SPERMIOGENESIS IN THE PULMONATE SNAIL, EUHADRA HICKONIS II. STRUCTURAL CHANGES OF THE NUCLEUS

In accordance with the characteristic shape of the nucleus and degree of condensation of the nuclear substance, spermiogenesis in Euhadra hickonis can be roughly divided into four stages. The chromatin in the highly polymorphic nucleus of the first stage, early spermatid, forms relatively thick (ca. 50 nm) fibrils which associate here and there into irregular clumps. In the next stage, the spermatid nucleus becomes conspicuously spherical, its contents appear more finely homogeneous and the irregular clumps of chromatin are few. In the third stage, the nucleus gradually takes on an ellipsoidal shape as the antero-posterior axis shortens. The anterior part of its envelope becomes structurally modified in preparation for the adherence to it of the developing acrosome, and an implantation fossa forms posteriorly at the center of a second area where the nuclear envelope has been modified. The diameter of the chromatin fibrils again increases and those near the implantation fossa become oriented perpendicular to the nuclear envelope.
As the nucleus elongates in the fourth stage, a concentric sheath of microtubules closely surrounds it. These appear to depolymerize as the nuclear elongation proceeds, so that they are no longer present in the head region of the mature spermatozoon. The diameter of the chromatin fibrils increases to about 10 nm and they become oriented parallel to the long axis of the cell. With the decrease in the nuclear volume the fibrils unite laterally to form longitudinal sheets, and these finally merge in the mature spermatozoon into a mass of very dense chromatin without perceptible internal structure.     

Analyse de la structure chromatinienne du sperme par la cytométrie en flux à l’acridine orange. — intérêt clinique

During spermatozogenesis, sperm nucleosomal DNA type, linked to histones, is transformed into a special form bound to small basic proteins, the protamines. This process allows sperm nucleus to condense and mature. This maturation process is achieved in the epididymis. When a spermatozoon enters an oocyte, protamines are released and replaced by histones, enabling the sperm nucleus to expand into a male pronucleus. Flow cytometry using acridine orange staining is an objective and quantitative technique well-adapted for the investigation of the chromatin structure at the level of each spermatozoon, as well as the whole ejaculate. This technique allows tracing the young sperm cell nuclei from the diploid stage, through tetraploid until the final haploid stage in mature spermatozoa. It allows also following the sperm maturation process during the epididymal transit. It detects various sperm chromatin condensation defects, hypocondensation, hypercondensation or other aberrations, as well as decondensation defects by using an in vitro assay. These defects may impair sperm fertilizing ability, even after sperm microinjection into the oocyte. Better understanding of sperm chromatin integrity and stability prerequisites might help us improving the quality of various technologies used in assisted medical procreation.     

Histones and nucleosomes in Cancer sperm (Decapod: Crustacea) previously described as lacking basic DNA-associated proteins: a new model of sperm chromatin

To date several studies have been carried out which indicate that DNA of crustacean sperm is neither bound nor organized by basic proteins and, contrary to the rest of spermatozoa, do not contain highly packaged chromatin. Since this is the only known case of this type among metazoan cells, we have re-examined the composition, and partially the structure, of the mature sperm chromatin of Cancer pagurus, which has previously been described as lacking basic DNA-associated proteins. The results we present here show that: (a) sperm DNA of C. pagurus is bound by histones forming nucleosomes of 170 base pairs, (b) the ratio [histones/DNA] in sperm of two Cancer species is 0.5 and 0.6 (w/w). This ratio is quite lower than the proportion [proteins/DNA] that we found in other sperm nuclei with histones or protamines, whose value is from 1.0 to 1.2 (w/w), (c) histone H4 is highly acetylated in mature sperm chromatin of C. pagurus. Other histones (H3 and H2B) are also acetylated, though the level is much lower than that of histone H4. The low ratio of histones to DNA, along with the high level of acetylation of these proteins, explains the non-compact, decondensed state of the peculiar chromatin in the sperm studied here. In the final section we offer an explanation for the necessity of such decondensed chromatin during gamete fertilization of this species.     

Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair

An important chromatin remodeling process is taking place during spermiogenesis in mammals and DNA strand breaks must be produced to allow the accompanying change in DNA topology. Endogenous DNA strand breaks are indeed detected at mid-spermiogenesis steps but are no longer present in mature sperm. Both in vitro and in vivo evidence suggests that the DNA-binding and condensing activities of a set of basic nuclear "transition proteins" may be crucial to the integrity of the chromatin remodeling process. We propose that these proteins are necessary for the repair of the strand breaks so that DNA fragmentation is minimized in the mature sperm.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

大银鱼卵膜孔结构的电镜观察

扫描电镜下，大银鱼成熟卵卵膜孔区域呈现奇异的放射沟脊状结构。在授精开始的３０秒内，测得卵膜附近约８６％的精子沿着凹沟进入精孔管；还拍摄到了通过卵孔中心的纵切面映象，通过电子计算机模拟，验证了卵膜孔的这种结构，为精子的云集和进入精孔管所提供有利条件，从而，于泥鳅之后又发现了一种真骨鱼，其精子入卵除化学因素外，还存在着不容忽视的机械动力因素。     

Poly(ADP-ribose) metabolism is essential for proper nucleoprotein exchange during mouse spermiogenesis

Sperm chromatin is organized in a protamine-based, highly condensed form, which protects the paternal chromosome complement in transit, facilitates fertilization, and supports correct gene expression in the early embryo. Very few histones remain selectively associated with genes and defined regulatory sequences essential to embryonic development, while most of the genome becomes bound to protamine during spermiogenesis. Chromatin remodeling processes resulting in the dramatically different nuclear structure of sperm are poorly understood. This study shows that perturbation of poly(ADP-ribose) (PAR) metabolism, which is mediated by PAR polymerases and PAR glycohydrolase in response to naturally occurring endogenous DNA strand breaks during spermatogenesis, results in the abnormal retention of core histones and histone linker HIST1H1T (H1t) and H1-like linker protein HILS1 in mature sperm. Moreover, genetic or pharmacological alteration of PAR metabolism caused poor sperm chromatin quality and an abnormal nuclear structure in mice, thus reducing male fertility.     

The murine heterochromatin protein M31 is associated with the chromocenter in round spermatids and Is a component of mature spermatozoa

In mature sperm the normal nucleosomal packaging of DNA found in somatic and meiotic cells is transformed into a highly condensed form of chromatin which consists mostly of nucleoprotamines. Although sperm DNA is highly condensed it is nevertheless packaged into a highly defined nuclear architecture which may be organized by the heterochromatic chromocenter. One major component of heterochromatin is the heterochromatin protein 1 which is involved in epigenetic gene silencing. In order to investigate the possible involvement of heterochromatin protein in higher order organization of sperm DNA we studied the localization of the murine homologue of heterochromatin protein 1, M31, during chromatin reorganization in male germ cell differentiation. Each cell type in the testis showed a unique distribution pattern of M31. Colocalization to the heterochromatic regions were found in Sertoli cells, in midstage pachytene spermatocytes, and in round spermatids in which M31 localizes to the centromeric chromocenter. M31 cannot be detected in elongated spermatids or mature spermatozoa immunocytologically, but could be detected in mature spermatozoa by Western blotting. We suggest that M31, a nuclear protein involved in the organization of chromatin architecture, is involved in higher order organization of sperm DNA.     

Sperm-chromatin maturation in the mouse. A cytochemical approach

Cytochemical techniques were used to study chromatin during spermiogenesis and sperm maturation in the mouse, starting from the stages at which the substitution of somatic histones by testis-specific proteins occurs. It was possible to distinguish and analyze the different temporal incidence of two processes involved in sperm maturation, i.e. chromatin condensation (a tridimensional highly compacted arrangement) and chromatin stabilization (a tough structure, which protects the genome DNA). The first process, involving a reduction in the nuclear size and a decrease in the amount of sperm DNA accessible to specific cytochemical reactions and stainings, was found to reach its maximum in caput-epididymidis spermatozoa, in which electron microscopy revealed that the sheared chromatin was mainly organized into 120-A-thick knobby fibers. No further changes were found in sperm up to their appearance in the fallopian tubes. On the contrary, chromatin stabilization, the onset of which occurs in the testis (at the late spermatid stage) via the formation of -S-S- cross-links, is completed in the vas deferens, where chromatin has a superstructure consisting of thicker fibers, with diameters of 210 and 350 A. The reductive cleavage of disulfides in vas-deferens spermatozoa does not completely destroy the superstructure of sperm chromatin, which could indicate 'coiling' of the basic knobby fiber. In fact, when the ion concentration was increased, the chromatin of vas-deferens spermatozoa appeared to be organized into fibers with diameters similar to those of the caput epididymidis. This unique organization of mature sperm chromatin should have an essential role in the fast swelling of spermatozoa during fertilization.     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

