Proteases and Their Cognate Inhibitors of the Serine and Metalloprotease Subclasses, in Testicular Physiology

The testis is a highly dynamic organ not only in the fetal stage but also during postnatal development and in adult life. It is composed of two major compartments: the interstitium with the steroidogenic Leydig cells, and the seminiferous tubules. The seminiferous tubules are surrounded by peritubular cells. Tubules are composed of Sertoli cells and germ cells at different developmental stages. Sertoli cells play key roles in spermatogenesis. They are target cells for follicle stimulating hormone (FSH) and testosterone, responsible for the initiation and maintenance of spermatogenesis. They form the tubules and provide structural and nutritional support for the developing germ cells.^1-4

The gonads emerge as an outgrowth and will develop either as a testis or an ovary, depending on the presence of the Sry gene located on the Y chromosome.^5-6 In response to Sry, Sertoli cells differentiate. They synthesize the Müllerian Inhibiting substance, and they aggregate to form the cords together with peritubular cells originating from the mesonephros. Subsequently, Leydig cells differentiate in the interstitial milieu and start producing testosterone.^7-10 At puberty, dynamic changes are associated with the transformation of the cords into tubules and the initiation of spermatogenesis. In adult life, germ cells migrate from the base to the apex of the tubule epithelium while differentiating further. Finally spermatids are released from the apex of the seminiferous epithelium into the tubular lumen, becoming spermatozoa. A new wave of spermatogenesis will initiate again.

Previous reports suggested that proteases or their inhibitors of the serine-, cysteine-, or metallo-protease family were involved in this spatiotemporal and highly orchestrated process, either during testis development^11-13 or at specific stages of spermatogenesis.^14-17 This chapter summarizes current knowledge about the occurrence and expression pattern of members of the metallo- and of the serine- family of proteases and inhibitors synthesized within the testis. We also report the various predicted functions for these molecules in the establishment and/or maintenance of the testicular architecture and in the process of spermatogenesis.

General Aspects of Proteases and Proteases Inhibitors

A number of important processes that regulate the activity and fate of many proteins are strictly dependent on proteolytic events. For example, proteases are involved in the ectodomain shedding of cell surface proteins, the activation or inactivation of cytokines, hormones and growth factors, the exposure of cryptic neoproteins exhibiting functional roles distinct from the parent molecule, degradation of multiple extracellular matrix components facilitating cell migration and invasion. Accordingly, proteases are fundamental in nearly all complex processes of tissue maintenance, repair, growth and development, and alterations in the structure and expression patterns of proteases underlie many pathological processes including cancer, arthritis, osteoporosis, neurodegenerative disorders and cardiovascular diseases. The completion of the human genome sequence has allowed the determination of more than 2% of all human genes are proteases or protease inhibitors, reflecting the importance of proteolysis in human biology.^18,19 The activity of proteases is regulated at multiple levels including the level of production, the activation of the protease which is generally synthesized in an inactive pro-form, and the production of specific inhibitors.

Proteases catalyse the hydrolysis of peptide bonds in proteins. They are of two types, the exopeptidases and the endopeptidases. The exopeptidases attack only peptide bonds localized at/or near the amino or carboxy terminal portion of peptide chains. The endopeptidases, also named the proteinases, catalyse the hydrolysis of internal bonds in polypeptides. They are divided into 5 classes. Aspartic and metzincins proteases use an activated water molecule as a nucleophile to attack the peptide bond of the substrate. In the cysteine, serine and threonine classes the nucleophile is a catalytic amino-acid residue (Cys, Ser or Thr, respectively) that is located in the active site from which the class name derives. Analysis of the full repertoire of proteases present in the human, mouse and rat genome indicated that serine, metzincins and cysteine proteases are the most abundant proteolytic enzymes in rat, mouse and human (Table 1).²⁰

Table 1

Disribution of proteases in human, mouse and rat genomes.

The Metzincins

Members of the metzincin superfamily are metalloproteinases that require zinc at their catalytic sites. Metzincins are distinguished by a conserved structural topology, a consensus motif containing three histidines that bind zinc at the catalytic site, and a conserved “Met-turn” motif that sits below the proteinase active site zinc ion. The metzincins can be further subdivided into four distinct families, two of which i.e., the Matrixins or Matrix Metalloproteinases (MMPs) and the adamalysin-related proteinases are abundantly expressed in the testis. The actions of these proteinases are inhibited by the Tissue Inhibitors of Metalloproteinases (TIMPs).

