D-Asp upregulates PREP and GluA2/3 expressions and induces p-ERK1/2 and p-Akt in rat testis

in Reproduction

Correspondence should be addressed to S Minucci; Email: sergio.minucci@unicampania.it

*(A Santillo and M Venditti contributed equally to this work)

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D-Aspartate (D-Asp) is an endogenous amino acid that plays a central role in the development of the central nervous system (CNS) and functioning of the neuroendocrine system. In line with its functions, it is abundantly present in the CNS and reproductive systems of vertebrates and invertebrates. It has been implicated in the biosynthesis and/or secretion of hormones and factors that are involved in various reproductive functions, such as GnRH from the hypothalamus and testosterone from the testis. We conducted an in vivo study consisting of acute (i.p. injection of 2 µmol/g body weight) and chronic (15 days drinking solution) administration of D-Asp to adult rats to understand the signaling pathways elicited by D-Asp in the rat testis. We found that D-Asp upregulated the expression of prolyl endopeptidase (PREP), a serine protease having a pivotal role in the regulation of mammalian spermatogenesis and spermiogenesis. Immunofluorescence analysis revealed its overexpression in Leydig cells, Sertoli cells and spermatogonia. Moreover, PREP was found to co-localize with GluA2/3, an AMPA receptor subunit, whose protein expression also increased after D-Asp treatments. Finally, we found a significant increase in ERK and Akt activities in the testis of rats treated with D-Asp. Since PREP is known to be involved in regulating GnRH levels and in germ cell differentiation, we hypothesize D-Asp to play a pivotal role in regulating hormone homeostasis and spermatogenesis through activation of PREP, AMPAR, ERK and Akt.

Supplementary Materials

    • Fig. 1: Immunofluorescence co-localization analysis for GluA2/3 and PREP in rat testis from controls (a), and at 5 h (b) and 15 days (c) after D-Asp treatment. a, b, c: GluA2/3 (green), PREP (red) and DAPI-fluorescent nuclear staining (blue). The co-localization of the two proteins is given by the yellow tint, in Leydig cells (asterisk) and spermatogonia (arrowheads). PREP localizes also in the Sertoli cells cytoplasm (arrows). The images in the insets were captured at X40 magnification, all the others at X20 magnification. Scale bars represent 20 μm, except for the insets, where they represent 10 μm.
    • Fig. 2: Immunofluorescence co-localization analysis for GluA2/3 and p-ERK1/2 in rat testis from controls (a) and at 30 min (b), 2 h (c) and 5 h (d) after D-Asp acute treatment. a, b, c, d: GluA2/3 fluorescence (green), p-ERK1/2 staining (red) and DAPI-fluorescent nuclear staining (blue). The co-localization of the two proteins is given by the yellow tint, in Leydig cells (asterisk) and spermatogonia (arrowheads). p-ERK1/2 localize also in the Sertoli cells cytoplasm and in spermatozoa. The images in the insets were captured at X40 magnification, all the others at X20 magnification. Scale bars represent 20 μm, except for the insets, where they represent 10 μm.
    • Fig. 3: Immunofluorescence co-localization analysis for GluA2/3 and p-Akt in rat testis from controls (a) and at 30 min (b), 2 h (c) and 5 h (d) after D-Asp acute treatment. a, b, c, d: GluA2/3 fluorescence (green), p-Akt staining (red) and DAPI-fluorescent nuclear staining (blue). The co-localization of the two proteins is given by the yellow tint, in Leydig cells (asterisk) and spermatogonia (arrowheads) and spermatocytes. p-Akt localize also in the Sertoli cells cytoplasmic protrusions. The images in the insets were captured at X40 magnification, all the others at X20 magnification. Scale bars represent 20 μm, except for the insets, where they represent 10 μm.
    • Fig. 4: Specificity controls of the used antibodies. A: Western blot analysis carried out on total protein extracts from control rat testis, using PREP antiserum alone (left lane) or preadsorbed 5fold excess with the epitope (right lane). B: Immunofuorescence staining in control rat testis. PREP specificity control was checked by preadsorbing primary antiserum with of the corresponding epitope. C: IgG isotype controls, obtained using rabbit IgG serum at the same concentration as antibodies (a-e); negative controls, obtained by omitting the primary antibody (f-j). a,f: controls; b,g: 30 min D-Asp; c,h: 2h D-Asp; d,i: 5h D-Asp; e,j: 15 days D-Asp. The scale bars in B an C represent 20 μm.

 

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