Nuclear morphogenesis during spermiogenesis in the nudibranch molluscHypselodoris tricolor (Gastropoda,opisthobranchia)

An electron microscope study was carried out on Hypselodoris tricolor spermatids to describe the development of the nuclear morphogenesis and investigate the possible cause(s) of the change in the shape of the spermatid nucleus during spermiogenesis. Three different stages may be distinguished in the course of the nuclear morphogenesis on the basis of the morphology and inner organization of the nucleus. Stage 1 spermatid nuclei are spherical or ovoid in shape and the nucleoplasm finely granular in appearance. Stage 2 nuclei exhibit a disc- or cup-shaped morphology, and the chromatin forms short, thin filaments. During stage 3, a progressive nuclear elongation takes place, accompanied by chromatin rearrangement, first into fibers and then into lamellae, both formations helically oriented. A row of microtubules attached to the nuclear envelope completely surrounds the nucleus. Interestingly, the microtubules always lie parallel to the chromatin fibers adjacent to them. Late stage 3 spermatids show the highest degree of chromatin condensation and lack the manchette at the end of spermiogenesis. Our findings indicate the existence of a clear influence exerted on the chromatin by the manchette microtubules, which appear to be involved in determining the specific pattern of chromatin condensation in Hypselodoris tricolor.     

Immunocytochemical and ultrastructural analyses of the function of the ubiquitin-proteasome system during spermiogenesis with the use of the inhibitors of proteasome proteolytic activity in the alga, Chara vulgaris

Spermiogenesis in Chara vulgaris and in animals share many common features, including exchange of nucleohistones into nucleoprotamines, remodeling and extreme condensation of chromatin, formation of flagellae and of microtubule manchette, and decrease in cytoplasm volume. In C. vulgaris, spermiogenesis is not preceded by meiosis since this alga is a haplobiont. In the present work we showed that in early spermiogenesis characterized by a significant metabolic activity of spermatids, the inhibitors of proteasomes did not visibly change their ultrastructure but significantly prolonged this process. At late stages of spermiogenesis, MG-132 and epoxomicin dramatically changed the structure of nuclei: regular fibrillar and lamellar structure of chromatin was disturbed and clusters of grains corresponding to aggresomes appeared, but the nucleus shape and cytoplasm structure were the same as in the controls. Immunocytochemical studies revealed that these inhibitors blocked disappearance of histones from nuclei while the structures corresponding to aggresomes were clusters of undegraded ubiquitinated histones, since they gave positive immunosignals indicating the presence of ubiquitin and histones.     

Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis

Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.     

Spermatogenesis in free living mite, Pergamasus viator Hala?. (Parasitidae, Mesostigmata). II. Spermiogenesis.

The paper presents the results of ultrastructural studies on the spermiogenesis in the mite, Pergamasus viator Hala?kova. The cysts containing 16 spermatids per cyst, are localized in the anterior part of the saccular gonad. The process of spermatid maturation has been divided into three stages: of the early spermatid, middle spermatid and late spermatid. The modifications of spermatid occuring during the spermiogenesis include: a change of cell shape, modifications of its organelles and formation of new structures like the superficial layer of cellular processes, striated bodies, granular bodies, flattened cisternae and canaliculi, central canaliculi, and stiff bands. Within the nucleus the chromatin condenses to threads or lamellae, to form subsequently several electron-dense granules. The remaining nucleoplasm is filled with an electron-dense material, which appears in the middle spermatid and gradually accumulates. The above modifications occuring in the course of spermiogenesis and their relation to the data available from the literature concerning the spermatogenesis of allied groups of animals are discussed in length.     

Fine structure of spermiogenesis with special reference to the spermatid morulae of the freshwater mussel Prisodon alatus (Bivalvia,Unionoidea)