Matrix Metalloproteinases (MMPs)

The MMPs are a family of extracellular matrix (ECM) degrading enzymes that share common functional domains and activation mechanism. These are Ca²⁺ and Zn²⁺-dependent endopeptidases that are active at neutral pH. They are synthesized as secreted or transmembrane proenzymes and processed to an active form by the removal of an amino-terminal propeptide. MMPs can be activated by chaotropic agents or by cleavage of the propeptide by members of the MMP family or other proteases such as the plasminogen activator of the urokinase-type. To date, more than 30 members of the MMP family have been identified. There are several distinct subgroups based on preferential substrates or similar structural domains: collagenases that are active against fibrillar collagen, gelatinases that have high activity against denatured collagens, stromelysins that degrade noncollagen components of the ECM, membrane-type MMPs (MT-MMPs) that are transmembrane molecules and other less characterized members. Much of the functions attributed to MMPs to date are the results of the cleavage products of ECM components. These include the release of bioactive ECM fragments which may alter the ECM microenvironment, changing the cell from an adhesive to a migratory phenotype. MMPs may also activate chemokines, cytokines and growth factors synthesized as inactive pro-forms, inactivate the SERPINs (SERine Protease INhibitors) and generate soluble forms of a transmembrane receptor through shedding of the ectodomain.^18-21 MMPs are controlled at a transcriptional level depending on MMPs and on the tissue or cell type considered. Such a control is exerted by hormones, growth factors and cytokines as well as basigin or EMMPRIN (Extracellular Matrix metalloproteinase Inducer) which belongs to the immunoglobulin superfamily.²²

The Adamalysin-Related Proteinases

This family includes the ADAMs which are cell-surface rather than secreted proteins that share a disintegrin and metalloproteinase domain. They are at least 32 ADAMs that have been cloned and sequenced, each containing a signal sequence followed in order by a pro-domain, a metalloproteinase or metalloproteinase-like domain, a disintegrin-like domain, a cysteine-rich domain, EGF-like repeats, a transmembrane domain, and a cytoplasmic tail. Accordingly, ADAMs potentially perform four distinct but complementary functions: proteolysis via the metalloproteinase domain, adhesion via the disintegrin domain, cell-cell fusion via a candidate hydrophobic fusion peptide in the cysteine-rich domain, and cell signaling via the intracellular domain. A large number of ADAMs show testis-specific expression and are mostly involved in sperm-egg recognition (Table 2).^23-25 ADAMTS are the soluble counterparts of the ADAMs. They do not contain a transmembrane domain, but instead contain thrombospondin-1 motifs that permit ECM-association. To date, 19 ADAMTSs have been identified in human tissues, and two of them display testis expression (ADAMTS-2 and -20; Table 2).

Table 2

General features of ADAMs and ADAMTS.

An Overview of the Repertoire in Testis

MMPs and TIMPs

The occurrence of these molecules is highly dependent on the species, the developmental age of the testis and its endocrine environment, as summarized in Tables 3 and 4.^{13,17,50- 61} The MMP family has greatly expanded these last 20 years and a tissue distribution has generally been perfomed for each newly discovered MMP member using adult rat or mouse testes. Although the MMP-18, MMPs 23-26 and MMP-28 are present in the testis,^50,62-65 there is no indication relative to their cellular localisation. In fact, most of the information available are on the gelatinases MMP-2 and MMP-9 probably because of the avaibility of a rapid and simple biological test i.e., the gelatin zymography. The human fetal testis is also the site of expression of MMPs and TIMPs.⁶⁶ In the mouse, MMPs 2 and 9 are detected in fetal testes together with the TIMPs 1-3 and EMMPRIN.¹³ FSH regulation has been explored using 20-day old rat Sertoli cells, and it was shown that FSH regulated MMP-2 but not MMP-9 or MMP-14, and TIMP-1 and TIMP-2.^{17,51,53-55,57-60,67,68} Cytokines largely involved in testicular physiology such as TNFα (tumor necrosis factor α), TGFβ3 (transforming growth factor β3) and FGF2 (basic fibroblast growth factor) have also been shown to regulate various MMPs and TIMPs in a culture or coculture model of testicular cells.^53-55,58,61 Leydig cells have been shown to express ADAM31,⁶⁹ as well as TIMP-2 throughout development.^70,71 It is yet unknown whether these proteins are under gonadotropin regulation in Leydig cells.

