent testis, apical ES replaces the desmosome and GJ found at the Sertoli–spermatid (steps 1–7) interface, serving as the only anchoring device to adhere developing haploid spermatids onto the Sertoli cell in the seminiferous epithelium until the release of sperm at spermiation (O’Donnell et al. 2011, O’Donnell 2014). Ultrastructurally, the apical and the basal ES look similar wherein an array of actin filaments that aligned as bundles are sandwiched in-between the Sertoli cell endoplasmic reticulum (ER) and the apposing Sertoli–spermatid plasma membranes (i.e. apical ES) vs Sertoli cell–cell plasma membranes (i.e. basal ES) (Fig. 1). Except that in the apical ES, there is only a single array of actin filament bundles vs two arrays of actin filament bundles in the basal ES (Fig. 1). When examined by electron microscopy at the ultrastructural levels, spermatids do not appear to contribute structurally to the apical ES. However, in late stage VII through early VIII of the epithelial cycle, the ES undergoes some structural modifications wherein giant endocytic vesicles are formed at the ES and designated tubulobulbar (TBC) complex, which carries the ‘old’ ES proteins to be recycled to assemble ‘new’ ES at the apical and basal sites (Vogl et al. 2013) (Fig. 1). This thus avoids de novo protein synthesis throughout the epithelial cycle of spermatogenesis due to the limited resources and space in the seminiferous epithelium to support the production of millions of sperm per day from the testis after puberty. However, at the molecular level, the apical ES is considerably different from the basal ES. For instance, elongating/elongated spermatids specifically express nectin 3 (Ozaki-Kuroda et al. 2002, Inagaki et al. 2006), JAM-C (Gliki et al. 2004), VE cadherin (Aivatiadou et al. 2007), and laminin-α3ß3γ3 trimeric ligand (Koch et al. 1999, Yan and Cheng 2006) as adhesion molecules, not found in Sertoli cells, and are crucial to support stability and integrity of spermatid-Sertoli cell adhesion. On the other hand, Sertoli cells express nectin 1, nectin 2, JAM-A, JAM-B, E-cadherin, N-cadherin, and α6ß1-integrin to form the functional adhesion protein complexes with the corresponding adhesion proteins expressed by spermatids at the apical ES (Palombi et al. 1992, Salanova et al. 1995, Mruk and Cheng 2004, Setchell 2008, Berruti and Paiardi 2014). More important, studies have shown that Rap1 (Ras-related protein 1), a small GTPase, serves as a crucial central organizer of cell architecture and junction stability at the apical ES through its effects on F-actin cytosekeleton, and with the participation of the upstream and downstream mediators of Rap1, which include Rho GTPase, Cdc42 GTPase, and cell polarity protein Par (partitioning-defective protein), as well as integral membrane proteins VE cadherin, nectin 3 and JAM-C based on studies in vivo using genetic models (Aivatiadou et al. 2007, 2009, Berruti and Paiardi 2014, Berruti et al. 2018).

Figure 1
Figure 1

A schematic drawing that illustrates the morphological features of the apical ES and basal ES in the seminiferous epithelium of adult rat testes. This schematic drawing illustrates some of the notable features of the cross-section of a stage VII tubule, composed to adjacent Sertoli cells and germ cells at different stages of their development, as discussed in this review. Ectoplasmic specialization (ES) is typified by the presence of an array of actin filament bundles sandwiched between cisternae of endoplasmic reticulum (ER) and the apposing plasma membranes of Sertoli cells and step 8–19 spermatids at the Sertoli-spermatid interface (apical ES), but with two arrays of actin filament bundles at the basal ES. The basal ES, together with the tight junction (TJ) and gap junction, along with the intermediate filament-based desmosome constitute the blood-testis barrier (BTB), which in turn divides the seminiferous epithelium into the basal and adluminal (apical) compartments. Also noted is the progressive transport of germ cells across the seminiferous epithelium from the base to the lumen of the tubule. In stage VII tubule, the concave (ventral) side of spermatid head, apical ES undergoes extensive remodeling, creating an ultrastructure known as the apical TBC (tubulobulbar complex). The apical ES represents numerous endocytic vesicles used to support endocytosis and recycling of apical ES protein (such as adhesion protein complexes of integrin-laminin, necin-afadin, or cadherin-catenin complex), which is facilitated by the extensive branching of the actin filament bundles at the site to destabilize apical ES to facilitate endocytic protein trafficking events. A similar ultrastructure known as basal TBC can also be found at the BTB to support BTB remodeling. The seminiferous epithelium is laid on the basement membrane, constituted mostly by type IV collagen, laminins (mostly laminin-α2 chains), heparan sulfate proteoglycans and entactin, to be followed by the collagen type I layer, and then the peritubular myoid cell layer.

