Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice

Abstract

Transition nuclear proteins (TPs), the major proteins found in chromatin of condensing spermatids, are believed to be important for histone displacement and chromatin condensation during mammalian spermatogenesis. We generated mice lacking the major TP, TP1, by targeted deletion of the Tnp1 gene in mouse embryonic stem cells. Surprisingly, testis weights and sperm production were normal in the mutant mice, and only subtle abnormalities were observed in sperm morphology. Electron microscopy revealed large rod-like structures in the chromatin of mutant step 13 spermatids, in contrast to the fine chromatin fibrils observed in wild type. Steps 12-13 spermatid nuclei from the testis of Tnp1-null mice contained, in place of TP1, elevated levels of TP2 and some protamine 2 (P2) precursor. Most of the precursor was processed to mature P2, but high levels of incompletely processed forms remained in epididymal spermatozoa. Sperm motility was reduced severely, and Ϸ60% of Tnp1-null males were infertile. We concluded that TP1 is not essential for histone displacement or chromatin condensation. The absence of TP1 may partially be compensated for by TP2 and P2 precursor, but this dysregulation of nucleoprotein replacement results in an abnormal pattern of chromatin condensation and in reduced fertility. The transformation of spermatids into spermatozoa (spermiogenesis) involves the most dramatic changes in chromatin structure and function that occur in any cell type. During the latter part of spermiogenesis, the nucleus elongates, transcription ceases, the histones are almost completely removed, and the chromatin appears as smooth fibers and then becomes highly condensed (1). In many animal and plant species, chromatin condensation is facilitated by the association of highly basic nuclear proteins, the protamines (2). The transition from histone-containing chromatin to the protamine-associated one seems to occur directly in fish and birds (2). However, in mammals (3), small, basic nuclear proteins appear when the histones are displaced and chromatin condensation is initiated; they are referred to as transition nuclear proteins (TPs), because they are subsequently replaced by protamines (3).Although other TPs exist (4), TP1 and TP2 are the predominant ones found in rodent spermatids (5). TP1, a 6.2-kDa protein, consists of Ϸ20% each arginine and lysine and lacks cysteine (6, 7). TP2, a 13-kDa protein, consists of Ϸ10% each arginine and lysine and 5% cysteine (5). TP1 is expressed abundantly in most mammals (6) and is highly conserved, showing cDNA nucleotide and amino acid sequence homologies of 90% across species (8). The TPs are localized exclusively to nuclei of condensing spermatids (3, 9), and in the rat, constitute Ͼ90% of basic chromosomal proteins of these nuclei (10).During human (11) and mouse (12) spermiogenesis, the TPs are replaced by two protamines, protamine 1 (P1) and protamine 2 (P2). Whereas P1 is synthesized as a mature protein, P2 is synthesized as the precursor. In mouse, mature P2 of 63 residues is derive...

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“…Indeed, the formation of a mature, compacted sperm nucleus (characterized by the replacement of nuclear histones with transition proteins and then protamines) and the development of the sperm flagellum, both originate during spermiogenesis. Moreover, experimental studies have demonstrated that targeted disruption of nuclear chromatin compaction (using protamine or transition protein insufficiency models) is associated with the development of an abnormal flagellum and defective motility [16,36]. Another potential explanation for the observed association between DNA integrity and motility lies in the inherent susceptibility of human spermatozoa to oxidative stress.…”

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“…Moreover, the expression patterns of all postmeiotic genes examined, such as Tp1 (transition protein 1), Tp2 (transition protein 2), Prm2 (protamine 2), Gapdhs (glyceraldehyde 3-phosphate dehydrogenase-S, testis specific), Trf2 (TBP-related factor 2), and Crem (cyclic AMP-responsive element modulator) were comparable in Cib1 ϩ/ϩ and Cib1 Ϫ/Ϫ mice ( Fig. 4A; Tp1 and Gapdhs are not shown) (1,41,47,49). This is in sharp contrast to Crem knockout or mutant mice and Trf2 knockout mice, which have marked defects during the round spermatid stages and altered expression of the aforementioned genes (3,20,28,41,49).…”