Determinants of sperm nuclear shaping in the genus Xenopus

The morphogenesis of sperm nuclei was investigated in six different species or subspecies of the genus Xenopus (Pipidae, Anura). The sequence of nuclear morphogenesis was similar in each species used in this study. Electrophoretic comparison of the basic chromatin proteins from late spermatids and sperm of each species demonstrated that the complements of histones and spermatid-sperm-specific basic proteins were extremely diverse suggesting that shape was not determined by specific basic proteins or mechanisms of histone removal. This conclusion was reinforced by the observation that Xenopus sperm DNA decondensed by 2.0 M NaCl remained contrained in residual structures which resembled intact sperm nuclei. These observations suggested that morphogenesis of sperm nuclei is directed by proteins or RNA molecules which are not directly responsible for chromatin condensation.     

Poly(ADP-ribose) polymerases PARP1 and PARP2 modulate topoisomerase II beta (TOP2B) function during chromatin condensation in mouse spermiogenesis

To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling.     

THE FINE STRUCTURE AND CHEMICAL COMPOSITION OF NUCLEI DURING SPERMIOGENESIS IN THE HOUSE CRICKET : I. Initial Stages of Differentiation and the Loss of Nonhistone Protein

The early stages of nuclear differentiation in spermatids of the house cricket are described with regard to the fine structural elements and chemical components which occur. Particular attention is given to the loss of nonhistone protein from the nucleus and its relation to chromatin structure. Granular elements about 25 to 80 mµ in diameter, and fibers about 8 mµ in diameter occur in the earliest spermatid nucleus. The fibers are found in diffuse and condensed chromatin while granules are found only in diffuse material. DNA and histone parallel the chromatin fibers in distribution, while nonhistone protein and RNA parallel the granules in distribution. The granules and most of the nonhistone protein are lost, simultaneously, after the early spermatid stage. The protein loss occurs without detectable change in the structure of chromatin fibers. Chromatin fibers first show a structural change in mid spermiogenesis, when they become thicker and very contorted. Unusually thin fibers (about 5 mµ) also appear in mid spermatid nuclei; they are apparently composed of nonhistone protein and free of DNA and histone.     

DNA and histone methylation in plants

Heritable patterns of gene activity and gene silencing arise by the formation and the propagation of specific chromatin states that restrict or permit gene expression. In mammals and in plants, restrictive heterochromatin is associated with the hypermethylation of DNA at CG sites and with the specific modification of histones, such as the methylation of histone H3 at lysine 9 (H3K9(Me)). In addition to CG methylation, plant nuclear DNA packaged in restrictive chromatin is also usually methylated in cytosines outside a CG sequence context. The functional relationship between an unexpectedly complex plant DNA-methylation system and histone modifications that lead to chromatin compaction and gene silencing is under intense scrutiny. The results of recent studies indicate intriguing links between chromatin remodeling, histone methylation, DNA methylation and RNA interference.     

Ultrastructural localization of phosphatases in the testes of the domestic fowl: Acid phosphatase and thiamine pyrophosphatase

Summary ACPase and TPPase activity has been examined in the germinal epithelium of the testes in the domestic fowl. ACPase activity in spermatogonia and spermatocytes was confined to the Golgi complex. In spermatids ACPase activity was seen in the endoplasmic reticulum and nuclear envelope in the phase I and especially in the phase II (the elongating phase). This activity gradually decreased during the next phase III, and had disappeared in the final phase IV. The membrane body showed ACPase reaction in the small peripheral vacuoles and cisternal structures surrounding large central vacuoles. ACPase was also present in vesicles surrounding the developing tail. Late spermatids showed an abundance of autophagic vacuoles which had a complex array of ACPase positive delimiting membranes. In Sertoli cells ACPase activity was predominant in the lysosomes. TPPase activity was seen in the cisternae of the Golgi complex in spermatogonia and spermatocytes. In spermatids activity was present in the endoplasmic reticulum during the phase II, but it is lost in later stages. The smaller vacuoles and cisternal structures in the membrane body also showed reaction products. According to the present results it is thought likely that the smaller vacuoles and cisternal structures of the membrane body are of endoplasmic reticulum origin. The autophagic vacuoles in spermatids and the lysosomes of Sertoli cells are considered responsible for the degradation of residual bodies cast off by spermatids.     

