Elsevier

Differentiation

Volume 106, March–April 2019, Pages 23-34
Differentiation

Matrix metalloproteinase-dependent regulation of extracellular matrix shapes the structure of sexually differentiating mouse gonads

https://doi.org/10.1016/j.diff.2019.01.006Get rights and content

Highlights

  • Inhibition of MMPs in developing gonads leads to accumulation of ECM.

  • Inhibition of MMPs causes decrease of testis-markers expression.

  • Accumulation of ECM in developing gonads disrupts cell adhesion.

  • Accumulation of ECM disrupts gonad structure formation.

  • Activation of MMPs leads to dispersion of gonad cells.

Abstract

The extracellular matrix (ECM) proteins play an important role in the establishment of the sex-dependent structure of developing gonads. The matrix metalloproteinases (MMPs) are the major players in the regulation of ECM. Our hypothesis was that the MMPs-dependent regulation of EMC is crucial for the establishment of the correct, either testis or ovary, structure of developing gonad. We cultured developing mouse gonads in vitro in the presence of the MMPs inhibitors (α-2-macroglobulin, leupeptin, phosphoramidon) or the MMPs activator, APMA (4-aminophenylmercuric acetate). These inhibitors and activator inhibit/activate, to a different degree, matrix metalloproteinases, but the exact mechanism of inhibition/activation remains unknown. We found that the MMP inhibitors increased accumulation of ECM in the developing gonads. The α-2-macroglobulin had the weakest, and the phosphoramidon the strongest effect on the ECM and the structure of the gonads. The α-2-macroglobulin caused a slight increase of ECM and did not disrupt the gonad structure. Leupeptin led to the strong accumulation of ECM, resulted in the formation of the structures resembling testis cords in both testes and ovaries, and caused increase of apoptosis and complete loss of germ cells. Phosphoramidon caused the strongest accumulation of ECM, which separated individual cells and completely prevented intercellular adhesion both in the testes and in the ovaries. As a result of aberrant morphology, the sex of the phosphoramidon-treated gonads was morphologically unrecognizable. The APMA - the activator of MMP caused ECM loss, which led to the loss of cell adhesion, cell dispersion and an aberrant morphology of the gonads. These results indicate that the ECM accumulation is MMPs-dependent and that the correct amount and distribution of ECM during gonad development plays a key role in the formation of the gonad structure.

Introduction

During sexual differentiation of the gonads, the bipotential gonads differentiate into the testis or ovary. Although this processes has been well studied in mice, the molecular and cellular machinery governing the development of testes and ovaries is very complex and still requires further studies. Gonad primordia, termed genital ridges, appear in mice soon before 10.5th day of embryonic life (E10.5) (Hu et al., 2013; reviewed by Piprek et al., 2016). Between stage E10.5 and E12.5, the still undifferentiated gonads initiate the expression of sex-determining genes (Bullejos and Koopman, 2001; Kobayashi et al., 2005). Depending on the genetic sex, the male or female sex-determining pathway prevails and determines the structure and fate of the gonad (Kim et al., 2006; Chassot et al., 2008; reviewed by Piprek, 2009a,b). The first differences in the structure between male and female gonads appear around stage E12.5 (Schmahl et al., 2000; Nel-Themaat et al., 2009; reviewed by Piprek, 2010). A day later, i.e. at E13.5, the gonads are already sexually differentiated, and their sex can be easily distinguished histologically (Nel-Themaat et al., 2009). In the differentiating testes, the somatic cells derived from the coelomic epithelium proliferate leading to the extensive growth of the male gonad (Schmahl et al., 2000). The presumptive Sertoli cells enclose germ cells forming elongated testis cords surrounded by the basement membrane (Svingen and Koopman, 2013). The cells migrating from the adjacent mesonephros give rise to mainly the endothelial cells of the gonad vasculature (Brennan et al., 2002). The subpopulation of the mesonephros-derived cells, and the cells derived from the coelomic epithelium form the interstitium, which separate the testis cords, and thus, shape the testis structure (Tilmann and Capel, 1999; DeFalco et al., 2011). The interstitium contains steroidogenic fetal Leydig cells (FLCs) and abundant extracellular matrix (ECM). The development of the ovary takes a different path. Although the germ cells in developing ovary also become surrounded by the somatic cells (pre-follicular cells) (Albrecht and Eicher, 2001), the elongated cords do not develop. The ovigerous cords are built of many small and irregularly shaped clusters of the somatic and germ cells, known as the germ cell nests, embedded in the ovarian stroma (Lei and Spradling, 2013). Later in development, the ovigerous cords split into ovarian follicles (Pepling and Spradling, 2001; Pepling et al., 2010).