Citation: Reproduction 159, 3; 10.1530/REP-19-0288

Role of F5-peptide in junction dynamics in the testis

In mammalian tissues, laminin chains are usually restrictively expressed at the basal lamina, which is the homogeneous extracellular matrix (ECM) substance deposited at the base of virtually all cell epithelia. In the testis, the basement membrane is a modified form of ECM, which is derived from Sertoli cells and contributed, at least in part, by peritubular myoid cells, appearing as a homogenous layer of ~0.15 µm thick (Dym 1994, Siu and Cheng 2004a , 2008) in the tunica propria (Fig. 1). In rodent testes, the basement membrane is constituted largely by type IV collagen and laminins (e.g., laminin-α2 chain), and also heparin sulfate proteoglycan (Hadley and Dym 1987) and entactin (Lian et al. 1992). Interestingly, laminin-γ3 is one of the first laminin chains shown to be deposited outside the basement membrane in the mouse testis, at the apical ES (Koch et al. 1999). Subsequent studies have shown that laminin-γ3 (Siu and Cheng 2004b ) form a functional trimeric ligand with laminin-α3 and laminin-ß3 at the apical ES (Yan and Cheng 2006). More important, this trimeric laminin-α3ß3γ3 ligand contributed by spermatids (Yan and Cheng 2006) creates a bona fide cell adhesion protein complex with α6ß1-integrin contributed by Sertoli cells (Palombi et al. 1992, Salanova et al. 1995, 1998) at the apical ES (Mulholland et al. 2001, Siu and Cheng 2004b , Yan and Cheng 2006) (Fig. 2). Studies using different genetic models have also demonstrated the physiological significance of laminin chains in the mammalian body (Table 1). Other studies have shown that biologically active fragments are generated from laminin chains via the action of MMPs (matrix metalloproteinases) in the basal lamina of other epithelia which are capable of regulating cell adhesion, cell migration, vascular permeability, cell apoptosis, cell proliferation and cell differentiation (Sato et al. 1994, Tang and Saito 2018a ,b , Yan et al. 2007) (Table 2). Studies in the testis have shown that domain IV released from laminin-γ3 chain, likely via the action of MMP-2 (Siu and Cheng 2004b ), is capable of perturbing Sertoli cell TJ-permeability barrier function based on studies in vitro (Yan et al. 2008). Subsequent studies have mapped the biologically active domain to a stretch of peptide sequence of about 50 amino acid residues in the domain IV of laminin-γ3 chain designated F5-peptide (Su et al. 2012) (Fig. 2). Overexpression of this F5-peptide (following cloning of its cDNA into the mammalian expression vector pCI-neo) in Sertoli cells cultured in vitro or the testis in vivo is able to induce reversible Sertoli cell BTB disruption (Su et al. 2012, Gao et al. 2016). More important, F5-peptide is rapidly taken up by Sertoli cells in the testis in vivo via the drug transporter S1c15a1 (Su et al. 2015), illustrating that generation of the F5-peptide from laminin-γ3 chain via the action of MMP-2 outside the Sertoli cells in the seminiferous epithelium can also be taken up by Sertoli cells through the drug transport S1c15a1, which, in turn, modulates Sertoli cell permeability barrier function. Furthermore, studies have shown that the F5-peptide exerts its disruptive effects in Sertoli cells by modifying F-actin organization across the cell cytosol, but also capable of perturbing F-actin organization across the seminiferous epithelium through disruptive changes in the spatiotemporal expression of p-FAK-Y407 (Su et al. 2012), which was earlier shown to be a crucial regulator of BTB integrity (Lie et al. 2012). Thus, it is not entirely unexpected that overexpression of F5-peptide in the testis also induces germ cell exfoliation, in particular elongating/elongated spermatids, in the testis (Su et al. 2012, Gao et al. 2016) since the apical ES utilizes F-actin for attachment of its adhesion protein complexes, and it is considered to be an atypical adherens junction (Mulholland et al. 2001, Vogl et al. 2008, Wong et al. 2008). In this context, it is of interest to note that p-FAK-Y407 (Lie et al. 2012, Gao et al. 2016) and its closely related activated form p-FAK-Y397 (Siu et al. 2003b , Wan et al. 2013) are known regulators of basal ES/BTB and also apical ES function, but they have differential function on cell adhesion in the testis. For instance, p-FAK-Y407 confers both basal ES/BTB function and apical ES integrity, whereas p-FAK-Y397 confers apical ES integrity but it induces basal ES/BTB disruption (Lie et al. 2012). This conclusion was reached based on an in vitro study using primary cultures of Sertoli cells for the overexpression of their full-length cDNAs (i.e., wild-type (WT)) vs different constitutively active (i.e., phosphomimetic mutants) and constitutively inactive (i.e., non-phosphorylable mutants) (Lie et al. 2012). For instance, overexpression of p-FAK-Y397E mutant (a phosphomimetic and constitutively active mutant of p-FAK-Y397) in adult rat testes in vivo promotes apical ES function by delaying spermiation since F-actin remained robustly expressed at the apical ES to support elongated spermatid adhesion (Wan et al. 2013). On the other hand, overexpression of p-FAK-Y407E mutant (a phosphomimetic and constitutively active mutant of p-FAK-Y407) in rat or primary human Sertoli cells promotes the Sertoli cell TJ-permeability barrier, capable of blocking the PFOS (perfluorooctanesulfonate, an environmental toxicant known to mediate TJ-barrier disruption (Wan et al. 2014))-induced TJ-barrier disruption (Wan et al. 2014, Chen et al. 2017a ). Collectively, these findings illustrate that the testis is producing the regulatory F5-peptide locally at the apical ES site by potentiating the degeneration of apical ES to facilitate sperm release at spermiation (Gao et al. 2016). This cellular event is mediated through the signaling protein p-FAK-Y407 (Su et al. 2012, Gao et al. 2016), probably also p-FAK-Y397, downstream. Additionally, the F5-peptide also acts as an autocrine factor to affect other functions, such as by inducing BTB remodeling at the basal ES near the basement membrane. In this context, it is of interest to note that based on this physiological activity of the F5-peptide wherein it can effectively modify the transport function of the Sertoli cell BTB as noted in earlier studies (Su et al. 2012, Gao et al. 2016), the F5-peptide can serve as an adjuvant to modify the BTB permeability/transport function, thereby improving bioavailability of a drug considerably if co-administering into male rodents. In fact, a recent report has confirmed this possibility since it was shown that co-administration of F5-peptide through its overexpression with a drug (e.g. the non-hormonal male contraceptive adjudin, earlier shown to have poor bioavailability (Cheng et al. 2005)) considerably improved the efficacy of adjudin to induce reversible male contraception in adult rats (Chen et al. 2019). In brief, future investigations are warranted to examine if the F5-peptide can modify other blood–tissue barriers (e.g. the blood–brain barrier, BBB) so that it can facilitate the transport of therapeutic drugs across the BBB to treat illnesses, such as glioma, in the brain.