Chromosome behaviour during meiotic prophase in the solanaceae

The molecular structure of chromatin during dogfish spermiogenesis was examined by electron microscopy after the dispersion of nuclei at low ionic strength. In early and late stages of differentiation (round and elongating spermatids), chromatin is globular, although basic nuclear proteins are different from those present in somatic nuclei. Three protein fractions are complexed with DNA in sperm nuclei. These fractions appear at the end of differentiation (elongated spermatids), subsequently undergoing a modification of their solubilization properties; only one protein fraction remains acid-soluble. Dispersed chromatin from sperm nuclei again shows a beads-on-a-string configuration both in the presence of the three specific sperm proteins and when the acid soluble fraction is extracted. Variations of the mean diameter of chromatin subunits during spermiogenesis appear rather limited compared to extensive modifications of chromatin superstructures.     

The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.     

Cycle specific association of nascent chromatin with nuclear envelope components in Physarum polycephalum.

The action of heparin on isolated nuclei derived from different phases of the mitotic cycle in plasmodia of Physarum polycephalum was studied. Heparin addition at two-fold excess over DNA concentration to nuclei in Mg-free low ionic strength buffer (10 mM TRIS-HC1, 10 mM Na2 HPO4, pH = 8) releases 60-80% of chromatin from S, G2, and mitotic phase nuclei. The RNA/protein ratio of herparin-solubilized cromatin is constant through S and G2 phases, but rises about two-fold at early prophase coincident with nucleolar breakdown. Purified nuclear envelopes were obtained from heparin-treated nuclei by sedimentation according to Bornens procedures (Nature 244, 28, 1973), and examined by transmission electron microscopy. Residual chromatin is seen at all stages with fine network of DNA fibrils in contact with the envelop. Regardless of time in S, 80% of 3H-labeled DNA was released into soluble chromatin with identical 3H/14C ratios. The residual chromatin in nuclear envelopes exhibited a preferential association of early S-DNA in nuclei engaged in early S replication, and late S preferential association in nuclei engaged in late S replication.     

Changes in intranuclear chromatin architecture induce bipolar nuclear localization of histone variant H1T2 in male haploid spermatids

Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell packing the DNA into the condensed spermatid nucleus. H1T2 is a histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis that specifically localizes to a chromatin domain at the apical pole under the acrosome. We explored the mechanisms determining polar localization of H1T2 in spermatids. In acrosome-deficient round spermatids of hrb -/- and gopc -/- mice, H1T2 localization is not altered, indicating that proper acrosome development is not required for specifying nuclear polarity. In contrast, in late round spermatids from trf2 -/- or hmgb2 -/- mice, a bipolar H1T2 localization was observed revealing that polarity is modified by loss of proteins specifying chromatin architecture. Our results show that intranuclear chromatin organization is critical for correct polar localization of H1T2 and that H1T2 can be a useful molecular marker revealing chromatin disorganization in spermatids.     

Cytological steps during spermiogenesis in the house sparrow (Passer domesticus,Linnaeus)

Spermiogenesis of the domestic sparrow was investigated with the light and electron microscopes and a step by step classification is proposed. Three cell populations corresponding to early, mid and late spermatids were easily divided according to their positions in the seminiferous epithelium. In addition to this initial separation, six steps were recognized, based on nuclear morphology and the degree of chromatin condensation, in association to their acrosomal and flagellar development. Early spermiogenesis is the period previous to chromatin condensation. The first step can be recognized by the extending flagellum and the second by the pro-acrosome development in contact with the nucleus. During the third or intermediate step, chromatin condenses and the cell becomes polarized with the pro-acrosomic vesicle and the tail occupying opposite sides of the nucleus. Late spermiogenesis, including steps IV-VI, is marked by complete chromatin condensation. The final cellular modifications lead to the formation of a spiraled spermatozoon. This shape is due to the twisting of the acrosome and nucleus, as well as the helical arrangement of mitochondria around the axoneme along most of the flagellum, making an exceptionally long middle piece.     

