PIAS (protein inhibitor of activated STAT) proteins interact with and modulate the activities of various transcription factors. In this work, we demonstrate that PIAS proteins x␣, x␤, 1, and 3 interact with the small ubiquitin-related modifier SUMO-1 and its E2 conjugase, Ubc9, and that PIAS proteins themselves are covalently modified by SUMO-1 (sumoylated). PIAS proteins also tether other sumoylated proteins in a noncovalent fashion. Furthermore, recombinant PIASx␣ enhances Ubc9-mediated sumoylation of the androgen receptor and c-Jun in vitro. Importantly, PIAS proteins differ in their abilities to promote sumoylation in intact cells. The ability to stimulate protein sumoylation and the interaction with sumoylated proteins are dependent on the conserved PIAS RING finger-like domain. These functions are linked to the activity of PIASx␣ on androgen receptor-dependent transcription. Collectively, our results imply that PIAS proteins function as SUMO-1-tethering proteins and zinc finger-dependent E3 SUMO protein ligases, and these properties are likely to explain their ability to modulate the activities of various transcription factors.Members of the recently identified PIAS (protein inhibitor of activated STAT) protein family have been found to interact with several distinct nuclear proteins. PIAS1 and PIAS3 bind to STAT1 and STAT3, respectively, and inhibit their action (4, 28). PIASx␣/ARIP3 (androgen receptor [AR]-interacting protein 3) was first characterized as an AR-interacting protein, and it modulates the transcriptional activity of the receptor (34). Other PIAS proteins have recently been demonstrated to function as coregulators for AR and other steroid receptors (25, 57). PIASx␤/Miz1 (Msx-interacting zinc finger) associates with a homeodomain-containing Msx2 protein (61), and GBP (Gu/RNA helicase II-binding protein), which is nearly identical to PIAS1, interacts with Gu/RNA II-helicase (59). More recently, PIAS proteins have been identified in many yeast two-hybrid screens, including PIASx␣/ARIP3 from its interaction with mouse disabled 2 (mDab2) (3) and DJ-1 protein (54), PIAS1 from its interaction with p53 (9), and PIAS3 from its interaction with high-mobility-group protein HMGI-C (63) and zinc finger protein Gfi-1 (43). In addition to PIASx (PIASx␣/ARIP3 and PIASx␤/Miz1), SUMO-1 (small ubiquitinrelated modifier 1) was identified as a p73␣-interacting protein by Minty et al. (32), who suggested that PIASx was isolated via the interaction with Smt3p (yeast SUMO) covalently linked to p73␣. We have also detected SUMO-1 as a major PIASx␣/ ARIP3-interacting protein in yeast (unpublished results).Members of the SUMO protein family, also known as Sentrin, GMP1, PIC1, and Ubl1 (31, 37, 62), are present in protozoa, metazoa, plants, and fungi. SUMO proteins from metazoa can be divided into two families: the SUMO-1 family and the SUMO-2 and -3 family (31, 37, 62). SUMO-2 and SUMO-3 are very similar at the amino acid level (97% identity for the human proteins), but they are only ϳ50% identical to SUMO-1. SUMO-1 is...

The chromatoid body is a perinuclear, cytoplasmic cloud-like structure in male germ cells whose function has remained elusive. Here we show that the chromatoid body is related to the RNA-processing body of somatic cells. Dicer and components of microRNP complexes (including Ago proteins and microRNAs) are highly concentrated in chromatoid bodies. Furthermore, we show that Dicer interacts with a germ cell-specific chromatoid body component, the RNA helicase MVH (mouse VASA homolog). Thus, chromatoid bodies seem to operate as intracellular nerve centers of the microRNA pathway. Our findings underscore the importance of posttranscriptional gene regulation and of the microRNA pathway in the control of postmeiotic male germ cell differentiation.Argonaute ͉ microRNA ͉ spermatogenesis ͉ RNA processing

The chromatoid body, a unique cloud-like structure of male germ cells, moves dynamically in the cytoplasm of haploid spermatids, but its function has remained elusive for decades. Recent findings indicate that microRNA and RNA-decay pathways converge to the chromatoid body. This highly specialized structure might function as an intracellular focal domain that organizes and controls RNA processing in male germ cells.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.

