The Loss of Polysialic Acid Impairs the Contractile Phenotype of Peritubular Smooth Muscle Cells in the Postnatal Testis

In the testis, the germinal epithelium of seminiferous tubules is surrounded by contractile peritubular cells, which are involved in sperm transport. Interestingly, in postnatal testis, polysialic acid (polySia), which is also an essential player for the development of the brain, was observed around the tubules. Western blotting revealed a massive decrease of polySia from postnatal day 1 towards puberty, together with a fundamental reduction of the net-like intertubular polySia. Using polysialyltransferase knockout mice, we investigated the consequences of the loss of polySia in the postnatal testis. Compared to postnatal wild-type animals, polySia knockouts showed slightly reduced smooth muscle actin (SMA) immunostaining of peritubular smooth muscle cells (SMCs), while calponin, marking more differentiated SMCs, dramatically decreased. In contrast, testicular SMA and calponin immunostaining remained unchanged in vascular SMCs in all genotypes. In addition, the cGMP-dependent protein kinase PKG I, a key enzyme of SMC relaxation, was nearly undetectable in the peritubular SMCs. Cell proliferation in the peritubular layer increased significantly in the knockouts, as shown by proliferating cell nuclear anti (PCNA) staining. Taken together, in postnatal testis, the absence of polySia resulted in an impaired differentiation of peritubular, but not vascular, SMCs to a more synthetic phenotype. Thus, polySia might influence the maintenance of a differentiated phenotype of non-vascular SMCs.


Introduction
The testis is responsible for the production of male germ cells and testosterone [1]. The major components of the testis are the seminiferous tubules, with their germinal epithelium [1] and the surrounding peritubular lamina propria [2][3][4]. In rodents, this structure consists only of a single layer of contractile (myoid) cells. These cells are suggested to affect both transport of spermatozoa and germ cell maturation, in addition to further paracrine and immunological functions [3,4]. Among seminiferous tubules, the so-called interstitial tissue is visible, which includes the testosterone-producing Leydig cells, blood vessels, immune cell populations and connective tissue components [5]. The entirety of the tubules is surrounded by the capsular tunica albuginea, which also comprises contractile cells [6,7].
In addition to the reproductive tract and the brain, polySia also appears during the development of other organs, such as the heart, lung, kidney, and liver [20][21][22][23][24]. It seems to be that the loss of one of the polysialyltransferases can be partly compensated by the other one, since knockout mice without ST8SiaII or ST8SiaIV show only a relatively mild phenotype [25,26]. Additionally, in fish, a loss of one polysialyltransferase seems to be compensated, since in the course of evolution, several teleost lineages lost one of the polysialyltransferases [27,28]. In contrast, the simultaneous loss of both polysialyltransferases leads to remarkable neurodevelopmental alterations and higher postnatal mortality rates in double knockout mice [9][10][11][12]. In addition, polySia negative mice showed a reduction of fatty tissue and limb muscles, which comes along with growth retardation [9]. However, in other organs, such as heart, lung, stomach, pancreas, gut, spleen and kidney, no significant pathological changes were observed [9]. Regarding the male reproductive tract, the biological role and impact of polySia on the formation of the contractile areas are unknown so far.
The best-known cellular functions of polySia are mainly related to the modulation of cell-cell interactions and migration mechanisms [12,18]. Due to its negative charge, the interaction between adhesion molecules can be inhibited, resulting, e.g., in an increased migration capacity of polysialylated cells [29,30]. In addition to its impact on cellular interaction, polySia can bind several growth factors, such as brain-derived neurotrophic factor (BDNF), nerve growth factor, neurotrophin-3, neurotrophin-4, vascular endothelial growth factor, and basic fibroblast growth factor 2 (bFGF) [31][32][33]. The interaction can modulate the activity of such growth factors by altered sensitivity to proteases and/or binding efficiency with their respective receptors [31,32,34]. Thus, several cellular mechanisms can be modulated by polySia, which might also take place in polySia-positive areas of the testis during postnatal development.
To examine the impact of polySia on the contractile elements of the testis, we investigated the phenotype of smooth muscle cells (SMCs) in polySia knockout mice during postnatal development, demonstrating that the phenotype of peritubular SMCs, different to vascular SMCs, changed in the absence of polySia.
In the case of mAb 735, in parallel, sections were treated with endoneuraminidase (endoN) (10 µg/mL) overnight at 37 • C. EndoN cleavages polySia chains into oligomers consisting of up to 7 sialic acid residues [35][36][37]. However, the mAb 735 needs a minimum chain length of 8 for binding [38]. Thus, the combination of endoN and the mAb 735 is a powerful combination to control the immunostaining against polySia. The mAb 735 and endoN were provided by Martina Mühlenhoff (MHH, Hannover, Germany) [35,39].
All polySia slides were additionally counterstained with hematoxylin. Furthermore, slides were processed without a primary antibody. These slides were only treated with PBS/0.2% bovine serum albumin. Some of the polySia-stained tissues had already been used for the analysis of the polysialylation status in the epididymis and tunica albuginea testis [8]. All pictures were taken with a Zeiss Axioskop 2 plus (Carl Zeiss Vision, Munich, Germany) and processed with Axio Vison Software (Carl Zeiss Vision).