It has been shown that in mouse, rat, cattle, chicken, slider (Trachemys scripta) and the African clawed frog (Xenopus laevis) (Paranko et al., 1983; Yao et al., 2004; Hummitzsch et al., 2013; Piprek et al., 2017a, 2018) the ECM plays important role in gonad development. The ECM contains many different proteins including collagens, laminins, fibronectin, and proteoglycans (reviewed by Yue, 2014). The amount and distribution of ECM depends on two processes: i.) synthesis of the ECM components and their deposition between cells, ii.) degradation of the ECM components by the extracellular matrix enzymes (ECM enzymes). Two main groups of ECM enzymes involved in the ECM formation/degradation are matrix metalloproteinases (MMPs: MMP1 to MMP28) that digest ECM components, and inhibitors of MMPs (TIMPs), which inhibit MMPs (Birkedal-Hansen, 1993; Stamenkovic, 2003; Arpino et al., 2015). We hypothesize that a balance between the formation and degradation of ECM components plays an important role in the regulation of the amount and distribution of ECM.

The knowledge on the role of the ECM in gonad development, especially during the sexual differentiation, is very limited. We showed recently that in the mouse, between E11.5 and E13.5 (i.e. during the period of sexual differentiation) many genes encoding ECM components and MMPs are expressed differentially in the male and female gonads (Piprek et al., 2018). Considering the high number of ECM enzymes, the machinery of ECM remodeling in developing gonads is probably very complex. Because the structure of the gonads is different between sexes, the ECM has different distribution in the testes and ovaries; presumably the sex-determining pathways (responsible for the gonad fate) also regulate the sex-specific distribution of ECM. Indeed it has been shown that in the mouse, the TIMP3, an enzyme inhibiting MMPs, is upregulated by male sex-determining pathway (Nishino et al., 2002). Moreover, gonads develop in the close proximity of the mesonephros. Between these two organs there is the vascular plexus. The vascular plexus disintegrates, and mesonephric cells derived from the disintegrating vascular plexus contribute to the endothelium and interstitium of the gonad, which is crucial for the patterning of testis cords (Coveney et al., 2008). The ECM enzymes are probably involved in the disintegration of vascular plexus and thus they facilitate the migration of the mesonephros-derived cells to the gonads. Several studies showed the role of ECM enzymes in kidney development (Ota et al., 1998; Tanney et al., 1998; Lelongt et al., 2005), however, a role of mesonephros in sexual differentiation of gonads remains unknown. It is known that tubular system of mesonephros joins rete testis later in development, however, molecular mechanisms driving this process are obscure (Joseph et al., 2009; Davidson et al., 2018).

Because the ECM is differentially patterned in developing testes and ovaries, and the genes encoding ECM components and enzymes responsible for ECM remodeling are differentially expressed, we hypothesized that the ECM and its enzymes are important factors controlling sexual differentiation of the gonads. The aim of this study was to explore how the structure of differentiating mouse testes and ovaries changes upon inhibition or activation of ECM regulating enzymes. Fetal gonads isolated at E11.5, i.e. just before the onset of sexual differentiation, were cultured in a medium supplemented with the inhibitors of MMPs (α-2-macroglobulin, leupeptin, or phosphoramidon) or with the activator (APMA, 4-aminophenylmercuric acetate) (Table 1). The gonads were analyzed after 3 days in culture using histological techniques, immunohistochemistry and gene expression analysis.

Section snippets

Animals and genotyping

The gonads were isolated from the C57bl/6 mouse strain. The study was approved by the I Local Commission for Ethics in Experiments on Animals. The animals were bred and housed in the Animal Facility at the Jagiellonian University (Krakow, Poland). The number of studied animals is presented in Table 1. Timed matings were performed by placing a male with 2 females overnight. The following morning, females were checked for the presence of the vaginal plug, and the pregnancies were estimated as

Activity of MMPs in the gonads after incubation in the presence of inhibitors and activator

Zymography analyses showed that α-2-macroglobulin, leupeptin and phosphoramidon inhibited MMP2, MMP3 and MMP9 in the gonads after 3 days of in vitro culture (Fig. 1). Phosphoramidon inhibited MMP2, MMP3 and MMP9 to the higher degree than a α-2-macroglobulin and leupeptin did. As expected, APMA activated MMP2, MMP3 and MMP9 (Fig. 1). The results of these experiments are summarized in Table 3.

Development of gonads under control in vitro conditions

The histology of the freshly isolated XY and XX gonads, before the start of the in vitro culture, was

Conclusion

We showed that the modulators, both the inhibitors and the activator, of MMPs trigger important changes in the structure of sexually differentiating developing mouse gonads (Table 3). 1. MMPs inhibitors causes accumulation of ECM, which drives cells dispersion and disappearance of testis cords. 2. MMPs activator APMA causes ECM loss and a complete disruption of the gonad structure. Thus, both the excessive accumulation of ECM and its decrease or loss leads to a dramatic impairment of the tissue

Acknowledgments

The study was conducted within the project financed by the Polish National Science Centre (NCN) assigned on the basis of the decision number DEC-2013/11/D/NZ3/00184.

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