No abstract

Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell involving replacement of the somatic histones by protamines packing the DNA into the condensed spermatid nucleus and elimination of the cytoplasm during the elongation phase. We describe H1T2, an histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis. In round and elongating spermatids, H1T2 specifically localizes to a chromatin domain at the apical pole, revealing a polarity in the spermatid nucleus. Inactivation by homologous recombination shows that H1T2 is critical for spermiogenesis as male H1t2 ؊/؊ mice have greatly reduced fertility. Analysis of spermiogenesis in H1t2 mutant mice shows delayed nuclear condensation and aberrant elongation. As a result, mutant spermatids are characterized by the presence of residual cytoplasm, acrosome detachment, and fragmented DNA. Hence, H1T2 is a protein required for proper cell restructuring and DNA condensation during the elongation phase of spermiogenesis.chromatin ͉ mouse ͉ protamine ͉ acrosome M ammalian spermatogenesis involves the differentiation of diploid spermatogonia into spermatocytes and then, through two successive meiotic divisions, into haploid round spermatids. During spermiogenesis, the haploid round spermatids undergo an elongation phase, transforming them into mature spermatozoa. This process entails a major biochemical and morphological restructuring of the germ cell where the majority of the somatic histones are replaced, first by transition proteins, then protamines packing the DNA into the sperm cell nucleus.Differentiating male germ cells use specialized transcriptional regulatory mechanisms involving testis-specific paralogues of transcription factors such as TLF͞TRF2, ALF, and TAF7L (1-3). In addition, male germ cells contain specialized chromatin components such as H1T, a histone H1 variant specifically expressed in pachytene spermatocytes and early haploid cells (4), and HILS1, a histone H1-related protein specifically expressed in elongating and elongated spermatids (5). However, no spermatogenesis abnormalities are observed in mice lacking the H1t gene, perhaps because of redundancy with the somatic H1.1, which is also expressed at this stage (6-9). This finding contrasts with the reduced fertility and defective spermiogenesis in mice lacking transition proteins 1 or 2 or haploinsufficient for protamines 1 or 2 (10-12). Thus, whereas H1 histones have been proposed to play a role in the formation of higher-order chromatin structure, the physiological functions of the tissue-specific variants remain obscure. Here, we describe a male germ cellspecific H1-related protein, H1T2, that plays a critical role during the elongation phase of spermiogenesis. Materials and MethodsCloning and Inactivation of H1t2. The H1t2 cDNA was cloned from a mouse testis cDNA library by screening with the short mouse TEST640 EST probe as described (2, 13). 5Ј RACE using mouse testis RNA was performed to confirm the sequence o...

Spermatogenesis constitutes a remarkable program of cell differentiation, which involves dramatic changes in cell morphology, biochemistry and gene expression 1,2 . But the study of male germ cells is complicated by the exceptional organization of the seminiferous epithelium and by the lack of established cell lines that are able to recapitulate any of the multiple differentiation steps of the spermatogenesis program in vitro. Cell types with differences in their sedimentation properties or cell surface markers can be isolated from the whole tissue by various methods, but these methods do not allow accurate identification of all the differentiation stages. As a consequence of strict paracrine regulation by Sertoli cells, spermatogenesis proceeds in synchronized waves along the seminiferous tubules, and every given cross-section of the tubule contains only certain cell types in a specific combination (Fig. 1). The light absorption pattern of a seminiferous tubule, as seen under a dissection microscope, correlates with defined stages of the spermatogenic wave, which makes it possible to isolate specific stages on the basis of their transillumination properties 3-6 . The accuracy of the isolation of specific stages can be improved by combining it with phase-contrast microscopy of live cell preparations 7-10 . The staging of the spermatogenic cycle is best characterized in rat and mouse 11,12 . Here, we describe a method designed to identify, isolate and characterize mouse male germ cells at specific steps of differentiation by transillumination-assisted microdissection. The distinctive morphological features of germ cells, as detected by phase-contrast microscopy, are detailed. Although the method described here generates small quantities of cells and therefore does not allow for further isolation of the different cell types comprising each differentiation stage, the applications of this method are powerful and diverse. It enables the monitoring of spermatogenic differentiation events, and in combination with biochemical analyses, it allows the characterization of the molecular mechanisms governing these processes. This method can be used for the identification of the effects of cytotoxic and environmental factors on male reproductive function, as well as for the rapid and comprehensive diagnosis and characterization of sperm cell defects attributable to infertility. In combination with gene targeting models, it enables the characterization of the critical factors involved in the cell cycle, chromatin dynamics, spermatid differentiation, stem cell biology and fertility.

Primary ciliary dyskinesia (PCD) results from defects in motile cilia function. Mice homozygous for the mutation big giant head (bgh) have several abnormalities commonly associated with PCD, including hydrocephalus, male infertility, and sinusitis. In the present study, we use a variety of histopathological and cell biological techniques to characterize the bgh phenotype, and we identify the bgh mutation using a positional cloning approach. Histopathological, immunofluorescence, and electron microscopic analyses demonstrate that the male infertility results from shortened flagella and disorganized axonemal and accessory structures in elongating spermatids and mature sperm. In addition, there is a reduced number of elongating spermatids during spermatogenesis and mature sperm in the epididymis. Histological analyses show that the hydrocephalus is characterized by severe dilatation of the lateral ventricles and that bgh sinuses have an accumulation of mucus infiltrated by neutrophils. In contrast to the sperm phenotype, electron microscopy demonstrates that mutant respiratory epithelial cilia are ultrastructurally normal, but video microscopic analysis shows that their beat frequency is lower than that of wild-type cilia. Through a positional cloning approach, we identified two sequence variants in the gene encoding sperm flagellar protein 2 (SPEF2), which has been postulated to play an important role in spermatogenesis and flagellar assembly. A causative nonsense mutation was validated by Western blot analysis, strongly suggesting that the bgh phenotype results from the loss of SPEF2 function. Taken together, the data in this study demonstrate that SPEF2 is required for cilia function and identify a new genetic cause of PCD in mice.