Differential Expression of Kisspeptin System and Kisspeptin Receptor Trafficking during Spermatozoa Transit in the Epididymis.

Elena Mele,Raffaella D’Auria,Marika Scafuro,Riccardo Pierantoni,Silvia Fasano,Antonietta Santoro,Rosaria Meccariello,Marianna Marino,Andrea Viggiano

doi:10.3390/genes13020295

Elena Mele, Raffaella D’Auria + Show 7 more

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https://doi.org/10.3390/genes13020295

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Abstract

The hypothalamus–pituitary–testis axis controls the production of spermatozoa, and the kisspeptin system, comprising Kiss1 and Kiss1 receptor (Kiss1R), is the main central gatekeeper. The activity of the kisspeptin system also occurs in testis and spermatozoa, but currently the need of peripheral kisspeptin to produce gametes is not fully understood. Hence, we characterized kisspeptin system in rat spermatozoa and epididymis caput and cauda and analyzed the possible presence of Kiss1 in the epididymal fluid. The presence of Kiss1 and Kiss1R in spermatozoa collected from epididymis caput and cauda was evaluated by Western blot; significant high Kiss1 levels in the caput (p < 0.001 vs. cauda) and constant levels of Kiss1R proteins were observed. Immunofluorescence analysis revealed that the localization of Kiss1R in sperm head shifts from the posterior region in the epididymis caput to perforatorium in the epididymis cauda. In spermatozoa-free epididymis, Western blot revealed higher expression of Kiss1 and Kiss1R in caput (p < 0.05 vs. cauda). Moreover, immunohistochemistry revealed that Kiss1 and Kiss1R proteins were mainly localized in the secretory epithelial cell types and in contractile myoid cells, respectively. Finally, both dot blot and Elisa revealed the presence of Kiss1 in the epididymal fluid collected from epididymis cauda and caput, indicating that rat epididymis and spermatozoa possess a complete kisspeptin system. In conclusion, we reported for the first time in rodents Kiss1R trafficking in spermatozoa during the epididymis transit and Kiss1 measure in the epididymal fluid, thus suggesting a possible role for the system in spermatozoa maturation and storage within the epididymis.

Highlights

Spermatogenesis, the process of male gamete production, requires the occurrence of mitosis, meiosis, and differentiation of haploid round spermatids into spermatozoa [1]
Kiss1 and Kiss1 receptor (Kiss1R) proteins were analyzed by Western blot in spermatozoa collected from caput and cauda epididymis
Kiss1 signal was very faint in caput epididymis spermatozoa, and the detected protein levels significantly decreased in spermatozoa collected from cauda epididymis (p < 0.001) (Figure 1B)

Summary

Introduction

Spermatogenesis, the process of male gamete production, requires the occurrence of mitosis, meiosis, and differentiation of haploid round spermatids into spermatozoa [1]. And peripherally produced modulators direct spermatogenesis via endocrine, paracrine, and autocrine routes; in this respect, the hypothalamic gonadotropin releasing hormone (GnRH), pituitary gonadotropins, and gonadal sex steroids are the main actors in spermatogenesis onset, progression, and maintenance [1]. The kisspeptin system has been included among the main central modulators of reproduction in mammalian species due to its ability to regulate upstream activity of GnRH secreting neurons. The kisspeptin system comprises the cleavage product of the Kiss precursor (i.e. kisspeptin (Kp)-54, originally known as metastin; Kp-14; Kp-13; and Kp-10] and the membrane G protein-coupled receptor, Kiss1R, previously known as Gpr54 [2]. Natural or induced loss/gain of function due to mutations in Kiss1/Kiss1r genes cause hypogonadotropic hypogonadism and precocious puberty, respectively [3–8]; this system is involved in the sex steroid feedback mechanisms [9] and in the epigenetic modulation of reproduction [10–13]

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