Protein localization and gene expression of the nerve growth factor and its cognate receptors in different testicle morphotypes of the grey squirrel (Sciurus carolinensis) living in the Umbria region

The gray squirrel (Sciurus carolinensis) is an invasive species that constitutes a serious threat to the native Eurasian red squirrel conservation due to its ability to adapt well to new environments such as the Umbria region (1). The safeguarding of native species is implemented from diversified actions including knowledge of the reproductive characteristics of the alien species to limit its reproductive success. To this end, the nerve growth factor (NGF) and its high- and low-affinity receptors (NTRK1, NGFR) were studied in the testis of gray squirrels since several studies describe NGF as an important factor involved in the regulation of mammalian reproduction. The NGF system was already described in the ovaries of female gray squirrels living in the Umbria region (2) while there is no information on male squirrels. In humans and laboratory animals, NGF affects the process of testicular morphogenesis and spermiogenesis as demonstrated by the presence of NGF receptors in both Sertoli and germ cells in prepubertal and adult subjects. In this study, seventeen male squirrels were captured in a natural area close to Perugia city. The animals were anesthetized with dexmedetomidine ketamine, maintained with sevoflurane in 100% O2, and underwent external or internal orchiectomies (3). Squirrels were captured and treated in compliance with regulations regarding wildlife control laid down in Art. 19 of the Italian Law 157/92, “Rules for the protection of wild animals and homeotherms and for hunting”, Habitat Directive 92/43/CEE, and European Parliament Regulation n. 1143/2014. Collected testis were classified into 3 different morphotypes by histological procedures: active spermatogenesis, pubertal, and immature (1). NGF, NTRK1, and NGFR were evaluated in the testis of the three different morphotypes by immunohistochemistry, western blotting, and Real-Time PCR. Significant differences among group means were calculated by ANOVA followed by Tukey post hoc test. Western Blot revealed the presence of bands matching the expected size for NGF and its receptors. Immunohistochemistry showed the NGF localization in the Leydig cells in all morphotypes with more intense cytoplasmic staining in pubertal and spermatogenesis ones. NTRK1 was localized in the Leydig cells. NGFR immunostaining was observed in Sertoli cells of the pubertal morphotype and both the Sertoli cells and Leydig cells of the spermatogenesis morphotype. By Real-Time PCR, NGF significantly increased in pubertal with respect to both active spermatogenesis and immature morphotypes. The presence of NGF receptors indicates that the testis of gray squirrels is sensitive to NGF action while the overlapping localization of the molecule and its receptors indicate that NGF, produced by Leydig cells, can exert its function on the testis through an autocrine and paracrine mechanism. The results obtained suggest that in the grey squirrel population here investigated the NGF system is involved in the testicle development and function, probably contributing to the reproductive success of this species. [1] Maranesi et al. Reproductive Traits of an Invasive Alien Population of Grey Squirrel (Sciurus carolinensis) in Central Italy. Animals (Basel). 10(4):738, 2020. [2] Maranesi et al. Seasonal Expression of NGF and Its Cognate Receptors in the Ovaries of Grey Squirrels (Sciurus carolinensis). Animals (Basel). 10(9):1558, 2020. [3] Bufalari et al. Traditional gonadectomy vs gonadectomy by Ligasure (R) in grey squirrels (Sciurus carolinensis): Comparing surgical techniques. Reproduction in Domestic Animals, 54(S3):137, 2019.