Table 3

The relative mRNA levels for all mouse MMPs and TIMPs and several ADAMs in testicular tissue from newborn mice.

Table 4

Summary of the findings reported for MMPs and TIMPs in the testis.

Serine Proteases and Serpins

PAs were the first serine proteases identified within the testis.⁷² Plasminogen is also synthesized within the testis.⁷³ Originally, it was described that Sertoli cells were the site of synthesis of the two PAs, and FSH stimulates tPA while reducing uPA levels in the rat testis.^74,75 Expression of uPA is also under a retinoic acid control.^74,76 Pachytene and diakinetic spermatocytes exhibit immunoreactivity for tPA,⁷⁷ indicating that a tPA proteolytic event may occur at the spermatocyte surface level. It would be interesting to determine whether the immunoreactivity corresponded to a tPA binding protein or a tPA receptor present on germ cell surface. Annexin II is a good candidate, because it acts as a receptor for tPA and its mRNA is represented in a testis cDNA library.⁷⁸ By contrast, the receptor for urokinase has been identified on both Leydig cells and at Sertoli-germ cell contacts and/or germ cells,⁷⁹ indicating that proteolysis involving plasminogen may occur in the vicinity of Sertoli and germ cells and at the Leydig cell membrane. The receptor for uPA has also been identified on sperm but in that case, uPAR would be involved in sperm-egg recognition.⁸⁰ The binding of uPA to its receptor promotes cell adhesion by increasing the affinity of uPAR for vitronectin.⁸¹ It is thus of interest that vitronectin has been identified in the cytoplasm of Leydig cells⁸² and in germ cells,⁸³ and that PAI-1 is a Sertoli cell product as well as a peritubular product.^84,85 Indeed, PAI-1 might regulate cell adhesion or migration through competition with uPAR in binding to vitronectin.⁸¹ PAI-1 is downregulated by cAMP analogs and FSH in Sertoli cells, and up-regulated by locally produced cytokines (TGFβ1, FGF2 and TNFα).^84-88 In contrast, PAI-3 or serpina5 is up-regulated by FSH and testosterone.^89-91 Of interest is the recent finding that other serpins produced by Sertoli cells are also regulated by androgens, including eppin and the serpins a3n and a12n.⁹¹

Germ cells are also a source of various serine proteases and inhibitors including the activated Protein C⁹² and its inhibitor (serpina5),⁷⁹ the hepatocyte growth factor activator (HGFA) and its 2 specific inhibitors, the HAIs.⁹³ They also express the serpinb6b and testisin.^94,95 Testisin (also named TESP5) is a GPI-anchored protein expressed by premeiotic testicular germ cells and is a candidate tumor suppressor for testicular cancer.⁹⁵ Another TEStis Specific serine protease-1 TESSP-1 is a membrane-bound enzyme specifically expressed in type B spermatogonia and spermatocytes in the adult mouse.⁹⁶ It is not known whether these proteases act within the seminiferous epithelium or later in sperm-egg recognition events as shown with most ADAMs.²³

Although few studies have explored the contribution of Leydig cells to the testicular protease repertoire, it is of interest that Leydig cells are known to express various serine proteases and serpins, and for some of these proteases, Leydig cells are the unique testicular site for their expression. For example, the serine protease originally named Leydin is in fact neurotrypsin.^18,97 Leydig cells are also the source of kallikreins 21, 24 and 27.^98-100 Interestingly, LH-hCG was found to regulate several serine proteases and serpins identified in Leydig cells (including urokinase, matriptase-2, kallikrein-21, HAI-2 and PCI),⁹³ indicative that common transcriptional signals may drive the expression of these molecules. Furthermore, kallikreins are regulated by testosterone and estradiol.^98-100 Table 5 recapitulates most of the data available on serine proteases and SERPINs expressed in testis.

Table 5

Serine proteases and serpins expressed in testis.

What Potential Functions in Testicular Physiology ?