Measurement and Counting of the Proliferating Cells
Three different sections of each animal were immunostained with antibodies against PCNA, as described above. Using a measuring tool from AxioVision Software, the total area of the testis was determined in every section. To obtain the area of the seminiferous tubules, we subtracted the interstitial tissue from the total testis area. Using a corresponding tool from Image J 1.50e (public domain software, NIH, Bethesda, MD, USA), we counted the proliferating peritubular myoid cells. Thereafter, we calculated the ratio of counted peritubular cells per area of seminiferous tubules in each section.

Loss of Polysia Leads to Altered Expression Patterns of Distinct Protein Markers of Peritubular SMCs in Postnatal Testis
As previously described, in the postnatal murine testis, polySia-positive areas were found in the tunica albuginea [8]. In addition, strong peritubular staining ( Figure 1A) occurred around almost all tubules at postnatal day 1. The degradation of polySia by endoN abolished the staining, verifying the specificity of the polySia visualization ( Figure 1B). It is known that polysialylation decreases with increasing concentrations of collagen in contractile areas of the male reproductive tract and that polySia-positive areas are colocalized with SMA staining [8]. At postnatal day 10, only minor amounts of polySia were detectable ( Figure 1C). In line with the immunohistochemical visualization of polySia, Western blot analyses confirmed a decrease of polySia during postnatal development ( Figure 1D). The results suggest that the main postnatal polySia-dependent processes occur during the first week after birth.