Figure 2
Figure 2

A schematic drawing that illustrates the structural features of a functional laminin ligand constituted by three laminin chains, one each of α, ß and γ chains, and the functional domains of the laminin-γ3 chain. Studies have shown that the functional laminin-333 composed of laminin-α3, -ß3, and -γ3 chains is a putative cell adhesion protein expressed by elongated spermatids that form a bona fide adhesion complex with α6ß1-integrin expressed by Sertoli cells. However, domain IV of laminin-γ3 chain is now known to generate the F5-peptide endogenously possibly through the action of MMP-2, which is capable of inducing BTB remodeling but also potentiating the breakdown of apical ES at late stage VIII tubule to coordinate the cellular events of spermiation and BTB remodeling at stage VIII of the cycle (see text for details).

Citation: Reproduction 159, 3; 10.1530/REP-19-0288

Table 1

Selected mouse models of deletion of laminin chains and the associated phenotypes.

Laminin chain Mouse model Approach Animal/cell types Phenotypes References
α2 Laminin-α2 chain deficient dy3K/dy3K mice Laminin-α2 chain deficiency Mouse Male infertility due to defects in basement membrane because of laminin-α2 deficiency which also reduces laminin-γ3 chain Häger et al. 2005
α3 Inactivation of the Lama3 gene in basal keratinocytes of adult mice Targeted disruption of the laminin-α3 gene Mouse Skin inflammation and fibrosis Pesch et al. 2017
α4 Laminin-α4 null (Lama4−/−) mice Mice lacking the presynaptic organizer laminin-α4 gene Mouse Disruption of nerve endings development Samuel et al. 2012
β2 Insertion of a neo cassette in exon 3 (the second coding exon) of the laminin-β2 gene Disruption of laminin-β2 gene Mouse Alterations in morphology and function of the CNS Libby et al. 1999
β2 and γ3 Whole body gene knockout Ablation of laminin-β2 and laminin-γ3 genes Mouse Defects in brain development Radner et al. 2013
γ1 (laminin–511) Laminin-γ1 KO mice Specific deletion of laminin-γ1 gene in keratinocytes Mouse primary melanocytes Defects in melanocyte migration and differentiation Ustun et al. 2019
γ1 (laminin-211) Mice lacking laminin-γ1 expression in keratinocytes Specific deletion of laminin-γ1 in epidermis Mouse Defects in hair morphogenesis Fleger-Weckmann et al. 2016
Table 2

Biologically active fragments of laminin chains in selected study models.

