Coprs inactivation leads to a derepression of LINE1 transposons in spermatocytes

Abstract

Repression of retrotransposons is essential for genome integrity during germ cell development and is tightly controlled through epigenetic mechanisms. In primordial germ cells, protein arginine N-methyltransferase (Prmt5) is involved in retrotransposon repression by methylating Piwi proteins, which is part of the piRNA pathway. Here, we show that in mice, genetic inactivation of coprs (which is highly expressed in testis and encodes a histone-binding protein required for the targeting of Prmt5 activity) affects the maturation of spermatogonia to spermatids. Mass spectrometry analysis revealed the presence of Miwi in testis protein lysates immunoprecipitated with an anti-Coprs antibody. The observed deregulation of Miwi and pachytene pre-piRNAs levels and the derepression of LINE1 repetitive sequences observed in coprs-/- mice suggest that Coprs is implicated in genome surveillance mechanisms.

Keywords: Miwi; coprs; piRNA; spermatocyte; teratozoospermia; testis.

Figure 1

In mice, Coprs is localized in spermatogonia and its absence delays spermatid maturation. (A) Left panel: IHC staining of WT (n = 2) and KO (n = 2) testis sections with an anti‐Coprs antibody is presented. Bar = 1 mm. Right panel: images at higher resolution. Bar = 50 μm. (B) Flow cytometry analysis (left panels) and histograms (right panel) showing the distribution of the different mouse testis cell types. Sc: Sertoli cells; Rs/Re: round/elongated spermatids; SpB: type B spermatogonia; SpA: type A spermatogonia; L, Z, and P: leptotene, zygotene, and pachytene spermatocytes. Results are expressed as the percentage of each cell type relative to all counted cells and are the mean ± SEM (n = 3 WT; n = 4 KO). *P < 0.1 and **P < 0.05 (Student's t‐test). (C) RT‐qPCR analysis of the expression of the indicated genes in WT and KO testes. Expression was normalized to actin expression, and values are expressed in arbitrary units (a.u.) and are the mean ± SEM of RNA samples independently prepared (n = 3 WT; n = 4 KO). *P < 0.05 (Student's t‐test).

Figure 2

Identification of Miwi as a major protein immunoprecipitated with Coprs. Protein extracts from WT and coprs KO testes were immunoprecipitated with an anti‐Coprs antibody. Bands (*) present only in the WT lane were excised from the Colloidal Blue‐stained gel (left) and analyzed by mass spectrometry (right).

Figure 3

coprs KO affects Miwi expression level. (A) IHC analysis of WT and KO mouse testis sections with or without (negative control, ‐) an anti‐Miwi antibody; bar = 50 μm. (B) Left panel: western blot detection of Miwi expression in whole‐cell extracts from WT and KO testes with anti‐Miwi and anti‐Gapdh antibodies; right panel: quantification of Miwi expression with ImageJ after normalization to Gapdh expression. Values are expressed in arbitrary units (a.u.) and are the mean ± SEM of two independent experiments. (C) Miwi expression was assessed by qPCR. Data were normalized to actin level and expressed in arbitrary units (a.u.) (mean ± SEM of three independent mice/group). **P < 0.05 (Student's t‐test).

Figure 4

coprs KO deregulates the expression of pre‐piRNAs and LINE1. (A) The expression profile of the indicated pachytene piRNAs precursors (prepiR1, prepiR2, and prepiR3) was assessed by qPCR and normalized to actin level. Values are expressed in arbitrary units (a.u.) and are the mean ± SEM of three independent mice/group. (B) The RNA profile of the indicated transposons was assessed by qPCR and analyzed as in A); *P < 0.1; **P < 0.05; ***P < 0.001 (Student's t‐test).

Figure 5

Non‐Mendelian inheritance of the coprs‐floxed allele and analysis of different parameters in spermatozoa of WT and coprs KO mice. (A) Table showing the relative proportion of animals obtained from intercrosses between heterozygous coprs mice. (B) Histograms showing the mean ± SD of spermatozoa concentration and motility in semen samples from WT (n = 3) and coprs KO (n = 3) mice at 11 weeks of age. (C) Quantification of the morphology of spermatozoa (sp) from the same semen samples analyzed in (B) and expressed in percent represents the mean ± SD.

Figure 6

Low level of Coprs mRNA and teratozoospermia. (A) Transcription profiling of human sperm from individuals with normospermia (C: normally fertile) and teratozoospermia (T) (GEO data: E‐GEOD‐6872). (B) Coprs expression in semen mRNA samples from controls (C, n = 3) and patients with fertility problems (T, n = 6) was analyzed by RT‐qPCR. Data normalized to HPRT RNA and expressed in arbitrary units (a.u.) represent the mean ± SD; *P < 0.1 (Student's t‐test). (C) Clinical classification of each patient according to the % of typical spermatozoon forms (TF) evaluated using one of three classical methods, as indicated 33, 34, 35.