Loss of Polysia Leads to Altered Expression Patterns of Distinct Protein Markers of Peritubular SMCs in Postnatal Testis
As previously described, in the postnatal murine testis, polySia-positive areas were found in the tunica albuginea [8]. In addition, strong peritubular staining ( Figure 1A) occurred around almost all tubules at postnatal day 1. The degradation of polySia by endoN abolished the staining, verifying the specificity of the polySia visualization ( Figure 1B). It is known that polysialylation decreases with increasing concentrations of collagen in contractile areas of the male reproductive tract and that polySia-positive areas are colocalized with SMA staining [8]. At postnatal day 10, only minor amounts of polySia were detectable ( Figure 1C). In line with the immunohistochemical visualization of polySia, Western blot analyses confirmed a decrease of polySia during postnatal development ( Figure 1D). The results suggest that the main postnatal polySia-dependent processes occur during the first week after birth. Therefore, it was of interest to know the extent of testicular changes in mice lacking both polysialyltransferases (st8sia2 −/− ; st8sia4 −/− ) at postnatal day 9.5, i.e., directly after the first important week. For this, testis samples of double knockout mice were compared to the corresponding st8sia2 +/− ; st8sia4 −/− and wild-type mice.
In the first set of experiments, polySia was visualized demonstrating that the deletion of polysialyltransferases leads to reduced polySia levels in st8sia2 +/− ; st8sia4 −/− mice and a complete loss of polySia in double knockout mice ( Figure 2). Therefore, it was of interest to know the extent of testicular changes in mice lacking both polysialyltransferases (st8sia2 −/− ; st8sia4 −/− ) at postnatal day 9.5, i.e., directly after the first important week. For this, testis samples of double knockout mice were compared to the corresponding st8sia2 +/− ; st8sia4 −/− and wild-type mice.
In the first set of experiments, polySia was visualized demonstrating that the deletion of polysialyltransferases leads to reduced polySia levels in st8sia2 +/− ; st8sia4 −/− mice and a complete loss of polySia in double knockout mice ( Figure 2). To analyze, if this loss of polySia affects peritubular SMCs, SMA was visualized by immunohistochemistry. In all genotypes, an unambiguous signal was located at the tunica albuginea ( Figure 3A-C), as well as in blood vessels ( Figure 3G-I). However, the SMA To analyze, if this loss of polySia affects peritubular SMCs, SMA was visualized by immunohistochemistry. In all genotypes, an unambiguous signal was located at the tunica albuginea ( Figure 3A-C), as well as in blood vessels ( Figure 3G-I). However, the SMA staining in peritubular SMCs seemed to be slightly stronger in wild-type and st8sia2 +/− ; st8sia4 −/− testes in comparison to double knockout mice. The identification of blood vessels was confirmed by the endothelial cell marker CD31 (Supplementary Figure S2). To analyze, if this loss of polySia affects peritubular SMCs, SMA was visualized by immunohistochemistry. In all genotypes, an unambiguous signal was located at the tunica albuginea ( Figure 3A-C), as well as in blood vessels ( Figure 3G-I). However, the SMA staining in peritubular SMCs seemed to be slightly stronger in wild-type and st8sia2 +/− ; st8sia4 −/− testes in comparison to double knockout mice. The identification of blood vessels was confirmed by the endothelial cell marker CD31 (Supplementary Figure S2).  SMCs can be roughly classified in a synthetic and a contractile phenotype. With the development of the contractile machinery in SMCs, increasing numbers of contractionspecific proteins are expressed. Remarkably, SMA is expressed in all SMCs independently of the functional phenotype and is therefore used as a general marker of these cells. Thus, for a more detailed classification, more marker proteins have to be investigated. For this reason, calponin was used as further marker for muscle cells. In contrast to SMA, calponin is only expressed in more differentiated muscle cells [42,43]. As shown in Figure 4, the double knockout mice showed a dramatic decrease of the calponin expression in the peritubular muscle cells in comparison to the other two genotypes. However, calponin staining was unchanged in SMCs of the tunica albuginea ( Figure 4A-C) and blood vessels ( Figure 4G-I). Consequently, deletion of the polysialyltransferases comes along with a for a more detailed classification, more marker proteins have to be investigated. For this reason, calponin was used as further marker for muscle cells. In contrast to SMA, calponin is only expressed in more differentiated muscle cells [42,43]. As shown in Figure 4, the double knockout mice showed a dramatic decrease of the calponin expression in the peritubular muscle cells in comparison to the other two genotypes. However, calponin staining was unchanged in SMCs of the tunica albuginea ( Figure 4A-C) and blood vessels ( Figure  4G-I). Consequently, deletion of the polysialyltransferases comes along with a selective loss of calponin in the peritubular muscle cells. The results suggest that the dedifferentiation of SMCs is restricted to seminiferous tubules. In addition to the classical differentiation markers of SMCs, which predominantly belong to the group of structural proteins, we also tested PKG1 as a signaling molecule ( Figure 5). PKG1 is an important regulator of smooth muscle cell relaxation [42,43]. In addition to the classical differentiation markers of SMCs, which predominantly belong to the group of structural proteins, we also tested PKG1 as a signaling molecule ( Figure 5). PKG1 is an important regulator of smooth muscle cell relaxation [42,43]. Whereas PKG1 staining only showed minor alterations among the groups in vascular SMCs ( Figure 5G-I), a dramatic loss of PKG1 in peritubular muscle cells was already detectable in st8sia2 +/− ; st8sia4 −/− mice, and was completely absent in these cells in double knockout mice ( Figure 5F,I).
Thus, loss of polySia coincides with decreased expression levels of proteins, which are involved in contraction and relaxation. Interestingly, the polySia binding partner bFGF represents a growth factor, which drives the differentiation of SMCs to a synthetic phenotype [43]. When bFGF is complexed by polySia, the direct binding of bFGF to its receptor seems to be inhibited resulting, for instance, in reduced cell growth [31]. Moreover, a loss of polySia may also alter the dynamic system of further growth factors in the postnatal testis, leading to the observed switch of SMC phenotype. In light of the reduced numbers of SMCs with a contractile phenotype in polySia knockout mice, it is interesting to note that changes in the human peritubular lamina propria come along with disturbed spermatogenesis. In these patients, the peritubular areas become fibrotic, showing an accumulation of collagen in addition to decreased SMCs [2,4,44,45]. Thus, loss of polySia coincides with decreased expression levels of proteins, which are involved in contraction and relaxation. Interestingly, the polySia binding partner bFGF represents a growth factor, which drives the differentiation of SMCs to a synthetic phenotype [43]. When bFGF is complexed by polySia, the direct binding of bFGF to its receptor seems to be inhibited resulting, for instance, in reduced cell growth [31]. Moreover, a loss of polySia may also alter the dynamic system of further growth factors in the postnatal testis, leading to the observed switch of SMC phenotype.
In light of the reduced numbers of SMCs with a contractile phenotype in polySia knockout mice, it is interesting to note that changes in the human peritubular lamina propria come along with disturbed spermatogenesis. In these patients, the peritubular areas become fibrotic, showing an accumulation of collagen in addition to decreased SMCs [2,4,44,45].