Laminin ligand or chain Protease partner Cleavage/regulation Animal/cell types Biological effects References
α2 MMP-9, MMP-10, MMP-12, MMPs Induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction Glomerular podocytes Laminin-α2 chain fragments activate focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo Toss et al. 2019
α5 (LAMA5) MMP1 Proteolytic cleavage of laminin MDCK, 21D1, and 21D1−MMP1 cells Promotes angiogenesis of endothelial cells Gopal et al. 2016
β1 (Laminin-111) MMP2 MMP2 Cleaves the β1-Chain of Laminin-111 Embryonic stem cells Regulates cell adhesion and migration and interacts with mouse ESCs (embryonic stem cells) via α3β1-integrin receptors Horejs et al. 2014
γ2 (laminin-332) MMP-9 Proteolytic cleavage of laminin Stromal cells Modulates cell attachment, migration, differentiation and proliferation Silva et al. 2018
laminin-111 MMP-2, MMP-9 Proteolytic cleavage of laminin Malignant cells Facilitates metastatic spread by allowing tumor cells to penetrate tissues Kikkawa et al. 2013
laminin-111,-211, -411, and -511 Neutrophil elastase (NE) and matrix metalloproteinase 9 (MMP9) Proteolytic cleavage of laminin Dormant cancer cells Laminin remodeling to awaken cancer cells Albrengues et al. 2018
laminin-332 (laminin-5) MMP-2, MMP-14, bone morphogenetic protein-1 (BMP-1), and mammalian Tolloid (mTLD) Proteolytic cleavage of laminin γ2 TAM (tumor-associated macrophages)-like cells Promotes monocyte differentiation Kamoshida et al. 2014
Laminin MMP11 Proteolytic cleavage of laminin Zebrafish Regulates fibronectin levels Jenkins et al. 2016

Role of NC1-peptide in junction dynamics in the testis

In the rodent testis, besides laminins, heparan sulfate proteoglycans and entactin, type IV collagen is the major structural component of the basement membrane (Dym 1994, Lin 2004, Siu and Cheng 2004a , 2008). Type IV collagen is a triple helical structure consisting of three collagen α chains of α1, α2, α3, α4, α5, or α6, either a homotrimeric or a heterotrimer structure (Fig. 3), wherein collagen α3 (IV) is the predominant chain. Each collagen α3 (IV) chain consists of a short 7S domain (with a short signal peptide at its N-terminal end) of ~15 amino acids at its N-terminus, to be followed by a long collagenous domain of ~1400 residues of Gly-Xaa-Yaa repeats, and a C-terminal NC1 (non-collagenous 1) domain of ~230 residues (Timpl et al. 1981, Hudson et al. 1993) (Fig. 3). Collagens are scaffolding proteins known to provide structural support to epithelial and endothelial cells, such as Sertoli cells in the seminiferous epithelium. The significance of collagen chains to maintain cell and tissue integrity has been demonstrated in studies using genetic models (Table 3). Studies have shown TNFα released from Sertoli cells can trigger activation of MMP-9 to release the NC1 domain and other peptides through limited proteolysis (Siu et al. 2003a ), which are physiologically active peptides (Ortega and Werb 2002). For instance, NC1 domain of various collagen chains, called tumstatin, endostatin, or constatin, was shown to serve as ligand to regulate morphogenesis, cell adhesion, cell migration, proliferation, angiogenesis, and apoptosis in different cell types via its interactions with cell-surface receptors, such as integrins (Ackley et al. 2001, Ortega and Werb 2002, Hamano and Kalluri 2005, Assadian and Teodoro 2008, Sudhakar and Boosani 2008, Rebustini et al. 2009, Barczyk et al. 2010). These findings thus prompted us to investigate if NC1-peptide released in the basement membrane of the testis would have similar effects to the testis function. Indeed, when NC1-peptide from collagen α3 (IV) in the testis was cloned into pCI-neo for its overexpression, or through the use of expression vectors (such as pET30 Ek/LIC expression vector or pTracer-CMV2 expression vector) to obtain its recombinant protein from E. coli or human embryonic kidney cell line Lenti-X 293 cells, respectively, for studies in vitro or in vivo. Overexpression of NC1-peptide or the use of NC1 recombinant peptide was shown to perturb Sertoli cell TJ-permeability barrier function in vitro and in vivo (Wong and Cheng 2013, Chen et al. 2017b ). More importantly, NC1-peptide exerts its effects to induce BTB remodeling and apical ES degeneration to promote germ cell exfoliation through changes in the organization of actin and MT cytoskeletons (Chen et al. 2017b ). In fact, this unusual physiological activity of the NC1-peptide by perturbing the Sertoli BTB integrity in vivo was also found to perturb spermatogenic function, illustrating its potential use as a male contraceptive. However, much study is needed to unravel the underlying molecular mechanism by which the NC1-peptide perturbs the actin- and MT-based cytoskeletons to perturb basal ES/BTB and apical ES function, in particular the downstream signaling protein(s) and pathway(s). This information is crucial to understand its physiological role to support spermatogenesis. Nonetheless, our findings regarding the biological function of NC1 peptide to modulate spermatogenic function are consistent with reports in the literature (Table 4). These other studies have illustrated biologically active fragments are also produced from collagen chains in tissues which also serve as regulatory peptides to modulate multiple cellular functions (Table 4).

Figure 3
Figure 3

A schematic drawing that illustrates the structural features of a functional collagen (IV) monomer, comprising three collagen chains. Studies have shown that a functional collagen (IV) monomer is a trimeric structure, comprising either α1, α2, α3, α4, α5 or α6, which can be a homotrimeric or a heterotrimeric structure. Each collagen α chain, such as collagen α3 (IV) has an N-terminal non-collagenous 7S domain of ~15 amino acid residues behind the signal peptide, a middle collagenous domain of ~1,400 residues of GXY repeats, and a C-terminal non-collagenous (NC1) domain of ~230 amino acid residues, which is also the domain that was shown to be biologically active in the testis and also in other epithelia (see text for details).