ECM Matrix Remodeling

One of the most described roles for proteases and inhibitors relates to the degradation of extra-cellular matrix that forms a physical barrier for cells to invade. In a very comprehensive review¹¹⁰ on basement membrane and its testicular composition, ECM matrix remodeling is presented as a major event during organogenesis and growth whereas adulthood is characterized by a very low index in the turnover of extracellular matrix components. Furthermore, human pathological testes exhibit a hyalinisation of the seminiferous tubules that is accompanied by a lower sperm production ability and such a feature is also the hallmark of the testicular phenotype in the klinefeleter syndrome.^111,112 Thus, a physiological link is likely to exist between ECM and sperm production.

Testis Cord Formation

ECM consitutes a pathway along which cells may migrate, for instance during the migration of primordial germ cells. Indeed, PGCs express several integrins that may act as receptors for fibronectin and laminin that pave the PGC pathway toward the genital ridge.^113,114 Another very important involvement of ECM proteins during testis organogenesis consists of the formation of a basement membrane between the epithelializing Sertoli cells and the mesenchymal peritubular cells.^110,115-119 Originally, the genital ridge is composed of primordial germ cells and a thickened layer of coelomic epithelium. When the indifferent gonad has a XY genotype, SRY induces a cascade of gene expression which results initially in the migration of mesenchymal cells as well as endothelial cells from the adjacent mesonephros. No migration occurs in case of a XX gonad.^120,121 Such a migration is accompanied by extensive restructuring which underlines a crucial need for balancing the proteinases/inhibitors ratio. Accordingly, major sex-related differences in the ECM components distribution^{122- 126} and the expression of proteases and inhibitors have been reported.^{11- 13,127- 131} Knowledge in this area has greatly been enhanced with the development of microarray studies dedicated to the identification of genes expressed in a sex-dimorphic fashion during the initial phases of testicular differentiation.^130,131 Table 6 summarizes data on the proteinases and inhibitors.

Table 6

Fold-increase in proteinases and inhibitors in fetal testes versus time-matched fetal ovaries.

Investigations on the desert hedgehog (dhh) classified in the cysteine proteinases family,^19,20 have also greatly increased our understanding on the role of the basement membrane in testis compartimentation.^132-135 Sertoli cells are the source of Dhh whereas receptors are localized on peritubular cells and possibly Leydig cells. Interestingly, Dhh expression levels increase more than 45-fold at the time of testicular differentiation,^130,131 and Dhh-null testes exhibit disorganized cords with occasional germ cells seen outside cords and abnormal Leydig cell development. It was concluded that these defects likely stemmed from abnormal peritubular stimulation due to the lack of Dhh.^132-135 Therefore, it would be of high interest to determine which proteinases and inhibitors may lie downstream of the Dhh/receptor complex.

Testis Growth and Lumen Formation

The prepubertal period is characterized by a rapid growth of the testis, the transformation of the seminiferous cords into tubules and the initiation of spermatogenesis. Specifically, tight junctions are formed between neighboring Sertoli cells thus creating the blood-testis barrier, and cords developed a lumen becoming tubules. Accordingly, Sertoli cells reorganize their cytoskeleton to support additional spermatogenic cell types as spermatogenesis is initiated, and tubules increase in diameter as well as in length. Several in vitro observations raise the possibility that proteases and inhibitors in response to hormonal (mainly FSH) and local stimuli such as HGF and FGF2 are critically involved in these substantial prepubertal changes.

For instance, using prepubertal rat Sertoli cells cultured in a two-chambered assembly to mimick the Sertoli cell barrier, it was demonstrated that proteases were implicated in the changes in the Sertoli cell cytoskeleton elicited by FSH and in modulation of the formation and maintenance of the Sertoli cell barrier. The nature of the protease(s) is not fully identified, but α2-macroglobulin opposed its(their) action and increased integrity of the Sertoli cell barrier.^67,136 MMP-2 and tPA are good candidates because they are Sertoli cell products and under FSH control.^55,74,75 A second set of experiments was designed to examine cord and lumen formation by Sertoli cells cocultured with rat prepubertal peritubular cells or Sertoli cells cultured on a reconstituted ECM. These models have been extremely fruitful in evidencing cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components,¹³⁷ and in highlighting the role of laminin in the morphognetic cascade resulting in the formation of tubule-like structure.^138,139 Other experiments also suggested that ECM components regulated the expression of tight junction proteins and the formation of a lumen.^58,60 Inasmuch as MMPs and PAs degrade laminin, fibronectin and collagen IV i.e., the major ECM components of the testicular basement membrane, any remodeling necessary to support the rapid and extensive growth of the prepubertal testis is thus expected to involve a delicate interplay between proteases and inhibitors, and certain growth factors. Indeed, various growth factors promote formation of cord-like structures by Sertoli cells in vitro. For instance, FGF2 which mediates mesenchymal-epithelial interactions of peritubular cells and Sertoli cells in the rat testis, promoted de novo testis cord formation and enhanced MMP-9, the 30 kDa glycosylated form of TIMP3 and PAI-1 in the cocultures.⁶¹ It would be of interest to investigate the expression pattern of proteases and inhibitors in the HGF-treated cultures because HGF is a powerful morphogen for Sertoli cells cultured on a reconstituted basement membrane. It not only promotes cords but also their further remodeling into tubules.^107,140 Additionally, the antisense strategy would constitute an elegant means to link morphogen cytokines, ECM components, restructuring events and involvement of proteases.

