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PLoS One
2019 Jun 10;146:e0217941. doi: 10.1371/journal.pone.0217941.
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Expression of genome defence protein members in proliferating and quiescent rat male germ cells and the Nuage dynamics.
Rocha-da-Silva L
,
Armelin-Correa L
,
Cantão IH
,
Flister VJF
,
Nunes M
,
Stumpp T
.
Abstract
During epigenetic reprogramming germ cells activate alternative mechanisms to maintain the repression retrotransposons. This mechanism involves the recruitment of genome defence proteins such as MAEL, PIWIL4 and TDRD9, which associate with piRNAs and promote Line-1 silencing. MAEL, PIWIL4 and TDRD9 form the piP-bodies, which organization and dynamics vary according to the stage of germ cell epigenetic reprogramming. Although these data have been well documented in mice, it is not known how this mechanism operates in the rat. Thus, the aim of this study was to describe the distribution and interaction of MAEL, PIWIL4, TDRD9 and DAZL during rat germ cell development and check whether specific localization of these proteins is related to the distribution of Line-1 aggregates. Rat embryo gonads at 15 days post-conception (dpc), 16dpc and 19dpc were submitted to MAEL, PIWIL4, TDRD9 and DAZL immunolabelling. The gonads of 19dpc embryos were submitted to the double-labelling of MAEL/DAZL, TDRD9/MAEL and PIWIL4/MAEL. The 19dpc gonads were submitted to co-immunoprecipitation assays and fluorescent in situ hybridization for Line-1 detection. MAEL and TDRD9 showed very similar localization at all ages, whereas DAZL and PIWIL4 showed specific distribution, with PIWIL4 showing shuttling from the nucleus to the cytoplasm by the end epigenetic reprogramming. In quiescent 19dpc gonocytes all proteins colocalized in a nuage adjacent to the nucleus. DAZL interacts with PIWIL4 and MAEL, suggesting that DAZL acts with these proteins to repress Line-1. TDRD9, however, does not interact with DAZL or MAEL despite their colocalization. Line-1 aggregates were detected predominantly in the nuclear periphery, although did not show homogeneous distribution as observed for the nuage. In conclusion, the nuage in quiescent rat gonocytes show a very distinguished organization that might be related to the organization of Line-1 clusters and describe the association of DAZL with proteins responsible for Line-1 repression.
Fig 1. Immunodetection of MAEL (A, B and C) and TDRD9 (D, E and F) in rat embryo gonads.At 15dpc (A e D) and 16dpc (B e E) the nuage (arrowheads) are distributed more homogeneously around the gonocyte nucleus. At 19dpc (C and F) the nuage (arrowheads) is adjacent to a restricted portion of the gonocyte nucleus.
Fig 2. Immunodetection of PIWIL4 (A, B and C) and DAZL (D, E e F) in rat embryo gonads.At 15dpc (A) is present in the nucleus of the (arrowheads), whereas DAZL (D) is localized in the cytoplasm, (arrowheads). At 16dpc (B and E) both proteins are localized in the cytoplasm (arrowheads), but PIWIL4 distribution is more restricted to the region close to the nucleus (arrowheads), whereas DAZL is diffusely distributed throughout the cytoplasm (arrowhead). At 19dpc (C and F), as observed for MAEL and TDRD9, the nuage (arrowheads) is is adjacent to a restricted portion of the gonocyte nucleus.
Fig 3. Colocalization of DAZL/TDRD9 (A), DAZL/PIWIL4 (B) and DAZL/MAEL (C) in the 19dpc gonads.The colocalization can be observed by the yellow/orange color in merge.
Fig 4. Western Blotting for MAEL, DAZL, PIWI4 and TDRD9.A) DAZL, that has been used as the bait, was present in the whole gonad extract (E19) and is absent from the Co-IP yield (Co-IP). B) PIWIL4 was detected in the Co-IP yield (Co-IP) and in the whole gonads extract (E19). C) TDRD9 was detected in the whole extract (E19), but not in the Co-IP yield (Co-IP). D) MAEL, as observed for PIWIL4, was detected in the whole extract and in the Co-IP yield (Co-IP). E) ß-Actin was used as the reference protein. T: testis; M: muscle.
Fig 5. Western Blotting for MAEL and TDRD9.A) MAEL was detected in the whole extract (E19) and in the testis (Tes), but not in the Co-IP yield (Co-IP). B) As observed for MAEL, TDRD9 was detected in the whole extract (E19) and in the testis (T), but not in the Co-IP yield (Co-IP). C) ß-Actin was used as the reference protein.
Fig 6. Detection of Line-1 in the nuclear genome by fluorescent in situ hybridization (FISH) in the 19dpc gonocytes.Line-1 aggregates are observed more frequently in the periphery of the gonocyte nucleus (arrows). A: DAPI; B: Line-1; C: merge.
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