Citation: Reproduction 159, 3; 10.1530/REP-19-0288

Table 3

Selected mouse models of deletion of collagen chains and the associated phenotypes.

Collagen chain Mouse model Approach Phenotypes References
Collagen α3 (IV) Mice with a targeted deletion encoding the NC1 domain of the Col4α3 gene Deletion of the Col4α3 gene Renal failure due to lack of collagen α3α4α5 (IV) networks but retention of collagen α1α2α1 networks in kidney glomerular basement membrane Steenhard et al. 2012
Collagen α2 (V) Mice with Col5α2 knockout Tamoxifen-induced whole body Col5a2 gene KO Skin and adipose abnormalities such as skin thinking and marked loss of dermal white adipose tissue (WAT) and abdominal WAT, and vascular defects including aortic aneurysm and dissection Park et al. 2017
Collagen α1 (V) Targeted tendon and ligament Col5α1-null mouse model Targeted deletion of collagen α1 (V) in tendons and ligaments Reduced body size, grip weakness, and early onset of osteoarthritis; abnormal joint phenotypes with reduced collagen fibrils in joint stabilizing ligaments and tendons Sun et al. 2015
Collagen α1 (VI) Col6α1 null mice Mouse whole body Col6a1 gene KO Reduced tensile strength of the skin, and reduced biomechanical strength and stiffness of tendons due to abnormal collagen I fibrils. Collagen VI deficiency led to disruptive changes in matrix architecture and biomechanical properties Lettmann et al. 2014
Collagen α1 (XXV) also known as CLAC-P/collagen XXV Col25α1-deficient (KO) mice Mouse whole body Col25a1 gene KO Col25α1 mice died immediately after birth due to respiratory failure. Motor axons projected to target muscles but failed to elongate and branch within the muscle, to be followed by axon degeneration Tanaka et al. 2014
Table 4

Biological activities of collagen chains and their fragments in selected study models.

Collagen ligand/chain (or fragments) Animal/cell types Biological effects References
Collagen α2 (I) (COL1A2) Chondrosarcoma and fibrosarcoma cells Mediates the pro- and anti-migratory effects of TBX3 (T-box transcription factor 3) in cells Omar et al. 2019
Collagen VI fragments Epithelial and endothelial cells Maintains epithelial andendothelial cell polarity Willumsen et al. 2019
T3 peptide from collagen α3 (IV) Rats Exerts cardio-protective effects against ischemia/reperfusion injury in heart Yasuda et al. 2019
T3 peptide from collagen α3 (IV) Cardiac fibroblasts Stimulates proliferation and migration of cardiac fibroblasts Yasuda et al. 2017
Collagen α1 (XI) chain (COL11A1) Tumor and stromal cells Overexpression of COL11A1 induces malignancies and aggressive tumor cell behavior Toss et al. 2019
Endostatin (C-terminal 20 kDa fragment of collagen XVIII) Humans Inhibitors of angiogenesis, lymphangiogenesis, and cancer metastasis Walia et al. 2015

