UBQLN1 interacts with SPEM1 and participates in spermiogenesis

Abstract

Spermiogenesis represents the process through which haploid male germ cells differentiate from round spermatids into elongated spermatids and eventually the male gametes called spermatozoa. Haploid cell differentiation is unique to male germ cell development and many unique genes/proteins essential for this process have been discovered. SPEM1 is one of these spermiogenesis-essential proteins encoded by a testis-specific gene exclusively expressed in the developing spermatids. Inactivation of Spem1 in mice results in deformed spermatozoa characterized by "head-bent-back" abnormalities with 100% penetrance. Using yeast two-hybrid screening assays, we identified UBQLN1 as one of the SPEM1-interacting partners. UBQLN1 and SPEM1 were colocalized to the manchette of elongating spermatids. Since UBQLN1 functions through binding and directing poly-ubiquitinated proteins to the proteasome for degradation, interactions between UBQLN1 and SPEM1 suggest a role in the regulation of protein ubiquitination during spermiogenesis.

Fig.1

Identification of UBQLN1 as a SPEM1-interacting protein using yeast 2-hybrid (Y2H) assay. (A) In the Y2H assay, SPEM1 was used as bait and an adult mouse testis library was screened. Interactions between SPEM1 and any testicular proteins would activate the expression of reporter genes, leading to yeast cell growth on selective medium. (B) Yeast co-transformation assays to confirm interactions between SPEM1 and two independent clones expressing UBQLN1. On the non-selective plates (SD/-Leu/-Trp) all transformed yeast cells grew, indicating viability of the cells and non-toxicity of the plasmids. Only cells co-transformed with pGBKT7-SPEM1 and pGDT7-UBQLN1 grew on the selective plates (SD/-Leu/-Trp/-His/-Ade), demonstrating true interactions between UBQLN1 and SPEM1 in yeast cells.

Fig. 2

Pull-down assays to verify the UBQLN1-SPEM1 interactions in mammalian cells. FLAG-tagged SPEM1 and V5-tagged UBQLN1 were both expressed in the COS-7 cells, as shown by Western blot analyses (WB). In the immunoprecipitation (IP) assays, V5-tagged UBQLN1 was detected in the anti-FLAG immunoprecipitants, suggesting UBQLN1 indeed interacts with SPEM1 in mammalian cells.

Fig. 3

Expression profiles of Ubqln1 mRNA and protein. (A) Northern blot analyses of Ubqln1 mRNAs in 12 mouse organs. 28S and 18S rRNA bands were shown as loading and mRNA quality controls. (B) Northern blot analyses of Ubqln1 mRNA in developing testes in mice. Testes of postnatal days 7-42 (P7-P42) were analyzed, and 28S and 18S rRNA bands were shown as loading and mRNA quality controls. (C) Levels of Ubqln1 mRNA in purified testicular cell populations determined using a semi-quantitative PCR method. The hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene was used as loaning control. PCR cycle number for Ubqln1 and Hprt were 26 and 20, respectively, which were tested to be within the exponential range. (D) Western blot analyses of UBQLN1 in mouse developing testes. ACTIN was used as a loading control.

Fig. 4

Immunofluorescent detection of UBQLN1 in the adult mouse testes. Green signals represent the immunoreactivity of UBQLN1 and the cell nuclei were counter-stained with propidium iodide (PI). Stages of the seminiferous epithelial cycle were shown in Roman numerals. Arrows point to the distinct, usually double-dotted nucleoli of Sertoli cells. Arrow heads indicate the signals of UBQLN1 immunoreactivity in the cytoplasm of elongating and elongated spermatids. All panels are in the same magnification and the scale bar represents 20 μm.

Fig. 5

Immunofluorescent localization of UBQLN1 to the manchette of elongating spermatids. Green fluorescence represents the UBQLN1 immunoreactivity and red fluorescence indicates the immunoreactivity of β-Tubulin, a marker for the manchette. Cell nuclei were counterstained using DAPI. All panels are in the same magnification. Scale bar = 10μm.

Fig. 6

Immunofluorescent localization of SPEM1 to the manchette of elongating spermatids. Green fluorescence represents the SPEM1 immunoreactivity and red fluorescence reflects the immunoreactivity of β-Tubulin, a marker for the manchette. Cell nuclei were counterstained using DAPI. All panels are in the same magnification. Scale bar = 10μm.

Fig. 7

Upregulation of UBQLN1 levels in the Spem1^-/- testes. (A) A representative result of Western blot analyses of UBQLN1 levels in adult wild-type (WT), Spem1^+/- and Spem1^-/- testes. ACTIN was used as a loading control. (B) Quantitative analyses of the Western blot results on UBQLN1 levels in WT, Spem1^+/- and Spem1^-/- testes. Bars represent mean ± SEM (n=3).