Changes in the stainability and sulfhydryl level in the sperm nucleus during sperm-oocyte interaction in mice

During maturation in the epididymis, mouse sperm nuclei become difficult to stain with Giemsa and its component basic dyes. Mature sperm from the cauda epididymis can be stained only after DTT treatment. Stainable sperm such as those from the testis accumulate ³H NEM when examined by autoradiography, while unstainable sperm do not, indicating a close correlation between the basic dye binding capacity and SH levels in the sperm nuclei. During insemination of zonafree ovarian oocytes with a germinal vesicle (GV), mature sperm nuclei become stainable and capable of binding with ³H NEM. At the same time, sperm have established pronase-resistant contact with the oocyte. Similarly, sperm nuclei become stainable during fertilization when the sperm attachment to the egg becomes pronase resistant. However, these changes occur before sperm chromatin decondensation begins. Therefore, it is suggested that S-S bonds in sperm nucleoproteins are reduced when the sperm establish a stable contact with the egg plasma membrane, thus reversing sperm maturational changes. The reduction of S-S bonds may be a prerequisite for sperm chromatin decondensation.     

三疣梭子蟹精子的发生及超微结构研究

用透射电镜观察三疣梭子蟹的精子发生过程及精子的超微结构。发现精原细胞较大，卵圆形。核大而圆，染色质分散，附着于核膜之内侧。胞质少，内含线粒体和粗面内质网等结构。初级精母细胞比精原细胞略小，卵圆形，核内染色质凝聚成团块，散布于核质中，除线粒体外，胞质中尚含有很多内质网小泡和游离核糖体。次级精母细胞多边形，核卵圆形，染色质致密，线粒体等含量均下降。早期精细胞质中由内质网产生许多颗粒，这些颗粒合并成为大     

Biology of sperm chromatin structure and relationship to male fertility and embryonic survival

Embryonic mortality in mammals is typically thought to result from 'female factor' infertility. There is growing evidence, however, that the status of sperm chromatin (DNA) at the time of fertilisation can also influence embryonic survival. During the final stages of spermatogenesis (spermiogenesis) a number of unique biochemical, morphological and physiological processes take place that are associated with marked changes in the structure of sperm chromatin. In early stages of spermatogenesis, sperm DNA is associated with histone nucleoproteins and structured into classical nucleosome core particles similar to other somatic cells. As spermiogenesis proceeds, the histone nucleoproteins are replaced by transition proteins which are subsequently replaced by protamines. At the completion of spermiogenesis the chromatin of mature sperm has a toroidal structure that is tightly compacted and resistant to denaturation. The compaction is necessary to protect sperm chromatin during transit through the epididymis and female reproductive tract. Disruption to chromatin remodelling during spermiogenesis results in chromatin that is susceptible to denaturation. Inappropriate chromatin structure has been shown in a number of mammalian species to be related to male infertility, and specifically the failure of embryonic development. A range of techniques are available to assess chromatin status in sperm but arguably the most informative is the sperm chromatin structure assay (SCSA). The SCSA is a flow cytometric assay that uses the metachromatic properties of acridine orange to measure the susceptibility of sperm chromatin to acid-induced denaturation. A relationship has been demonstrated, primarily in men, between the SCSA outcome and the probability of continued embryonic development and the establishment of pregnancy after fertilisation. The contribution of sperm chromatin instability to reproductive wastage in both natural mating and assisted reproduction warrants further investigation as it may prove valuable as a means of decreasing the incidence of embryonic mortality. In this regard, it is possible that 'male factor' infertility may emerge as an even more important component in embryonic development.     

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.     