Role of LG3/4/5-peptide in junction dynamics in the testis

Studies have shown that besides type IV collagen, laminin α1, α2, α4, β1, β2, and γ1 chains are constituent components of the basement membrane, surrounding the base of seminiferous tubules in rodent testes (Koch et al. 1999, Häger et al. 2005). Interestingly, few reports are found in the literature that examine the physiological role of these laminin chains residing in the basement membrane in supporting spermatogenic function except for their structural roles in the testis (Virtanen et al. 1997, Aydos et al. 1998). In an earlier report using a genetic model wherein transgenic mice deficient in laminin α2 chain (also known as merosin) were found to be infertile, and their seminiferous tubules were devoid of germ cells due to apical ES disruption, which, in turn, led to germ cell exfoliation (Häger et al. 2005). Also, a disruption of laminin function in the basement membrane by passive immunization of anti-laminin IgG to guinea pigs was found to induce spermatogenesis arrest (Lustig et al. 2000). Collectively, these reports illustrate the biological significance of basement membrane laminin chains in supporting spermatogenic function. Furthermore, there are reports in the literature illustrating that biologically active fragments are generated from laminin chains via the action of MMPs which can modulate multiple cellular functions including cell adhesion, vascular permeability, cell apoptosis, cell proliferation and cell differentiation in multiple epithelia, tissues and organs other than the testis (Sato et al. 1994, Yan et al. 2007, Tang and Saito 2018a ,b ) (Table 2). We thus sought to examine if laminin α2 chain in the basement membrane would have similar biological effects on Sertoli cell BTB and spermatogenic function in the testis. As noted in Fig. 4, laminin α2 chain consists of an laminin N-terminal domain (LN) to be followed by two laminin 4a domain (L4a) and laminin 4b domain (L4b) interspaced with the laminin EGF-like domain a (LEa), LEb, and LEc an a long laminin coiled-coil (LCC) domain and five short C-terminal laminin globular (LG) domains at the C-terminus. Interestingly, using an in-house prepared antibody specific to the 80 kDa fragment (Wong and Cheng 2013) and two additional commercially available anti-laminin antibodies, we have shown that an 80 kDa fragment of laminin α2 chain containing the LG domains generated at the basement membrane, likely through proteolytical cleavage of MMP9, is detected at the apical ES (Gao et al. 2017b ). A study using taxol (also known as paclitaxel, an anti-mitotic drug, which binds to ß-tubulin by inhibiting MT growth through hyper-stabilization of MTs, depleting the plasticity of MTs to support changes in cell shape and function to confer cellular homeostasis (Press et al. 2019)) has shown that this 80 kDa fragment from the C-terminal region of laminin-α2 chain (i.e., LG3/4/5-fragment) is being transported from the basement membrane to the apical ES site through an MT-dependent transport mechanism (Gao et al. 2017b ). This observation is important because it supports the notion that a biologically active fragment can likely be generated from laminin-α2 chain in the basement membrane, which is then transported to the apical ES site to exert its regulating effects. Interestingly using a siRNA specific to laminin-α2 chain cloned into pGene-Clip hMGFP vector for laminin-α2 knockdown by transfecting Sertoli cell epithelium cultured in vitro with this clone, it was found to perturb the Sertoli cell TJ-permeability function by disrupting the organization of actin filaments and MTs across the cell cytosol. These findings thus support the notion that laminin-α2 chain is being used to promote Sertoli cell TJ-barrier function in the testis in vivo under physiological conditions. Interestingly, in a follow-up study, it was shown that a silencing of laminin-α2 by RNAi to perturb Sertoli cell TJ function by disrupting distribution of TJ- and basal ES proteins at the cell–cell interface involved an activation of mTORC1/rpS6 (Gao et al. 2017a ). For instance, an upregulation on the protein steady-state levels of p-rpS6-S235/S236 and p-rpS6-S240/S244 (the activated phosphorylated form of rpS6, which is also the downstream signaling protein of mTORC1 and a phosphorylatable protein translation regulator (Laplante and Sabatini 2012, Meyuhas 2015)) and a concomitant downregulation of p-Akt1-S473 and p-Akt2-S474, were detected (Gao et al. 2017a ). These findings thus indicate that the laminin-α2 chain, most likely through the LG3/4/5-peptide, promotes BTB and spermatogenic function through the mTORC1/rpS6 and Akt1/2 signaling pathway downstream. In this context, it is of interest to note that mTORC1/rpS6 and Akt1/2 signaling pathway has been shown to be involved in regulating BTB and spermatogenic function in the testis (Mok et al. 2012). For instance, an activation of mTORC1/rpS6, such as through the use of a quadruple phosphomimetic (i.e. constitutively active) mutant of rpS6 namely p-rpS6-mutant (i.e, p-rpS6-S235E/S236E and p-rpS6-S240E/S244E quadruple phosphomimetic mutant) was found to induce Sertoli BTB disruption in vitro (Mok et al. 2014, 2015) and in vivo (Li et al. 2018). Nonetheless, the precise molecular mechanism, in particular, the involvement of the mTORC1/rpS6/Akt1/2 signaling pathway in LG3/4/5-peptide-mediated BTB and spermatogenic function, requires investigation in future studies.

Figure 4
Figure 4

A schematic drawing that illustrates the structural features of laminin-α2 chain and the functional laminin-α2-based ligand. The left panel shows the structural features of the laminin-α2-based laminin ligand found in the basement membrane of rat testes. A functional laminin ligand is also a trimeric structure, composed of three chains, one chain each of the α (from α1 to α5), ß (from ß1 to ß3) and γ (from γ1 to γ3) chains. The right panel is the detailed structural domain of laminin-α2 chain. From the N-terminus, three distinctive globular domains of (i) laminin N-terminal domain (LN), (ii) L4a (laminin 4a domain) and (iii) L4b (laminin 4b domain) are noted. There are also three rod domains of epidermal growth factor (EGF): (i) LEa (laminin EGF-like domain a), (ii) LEb (laminin EGF-like domain b) and LEc (laminin EGF-like domain c). The long arm of laminin-α2 is comprised of laminin coiled-coil (LCC) domain and 5 C-terminal laminin globular (LG) domains of LG1, LG2, LG3, LG4 and LG5. There is a putative proteolytic cleavage site close to the N-terminus of LG3, and its cleavage by MMP-9 thus generate the LG3/4/5, corresponding to the 80 kDa laminin-α2 fragment that has the biological activity of promoting BTB integrity and possibly spermatogenic function in the testis (see text for details).

