LEM-domain proteins are lost during human spermiogenesis but BAF and BAF-L persist

Abstract

During spermiogenesis the spermatid nucleus is elongated, and dramatically reduced in size with protamines replacing histones to produce a highly compacted chromatin. After fertilisation, this process is reversed in the oocyte to form the male pronucleus. Emerging evidence, including the coordinated loss of the nuclear lamina (NL) and the histones, supports the involvement of the NL in spermatid nuclear remodelling, but how the NL links to the chromatin is not known. In somatic cells, interactions between the NL and the chromatin have been demonstrated: LEM-domain proteins and LBR interact with the NL and respectively, the chromatin proteins BAF and HP1. We therefore sought to characterise the lamina-chromatin interface during spermiogenesis, by investigating the localisation of six LEM-domain proteins, two BAF proteins and LBR, in human spermatids and spermatozoa. Using RT-PCR, IF and western blotting, we show that six of the proteins tested are present in spermatids: LEMD1, LEMD2 (a short isoform), ANKLE2, LAP2β, BAF and BAF-L, and three absent: Emerin, LBR and LEMD3. The full-length LEMD2 isoform, required for nuclear integrity in somatic cells, is absent. In spermatids, no protein localised to the nuclear periphery, but five were nucleoplasmic, receding towards the posterior nuclear pole as spermatids matured. Our study therefore establishes that the lamina-chromatin interface in human spermatids is radically distinct from that defined in somatic cells. In ejaculated spermatozoa, we detected only BAF and BAF-L, suggesting that they might contribute to the shaping of the spermatozoon nucleus and, after fertilisation, its transition to the male pronucleus.

Nuclear envelope dynamics during mammalian spermatogenesis: new insights on male fertility

“…Similar to LAP2 structure, LEMD1 is composed of a large nucleoplasmic domain comprising the LEM motif at the N‐terminus, a single transmembrane domain and a short perinuclear domain at the C‐terminus (Wagner & Krohne, ; Barton, Soshnev & Geyer, ). The expression of this NE protein is largely restricted to the testis, where six alternatively spliced isoforms can be found (Yuki et al ., ), and it is thought to have a role in chromatin remodelling during spermiogenesis (Table ; see Section III.2) (Elkhatib et al ., ).…”

“…In this study, testis‐specific LEMD1 was also found to exhibit an atypical nucleoplasmic localization in human post‐meiotic male germline cells, in which it is excluded from the acrosomal area of early round spermatids and progressively concentrates at the posterior region from late round spermatids to elongated spermatids, disappearing in mature spermatozoa (Table ; Fig. ) (Elkhatib et al ., ). Similarly, LAP2, presumably the β isoform, was reported to be initially present at the nuclear periphery of human early round spermatids and to gradually relocate to the nucleoplasm until spermatid elongation is initiated, when it is no longer detectable (Table ) (Elkhatib et al ., ).…”

“…) (Elkhatib et al ., ). Similarly, LAP2, presumably the β isoform, was reported to be initially present at the nuclear periphery of human early round spermatids and to gradually relocate to the nucleoplasm until spermatid elongation is initiated, when it is no longer detectable (Table ) (Elkhatib et al ., ). The finding that LEMD1 and LAP2β are potentially not retained in the NE of human spermatids is quite unexpected and deserves further study.…”

“…Relevant NE proteins include: four nuclear lamina proteins, namely lamins A/C, B1, B3 and C2; 10 INM proteins, specifically Dpy‐19‐like protein 2 (DPY19L2), lamina‐associated polypeptides 1 and 2 (LAP1 and LAP2), lamin B receptor (LBR), LAP2/emerin/MAN1 (LEM) domain‐containing protein 1 (LEMD1) and Sad‐1/UNC‐84 homology domain‐containing proteins 1–5 (SUN1–SUN5, among which is the SUN1η isoform); three ONM proteins, including Klarsicht/ANC‐1/Syne‐1 homology domain‐containing protein 5 (KASH5), nesprin‐1 and nesprin‐3; and a newly identified NE protein termed spermatogenesis‐associated 46 (SPATA46). Of note, the expression of lamin B3 (Furukawa & Hotta, ; Schutz et al ., ), lamin C2 (Furukawa et al ., ), DPY19L2 (Carson, Cheung & Scherer, ; Pierre et al ., ), LEMD1 (Yuki et al ., ; Elkhatib et al ., ), SUN1η, SUN3 (Gob et al, ), SUN4 (Shao et al ., ; Pasch et al ., ; Yang et al, ), SUN5 (Frohnert, Schweizer & Hoyer‐Fender, ; Jiang et al ., ; Shang et al ., ), KASH5 (Morimoto et al ., ; Horn et al ., ) and SPATA46 (Chen et al ., ) is largely restricted to male germline cells, suggesting highly specialized roles in the testis.…”

“…In recent decades, numerous studies have demonstrated that spermatogenesis requires extensive rearrangement of the NE network, including the composition of its specific proteome and the localization of these proteins. Interestingly, the list of NE proteins restricted to or highly enriched in mammalian male germline cells is rapidly increasing, comprising a total of 10 NE components at present (Furukawa, Inagaki & Hotta, ; Schutz et al ., ; Gob et al ., ; Morimoto et al ., ; Pierre et al ., ; Pasch et al ., ; Chen et al ., ; Shang et al ., ; Elkhatib et al ., ). Furthermore, there is growing evidence that mutations in human genes encoding NE proteins can have deleterious effects on the development of fertilization‐competent spermatozoa and lead to severe infertility phenotypes in men (Zhu et al ., , ).…”