Peritubular SMC Proliferation Is Affected in polySia-Negative Testis
The absence of PKG1 and the decreased amount of calponin suggest that the differentiation of peritubular cells is negatively influenced in polySia knockout mice. Next, it was investigated, whether or not the postulated structural and functional deficits of Cells 2021, 10, 1347 8 of 12 peritubular cells coincide with higher cell proliferation, i.e., that a synthetic phenotype of SMCs predominates. Using the proliferation marker PCNA, peritubular cells of all tubules of a cross section were analyzed ( Figure 6A). In addition to proliferating germ cells, PCNApositive peritubular cells are clearly visible (arrows in Figure 6B). Afterwards, all positive peritubular cells were marked by red dots prior to the counting process ( Figure 6C). Cells 2021, 10, x 8 of 12

Peritubular SMC Proliferation is Affected in polySia-Negative Testis
The absence of PKG1 and the decreased amount of calponin suggest that the differentiation of peritubular cells is negatively influenced in polySia knockout mice. Next, it was investigated, whether or not the postulated structural and functional deficits of peritubular cells coincide with higher cell proliferation, i.e., that a synthetic phenotype of SMCs predominates. Using the proliferation marker PCNA, peritubular cells of all tubules of a cross section were analyzed ( Figure 6A). In addition to proliferating germ cells, PCNApositive peritubular cells are clearly visible (arrows in Figure 6B). Afterwards, all positive peritubular cells were marked by red dots prior to the counting process ( Figure 6C).  Figure 7A is corroborated by statistical analysis, in which all groups are significantly different in the ratio of PCNA-positive peritubular cells to tubular area (Figure 7B). In st8sia2 +/− ; st8sia4 −/− mice, a slight, but statistically not significant, increase of proliferating peritubular cells was visible. In polySia double knockout mice, however, there was a strong increase of proliferating peritubular cells compared to the wild-type. As suggested above for the observed loss of the contractile phenotype, the increased number of proliferating cells also implies a possible role of polySia during growth factor dependent processes. Interestingly, polySia-binding not only modulates the enrichment and presentation of growth factors to their respective receptors, but also shows protective effects against their proteolytic cleavage [34]. Thus, the half-life and binding efficiency of growth factors, such as bFGF, might be impaired. In sum, these results suggest a dedifferentiation in the absence of polySia, since also an increasing number of proliferating cells is an important characteristic of the synthetic phenotype of SMCs (Figure 8).  Figure 7A is corroborated by statistical analysis, in which all groups are significantly different in the ratio of PCNA-positive peritubular cells to tubular area ( Figure 7B). In st8sia2 +/− ; st8sia4 −/− mice, a slight, but statistically not significant, increase of proliferating peritubular cells was visible. In polySia double knockout mice, however, there was a strong increase of proliferating peritubular cells compared to the wild-type. As suggested above for the observed loss of the contractile phenotype, the increased number of proliferating cells also implies a possible role of polySia during growth factor dependent processes. Interestingly, polySia-binding not only modulates the enrichment and presentation of growth factors to their respective receptors, but also shows protective effects against their proteolytic cleavage [34]. Thus, the half-life and binding efficiency of growth factors, such as bFGF, might be impaired. In sum, these results suggest a dedifferentiation in the absence of polySia, since also an increasing number of proliferating cells is an important characteristic of the synthetic phenotype of SMCs (Figure 8). Cells 2021, 10, x 9 of 12  mAb against PCNA. The positive cells were labeled with red dots. Supplemental Figure 3 shows the same sections with smaller dots, which allow an additional evaluation of the peritubular cell-specific morphology. (B) Based on the immunohistochemical labeling of proliferating peritubular cells, the density of positive cells in the seminiferous tubules was calculated. The values (5 wild-type, 3 st8sia2 +/− ; st8sia4 −/− and 5 double knockout animals; per animal 3 sections were analyzed) are summarized and standard deviation (SD) is displayed in addition to the means. Statistically significant differences are indicated: * p < 0.05; ** p < 0.01, ns: not significant. The PCNA-positive cells within the tubules were not considered in the study.

Conclusions
In sum, the outlined experiments demonstrate that in postnatal murine testes, • polySia is detectable around seminiferous tubules. • the strongest net-like intertubular polySia staining is detectable during the first week of development. • the loss of polySia leads to a development of peritubular SMCs towards a synthetic phenotype ( Figure 8).
Thus, polySia might influence the maintenance of a differentiated phenotype of nonvascular SMCs.