日本沼虾精子发生的研究

对日本沼虾精子发生全过程的电镜观察表明：精原细胞核染色质分散,胞质内有线粒休、内质网的分布。初级精母细胞核染色质块状,不均匀地分布于核中,内质同多小泡多。次级精母细胞核染色质大多分布于核膜内侧,内质网聚集成团,精细胞分化形成精子的早期,胞核增大,核侧形成内质同多小泡的聚合体;中期的核内染色质浓缩,同时形成空囊状结构,     

Morphological changes of sperm nuclei during spermatogenesis in the brown alga Cystoseira hakodatensis (Fucales, Phaeophyceae)

Morphological changes and chromatin condensation of sperm nuclei were observed during spermatogenesis in the fucalean brown alga Cystoseira hakodatensis (Yendo) Fensholt. Ultrastructural studies have shown that the mature spermatozoid has an elongated and concave nucleus with condensed chromatin. The morphological changes and the chromatin condensation process during spermatogenesis was observed. Nuclear size decreased in two stages during spermatogenesis. During the first stage, spherical nuclei decreased in size as they were undergoing meiotic divisions and the subsequent mitoses within the antheridium. During the second stage, the morphological transformation from a spherical into an elongated nucleus occurred. Afterwards, chromatin condensed at the periphery in each nucleus, and chromatin‐free regions were observed in the center of the nucleus. These chromatin‐free regions in the center of nucleus were compressed by the peripheral chromatin‐condensed region. As the result, the elongated and concave nucleus of the mature sperm consisted of uniformly well‐condensed chromatin.     

银鲫(Carassius auratus gibelio)成熟精子染色质的结构和碱性蛋白的研究

银鲫属行雌核发育的遗传系统,其精子入卵后不能形成雄性原核。如同其它种类的精子一样,在精子发生过程中其染色质形成高度浓缩的具有一定结构的DNA和碱性蛋白的复合物。为了研究分析银鲫成熟精子的碱性蛋白、染色质的结构及其与体细胞染色质的异同,我们用聚丙烯酰胺凝胶电泳分析了银鲫精子碱性蛋白的组分,并结合核酸酶解的结果,以电镜铺片和超薄切片法探讨了银鲫染色质的结构。聚丙烯酰胺凝胶电泳分析表明,银鲫精子的碱??性蛋白为含H_1、H_2a、H_2b、H_3及H_4五个主要组分的组蛋白,且与体细胞核组蛋白无明显差异。电镜铺片法观察精子染色质,可见染色质丝的近100A粗细的串珠状结构。小球菌核酸酶水解精子染色质,DNA的琼脂糖电泳呈“阶梯型”电泳条带,证实了银鲫成熟精子保存了核小体基本单元,在低渗处理后的精子超薄切片中,可以见到直径为300A左右的染色质纤维,这个结果提示了精子染色质与体细胞染色体有类似的二级结构。根据以上所得到的结果推测,行雌核发育的银鲫,其精子在入卵后不能原核化,与精子染色质的基本结构及碱性蛋白组分无明显直接关系。     

Interaction of sperm histone variants and linker DNA during spermiogenesis in the sea urchin

Several physical properties of sea urchin spermatid chromatin, which contains phosphorylated Sp H1 and Sp H2B histone variants, and mature sperm chromatin, in which these histones are dephosphorylated, were compared. Density, thermal stability, average nucleosomal repeat length, and resistance to micrococcal nuclease digestion are all increased in mature sperm relative to spermatid chromatin. Since the chromatins are identical in histone variant subtypes, the altered physical properties are not a consequence of changes in histone primary structure during spermiogenesis. The data are interpreted to mean that dephosphorylation of the N-terminal regions of Sp H1 and Sp H2B in late spermatid nuclei permits strong ionic binding of these highly basic regions to the extended linker, stabilizing the highly condensed structure of sperm chromatin.     

Structural components of sea urchin sperm nuclei

The sea urchin sperm nucleus rapidly loses its conoid morphology and becomes more voluminous and spherical upon its entry into the egg cytoplasm during fertilization. This investigation has attempted to determine what are the structural constraints placed upon the sperm nucleus, so that further investigations might determine the egg cytoplasmic factors that are responsible for modifying nuclear morphology. Isolated sperm nuclei were subjected to various extraction procedures in order to remove the majority of the proteins (histones) and also the DNA; subsequently, the residual structures were processed for and examined by electron microscopy. The data presented in this investigation demonstrate the removal of the sperm nuclear histones plus other nonhistone proteins has no effect on the conoid morphology of the sperm nucleus, yet this protein removal has a profound effect on the structure of the nuclear chromatin. It is also shown that removal of the majority of the nuclear DNA has no effect on the shape of the sperm nucleus. These results indicate that there are other components (possibly a nuclear matrix) associated with the sperm nucleus that are responsible for maintaining its conoid morphology.