Few data are yet available in vivo to support these in vitro experiments. However, a recent study highlighted the role of laminin during the prepubertal period. Indeed, it was shown that male mice deficient for laminin alpha2 chain exhibited abnormal testicular basement membranes and displayed a defect in the timing of lumen formation, resulting in production of fewer spermatids. Furthermore, the authors demonstrated that laminin alpha1 chain corrected male infertility caused by absence of laminin alpha2 chain.¹⁴¹

Lessons from Transgenic Mice

Inasmuch as proteases and inhibitors are extremely abundant and redundant in their spectrum of actions, it is not surprising that very little knockout mouse models have to date contributed to our understanding on their roles in testicular function (Table 7). However, it should be stated that most of the time no systematic analysis of the testes of the deficient mice had been undertaken unless the authors experienced reproductive difficulties. For example, male mice deficient in PAI-1,¹⁷⁵ TIMP-1¹⁷⁶ or cathepsin L¹⁷⁷ may still reproduce. However because several pieces of evidences supported a role for cathepsin L in germ cell movement during spermatogenesis, testes from deficient mice were carefully investigated and tubules were found to contain 32% fewer spermatids than the average tubule number of control mice.¹⁷⁷ Such a study should be done on the MT1-MMP-deficient male mice which showed “no signs of sexual maturation”, as stated in the original publication.¹⁷⁸ In other cases, the testis may not be the primary target as shown with male mice deficient in serpinE2 which are sterile because of altered semen protein composition.¹⁷⁹ The different cases reported in Table 7 should be more informative with respect to the role of proteinases in testicular physiology, provided that an extensive study of the male reproductive system is done. Indeed, male mice deficient for EMMPRIN are azoospermic. Specifically, spermatogenesis is arrested at the metaphase of the first meiotic division, and the lumens are filled with round degenerated cells.¹⁸⁰ Given that the expression of EMMPRIN correlates in time with the appearance of spermatocytes in the seminiferous epithelium, it is predicted that EMMPRIN is involved in the interactions between Sertoli cells and germ cells.^180,181 However, no studies have yet reported the expression pattern of the MMPs known to be under EMMPRIN control in the deficient testes. Male sterility is also observed in transgenic mice with inactive alleles for ADAMTS-2,¹⁸² overexpression of MMP-7¹⁸³ and deficiency for serpina5,¹⁶⁴ but the exact nature of the disorder remains to be fully characterized.

Table 7

Data obtained from the generation of transgenic mice with reduced fertility.

Conclusions and Future Directions

We herein provided a series of evidences highlighting that proteases may be active partners in establishing and maintaining testicular architecture, and in facilitating germ cell migration which constitutes a prerequisite for germ cell progression throughout the spermatogenic process. Therefore, it may be worth to revisit the phenotypes of transgenic male mice deficient for a proteinase or an inhibitor proven to be expressed at the time of translocation and/or spermiation. Furthermore inasmuch as various proteases, inhibitors, junctional components (e.g., claudins, occludins, JAMs) are under a complex hormonal regulation via gonadotropins and/or testosterone, and local regulatory control involving cytokines and growth factors, models with reduced testosterone bioavaibility or with limited FSH or LH action coupled to microarray studies, as those recently published^89,91 should be of tremendous benefit to fully understand the mechanisms that underpin the role of proteases and inhibitors in testis development and function.

Acknowledgements

M.G. Forest is thanked for her contribution in carefully reading the manuscript. J. Bois and M.A. Dicarlo are thanked for their help in typing the manuscript.

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