Citation: Reproduction 159, 3; 10.1530/REP-19-0288

A hypothetical model by which the endogenously bioactive peptides support spermatogenesis

Based on findings summarized above, it is notable that F5- and NC1-peptides induce BTB remodeling at the basal ES to support the transport of preleptotene spermatocytes across the immunological barrier (Fig. 5). At the same time, these two peptides also support apical ES degeneration to facilitate sperm release at spermiation (Fig. 5). Thus, the biological activity of these two peptide are crucial to coordinate these two cellular events that take place at the opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle, namely (i) BTB remodeling near the base of the epithelium to support preleptotene spermatocytes at the basal compartment to be transported across the BTB to enter the adluminal compartment to prepare for meiosis and (ii) spermiation (Fig. 5). On the other hand, the LG3/4/5-peptide is crucial to promote basal ES integrity and BTB assembly such as the ‘new’ BTB that required to be assembled behind the preleptotene spermatocytes under transport as noted in Fig. 5. Furthermore, LG3/4/5-peptide also promotes apical ES assembly when step 8 spermatids appear in stage VIII tubules (Fig. 5). In short, it is through the concerted effects of F5- and NC1-peptides, and their contrasting effects with the LG3/4/5-peptide, different cellular events that take place simultaneously across the seminiferous epithelium can be coordinated throughout the epithelial cycle of spermatogenesis (Fig. 5). It is obvious that the hypothetical model depicted in Fig. 5 will be updated in the years to come when more information is available, but this model provides a framework upon which experiments can be designed to examine the role of these regulating peptides in the testis to support spermatogenesis.

Figure 5
Figure 5

A hypothetical model that illustrates the three endogenously produced biologically active peptides that modulate junction remodeling events to support spermatogenesis during the epithelial cycle across the seminiferous epithelium. On the left panel, it is a schematic drawing that illustrates a stage VII tubule, whereas the right panel illustrates a stage VIII tubule. The production of (i) the F5-peptide from laminin-γ3 chains, and (ii) the NC1-peptide from collagen α3 (IV) chains promote BTB remodeling and apical ES degeneration to facilitate the transport of preleptotene spermatocytes across the immunological barrier and the release of sperm at spermation, respectively. The production of (iii) the LG3/4/5-peptide from laminin-α2 chains promotes BTB integrity by facilitating the assembly of the ‘new’ BTB that forms behind the preleptene spermatocytes while transforming into leptotene spermatocytes. Thus, the BTB remains ‘intact’ throughout the epithelial cycle even when preleptotene spermatocytes are being transported across the immunological barrier (see text for details).

Citation: Reproduction 159, 3; 10.1530/REP-19-0288

Concluding remarks and future perspectives

As briefly reviewed herein, it is increasingly clear that the constituent components at the ES and the BM including both laminin and collagen chains are more than just proteins that provide structural support to the ES and BM. Besides their scaffolding role, locally expressed MMPs, such as MMP2 and MMP9 at the apical ES and BM, can induce proteolytic cleavage to generate different biologically fragments to modulate cellular function locally, such as changes in junction permeability, cytoskeletal organization, and even distribution of regulatory proteins of the actin- and/or MT-based cytoskeletons at different stages of the epithelial cycle. This thus provides an efficient local mechanism to modulate different aspects of spermatogenic function in response to changes in the epithelial cycle of spermatogenesis as summarized in the hypothetic model depicted in Fig. 5. It is anticipated that this model will be updated in the years to come. Nonetheless, this model provides a working framework for investigators in the field. In this context, it is of interest to note that since Rap1 GTPase, based on studies using genetic models, has shown to play a central role in regulating spermatid adhesion and maintaining apical ES integrity in the seminiferous epithelium through the epithelial cycle in vivo via its effects on cell adhesion complexes at the apical ES, such as VE-cadherin, nectin 3 and JAM-C (Aivatiadou et al. 2007, 2009, Berruti and Paiardi 2014, Berruti et al. 2018), it is likely that Rap1 is working in concert with these bioactive peptides. This possibility should be carefully evaluated in future investigation since both Rap1 and the three bioactive peptides exert their regulatory effects through changes in the organization of F-actin and/or MT-based cytoskeletons.

Declaration of interest

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

Funding

This work was supported in part by grants from the National Institutes of Health (NICHD, R01 HD056034 to C Y C); National Natural Science Foundation of China (Grants 81971367 to L L, 81730042 to R G); and Department of Science and Technology of Zhejiang Province (Grant 2019C03035 to R G).

Author contribution statement

C Y C conceived the project and wrote the paper. H L, S L, S W, and L L researched on the topics and searched for relevant information in the literature at www.PubMed.com which were discussed in this review. H L and C Y C prepared the Tables. H L and C Y C prepared the figures. H L, S L, S W, L L and R G discussed the concepts evaluated in this review. All authors read and approved the final manuscript.