Spermiogenesis and sperm structure in the crabUca tangeri (Crustacea,Brachyura), with special reference to the acrosome differentiation

Summary Early spermatids of the crabUca tangeri consists of the nucleus of granular chromatin and the cytoplasm, which contains a proacrosomal vesicle in close association with membrane lamellae. In the mid spermatids an invagination of the acrosomal vesicle membrane gives rise to the formation of the perforatorium, a spindle-shaped tubule which encloses tubular membranous structures. The pair of centrioles located at the base of the acrosome is not directly involved in perforatorial differentiation. The acrosomal vesicle shows a heterogeneous content composed of the operculum, the thickened ring, and three layers of different materials concentrically arranged around the perforatorium. During the late spermatid stage the nuclear profile differentiates numerous slender arms and the chromatin arranges into fibers. Membranous tubules from the cytoplasm become incorporated into the tubular structures of the perforatorium. The mature spermatozoon has the typical structure of the branchyuran sperm, with a complex acrosome, cupped by the nucleus, and a thin cytoplasmic band intervening between the former main elements. The centrioles are degenerate. The nuclear arms are unusually numerous (more than 20) and lack microtubules or microtubular derivatives.     

Possible function of caudal nuclear pocket: degradation of nucleoproteins by ubiquitin-proteasome system in rat spermatids and human sperm.

Many temporarily functioning proteins are generated during the replacement of nucleoproteins in the nuclei of late spermatids and seem to be degraded in the nucleus. This study was designed to clarify the involvement of the ubiquitin-proteasome degradation system in the nucleus of rat developing spermatids. Thus, we studied the nuclear distribution of polyubiquitinated proteins (pUP) and proteasome in spermiogenic cells and sperm using postembedding immunoelectron microscopy. We divided the nuclear area of late spermatids into two regions: (1) a dense area composed of condensed chromatin and (2) a nuclear pocket in the neck region. The latter was located in the caudal nuclear region and was surrounded by redundant nuclear envelope. We demonstrated the presence of pUP in the dense area and nuclear pocket, proteasome in the nuclear pocket, and clear spots in the dense area of rat spermatids. Using quantitative analysis of immunogold labeling, we found that fluctuation of pUP and proteasome levels in late spermatogenesis was mostly synchronized with disappearance of histones and transitional proteins reported previously. In the nuclei of human sperm, pUP was detected in the dense area, whereas proteasome was in the nuclear vacuoles and clear spots. These results strongly suggest that pUP occur in the dense nuclear area of developing spermatids and that the ubiquitin-proteasome system is more actively operational in the nuclear pocket than dense area. Thus, the nuclear pocket might be the degradation site for temporarily functioning proteins generating during condensation of chromatin in late spermatids.     

拟异蚖和古蚖精子的超微形态和精子形成的研究(原尾目:古蚖科)

余山拟异蚖和3种古蚖的精子均为扁圆形,未见顶体,线粒体集中在一侧;核呈环形、边位、中部由膜状体分布其间.领结古蚖的早期精细胞为球形,染色质凝集成团,继而核中裂并沿细胞赤道逐渐围绕成环,染色质呈细沙状,胞间有“桥”相通.核膜一端开始内陷,出现黑点.待发育到中期精细胞,这些黑点逐渐形成奇特的管状核膜陷体;染色质变成短线形,随后排成4—5行.线粒体颗粒状,细胞间仍有“桥”连通.晚期精细胞的染色质凝集成粗带,最后形成光滑质密的核,而多余的核物质,一段一段从精子一端脱离,形成一串孢囊状体夹在精子之间,待精子成熟游离时,这些孢状体分散开来.从观察结果表明拟异蚖精子与古蚖的非常相近.