Polysialic acid is present in mammalian semen as a post-translational modification of the neural cell adhesion molecule NCAM and the polysialyltransferase ST8SiaII.

Fertilization in animals is a complex sequence of several biochemical events beginning with the insemination into the female reproductive tract and, finally, leading to embryogenesis. Studies by Kitajima and co-workers (Miyata, S., Sato, C., and Kitajima, K. (2007) Trends Glycosci. Glyc, 19, 85-98) demonstrated the presence of polysialic acid (polySia) on sea urchin sperm. Based on these results, we became interested in the potential involvement of sialic acid polymers in mammalian fertilization. Therefore, we isolated human sperm and performed analyses, including Western blotting and mild 1,2-diamino-4,5-methylenedioxybenzene-HPLC, that revealed the presence α2,8-linked polySia chains. Further analysis by a glyco-proteomics approach led to the identification of two polySia carriers. Interestingly, besides the neural cell adhesion molecule, the polysialyltransferase ST8SiaII has also been found to be a target for polysialylation. Further analysis of testis and epididymis tissue sections demonstrated that only epithelial cells of the caput were polySia-positive. During the epididymal transit, polySia carriers were partially integrated into the sperm membrane of the postacrosomal region. Because polySia is known to counteract histone as well as neutrophil extracellular trap-mediated cytotoxicity against host cells, which plays a role after insemination, we propose that polySia in semen represents a cytoprotective element to increase the number of vital sperm.

Polysialylation of NCAM correlates with onset and termination of seasonal spermatogenesis in roe deer.

Roe deer (Capreolus capreolus) are seasonal breeders and cyclic structural changes of roe bucks' testis come along with a totally arrested (winter) and a highly activated spermatogenesis (summer). For this reason, roe buck represents an interesting model to study general mechanisms of initiation and termination of spermatogenesis. We investigated if polysialic acid (polySia)-a linear homopolymer of α2,8-linked sialic acids, which could act as a negative regulator of cell-cell adhesion-might be involved in the activation and/or inactivation of spermatogenesis. To address this point, testis samples of adult male roe deer were collected at different time point of the year. Intriguingly, we observed that polySia attached to the neural cell adhesion molecule was enhanced during the onset of spermatogenesis in April. In addition, polySia was highly expressed in December. Predominantly, polySia was detectable between Sertoli cells and spermatogonia in the basal regions of testicular tubules and in the adluminal part of Sertoli cells. Interestingly, similar polySia distributions were observed during early testis development of other mammalians when gonocytes (pre-spermatogonia) and Sertoli cells represent the only cell populations in tubuli seminiferi. Thus, polySia is expressed during key steps of the "on/off mechanisms" of spermatogenesis and might represent one mediator of the interaction and communication between Sertoli cells and germ cell precursors.

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.

Polysialylation of NCAM characterizes the proliferation period of contractile elements during postnatal development of the epididymis.

Polysialic acid (polySia) attached to the neural cell adhesion molecule (NCAM) regulates inter alia the proliferation and differentiation via the interactions with neurotrophins. Since in postnatal epididymis neurotrophins and their receptors like the Low-Affinity Nerve Growth Factor Receptor p75 and TrK B receptor are expressed, we wanted to analyze if the polysialylation of NCAM is also involved during the development of the epididymis. To this end, we monitored the developmental changes in the expression of the polysialyltransferases and NCAM polysialylation using murine epididymis at different time points during postnatal development. Our results revealed that during postnatal development of the epididymis both polysialyltransferases, ST8SiaII and ST8SiaIV, were expressed and that the expression levels dropped with increasing age. In agreement with the expression levels of the polysialyltransferases the highest content of polysialylated NCAM was present during the first 10 days after birth. Interestingly, proliferating smooth muscle cell populations prevalently expressed polysialylated NCAM. Furthermore, we observed that inverse to the decrease in polysialylation of smooth muscle cells a strong up-regulation of collagen takes place suggesting a functional relationship since collagen was recently described to induce the turnover of polysialylated NCAM via an induction of endocytosis in cellulo. The same time course of polySia and collagen synthesis was also observed in other regions of the male reproductive system e.g. vas deferens and tunica albuginea (testis). Together, we identified a spatio-temporal expression pattern of polySia-NCAM characterized by high proliferation rate of smooth muscle cells and low collagen content.

Polysialylation takes place in granulosa cells during apoptotic processes of atretic tertiary follicles.

In the neuronal system, polysialic acid (polySia) is known to be involved in several cellular processes such as the modulation of cell-cell interactions. This highly negatively-charged sugar moiety is mainly present as a post-translational modification of the neural cell adhesion molecule (NCAM). More than 20 years ago, differently glycosylated forms of NCAM were detected in the ovaries. However, the exact isoform of NCAM, as well as its biological function, remained unknown. Our analysis revealed that granulosa cells of feline tertiary follicles express the polysialylated form of NCAM-140. Unexpectedly, polySia was only expressed in the granulosa layers of atretic follicles and not of healthy follicles. By contrast, only the un-polysialylated form of NCAM was present on the membrane of granulosa cells of healthy follicles. To study a possible cellular function of polySia in feline follicles, a primary granulosa cell culture model was used. Interestingly, loss of polySia leads to a significant inhibition of apoptosis, demonstrating that polySia is involved during atretic processes in granulosa cells. Thus, polySia might not only directly influence regeneration processes as shown, for example, in the neuronal system, but also apoptosis.