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    Figure 1

    A schematic drawing that illustrates the morphological features of the apical ES and basal ES in the seminiferous epithelium of adult rat testes. This schematic drawing illustrates some of the notable features of the cross-section of a stage VII tubule, composed to adjacent Sertoli cells and germ cells at different stages of their development, as discussed in this review. Ectoplasmic specialization (ES) is typified by the presence of an array of actin filament bundles sandwiched between cisternae of endoplasmic reticulum (ER) and the apposing plasma membranes of Sertoli cells and step 8–19 spermatids at the Sertoli-spermatid interface (apical ES), but with two arrays of actin filament bundles at the basal ES. The basal ES, together with the tight junction (TJ) and gap junction, along with the intermediate filament-based desmosome constitute the blood-testis barrier (BTB), which in turn divides the seminiferous epithelium into the basal and adluminal (apical) compartments. Also noted is the progressive transport of germ cells across the seminiferous epithelium from the base to the lumen of the tubule. In stage VII tubule, the concave (ventral) side of spermatid head, apical ES undergoes extensive remodeling, creating an ultrastructure known as the apical TBC (tubulobulbar complex). The apical ES represents numerous endocytic vesicles used to support endocytosis and recycling of apical ES protein (such as adhesion protein complexes of integrin-laminin, necin-afadin, or cadherin-catenin complex), which is facilitated by the extensive branching of the actin filament bundles at the site to destabilize apical ES to facilitate endocytic protein trafficking events. A similar ultrastructure known as basal TBC can also be found at the BTB to support BTB remodeling. The seminiferous epithelium is laid on the basement membrane, constituted mostly by type IV collagen, laminins (mostly laminin-α2 chains), heparan sulfate proteoglycans and entactin, to be followed by the collagen type I layer, and then the peritubular myoid cell layer.

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    Figure 2

    A schematic drawing that illustrates the structural features of a functional laminin ligand constituted by three laminin chains, one each of α, ß and γ chains, and the functional domains of the laminin-γ3 chain. Studies have shown that the functional laminin-333 composed of laminin-α3, -ß3, and -γ3 chains is a putative cell adhesion protein expressed by elongated spermatids that form a bona fide adhesion complex with α6ß1-integrin expressed by Sertoli cells. However, domain IV of laminin-γ3 chain is now known to generate the F5-peptide endogenously possibly through the action of MMP-2, which is capable of inducing BTB remodeling but also potentiating the breakdown of apical ES at late stage VIII tubule to coordinate the cellular events of spermiation and BTB remodeling at stage VIII of the cycle (see text for details).

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    Figure 3

    A schematic drawing that illustrates the structural features of a functional collagen (IV) monomer, comprising three collagen chains. Studies have shown that a functional collagen (IV) monomer is a trimeric structure, comprising either α1, α2, α3, α4, α5 or α6, which can be a homotrimeric or a heterotrimeric structure. Each collagen α chain, such as collagen α3 (IV) has an N-terminal non-collagenous 7S domain of ~15 amino acid residues behind the signal peptide, a middle collagenous domain of ~1,400 residues of GXY repeats, and a C-terminal non-collagenous (NC1) domain of ~230 amino acid residues, which is also the domain that was shown to be biologically active in the testis and also in other epithelia (see text for details).

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    Figure 4

    A schematic drawing that illustrates the structural features of laminin-α2 chain and the functional laminin-α2-based ligand. The left panel shows the structural features of the laminin-α2-based laminin ligand found in the basement membrane of rat testes. A functional laminin ligand is also a trimeric structure, composed of three chains, one chain each of the α (from α1 to α5), ß (from ß1 to ß3) and γ (from γ1 to γ3) chains. The right panel is the detailed structural domain of laminin-α2 chain. From the N-terminus, three distinctive globular domains of (i) laminin N-terminal domain (LN), (ii) L4a (laminin 4a domain) and (iii) L4b (laminin 4b domain) are noted. There are also three rod domains of epidermal growth factor (EGF): (i) LEa (laminin EGF-like domain a), (ii) LEb (laminin EGF-like domain b) and LEc (laminin EGF-like domain c). The long arm of laminin-α2 is comprised of laminin coiled-coil (LCC) domain and 5 C-terminal laminin globular (LG) domains of LG1, LG2, LG3, LG4 and LG5. There is a putative proteolytic cleavage site close to the N-terminus of LG3, and its cleavage by MMP-9 thus generate the LG3/4/5, corresponding to the 80 kDa laminin-α2 fragment that has the biological activity of promoting BTB integrity and possibly spermatogenic function in the testis (see text for details).

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    Figure 5

    A hypothetical model that illustrates the three endogenously produced biologically active peptides that modulate junction remodeling events to support spermatogenesis during the epithelial cycle across the seminiferous epithelium. On the left panel, it is a schematic drawing that illustrates a stage VII tubule, whereas the right panel illustrates a stage VIII tubule. The production of (i) the F5-peptide from laminin-γ3 chains, and (ii) the NC1-peptide from collagen α3 (IV) chains promote BTB remodeling and apical ES degeneration to facilitate the transport of preleptotene spermatocytes across the immunological barrier and the release of sperm at spermation, respectively. The production of (iii) the LG3/4/5-peptide from laminin-α2 chains promotes BTB integrity by facilitating the assembly of the ‘new’ BTB that forms behind the preleptene spermatocytes while transforming into leptotene spermatocytes. Thus, the BTB remains ‘intact’ throughout the epithelial cycle even when preleptotene spermatocytes are being transported across the immunological barrier (see text for details).

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