Within the testicular cysts of the mussel Prisodon alatus are numerous somatic host cells described as Sertoli cells (SC), each containing a variable number of young spermatid morulae. Among them, several free spermatid morulae, spermatids, and spermatozoa were observed. Each free spermatid morula is surrounded by an external membrane. The early spermatids enclosed within the morulae have dense and homogeneous chromatin, and the cytoplasm occupies little space around the nucleus. Later, during spermiogenesis, the SC show lysis and disrupt to liberate the spermatid morulae. The membrane of the free morula is then disrupted, releasing the young spermatids. The SC disappear just after the appearance in the testis of a large number of free young spermatids. The nucleus of each free spermatid becomes gradually smaller and denser by the appearance of a granular pattern of condensed chromatin. During the maturation phase of the spermatids, the cytoplasm becomes more voluminous, and mitochondria and centrioles are more evident. Then, flagellogenesis occurs, and the nucleus gradually condenses into thicker strands. In the mature sperm, the apical zone has a disc-shaped acrosomal vesicle and the midpiece contains five mitochondria and two centrioles located at the same level. The flagellum has the common 9+2 microtubular pattern. The results are discussed with particular reference to Sertoli cells and clusters of spermatid morulae with those of species of closely related taxa in the bivalves. J. Morphol. 238:63–70, 1998. © 1998 Wiley-Liss, Inc.     

An in vitro, short-term culture method for mammalian haploid round spermatids amenable for molecular manipulation

Extensive chromatin remodeling is a characteristic feature of mammalian spermiogenesis. To date, methods for the molecular manipulation of haploid spermatids are not available as there is a lack of a well‐established culture system. Biochemical experiments and knockout studies reveal only the final outcome; studying the incremental details of the intricate mechanisms involved is still a challenge. We have established an in vitro culture system for pure haploid round spermatids isolated from rat testes that can be maintained with good viability for up to 72 hr. Changes in cell morphology and flagellar growth were also studied in the cultured spermatids. Further, we have demonstrated that upon treatment of cells with specific histone deacetylase inhibitors, sodium butyrate and trichostatin A, there is an increase in the hyperacetylation status of histone H4, mimicking an important event characteristic of histone replacement process that occurs during later stages of spermiogenesis. We have also tried various methods for introducing DNA and protein into these round spermatids in culture, and report that while DNA transfection is still a challenging task, protein transfection could be achieved using Chariot? peptide as a transfection reagent. Thus, the method described here sets a stage to study the molecular roles of spermatid‐specific proteins and chromatin remodelers in the cellular context. Mol. Reprod. Dev. 79:19–30, 2012. © 2011 Wiley Periodicals, Inc.     

Spatiotemporal Organization of AT- and GC-rich DNA and Their Association With Transition Proteins TP1 and TP2 in Rat Condensing Spermatids

Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12–15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GC-preference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4′,6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC- and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis. (J Histochem Cytochem 57:951–962, 2009)     

Occurrence of calreticulin during the exchange of nucleohistones into protamine-type proteins in Chara vulgaris spermiogenesis

During spermiogenesis of an alga Chara vulgaris, which resembles that of animals, nucleohistones are replaced by protamine-type proteins. This exchange takes place in a spermatid nucleus during the key V spermiogenesis stage, in which rough endoplasmic reticulum is the site of protamine-type protein synthesis and is also the pathway guiding the proteins to their destination, nucleus. In the present work, it was shown that a chaperon protein, calreticulin (CRT), abundantly present at this significant V stage of spermiogenesis in a few cellular compartments, i.e., a nucleus, lumen of cisternae, and vesicles of significantly swollen ER as well as outside these structures, e.g., in Golgi apparatus, could have taken part in the process of exchange of nuclear proteins. Colocalization of two proteins, protamine-type proteins, crucial for reproduction, and CRT, was especially visible in a nucleus, mainly on its peripheries where condensed chromatin was present. Localization of protamine-type proteins and CRT in nucleus is in agreement with our previous results showing that protamine-type proteins were twofold more labelled in the peripheral area in comparison to the nucleus center occupied by noncondensed chromatin. The role of CRT in the reproduction of both plants and animals is also discussed.     

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.     

Insights into role of bromodomain, testis-specific (Brdt) in acetylated histone H4-dependent chromatin remodeling in mammalian spermiogenesis

Mammalian spermiogenesis is of considerable biological interest especially due to the unique chromatin remodeling events that take place during spermatid maturation. Here, we have studied the expression of chromatin remodeling factors in different spermatogenic stages and narrowed it down to bromodomain, testis-specific (Brdt) as a key molecule participating in chromatin remodeling during rat spermiogenesis. Our immunocytochemistry experiments reveal that Brdt colocalizes with acetylated H4 in elongating spermatids. Remodeling assays showed an acetylation-dependent but ATP-independent chromatin reorganization property of Brdt in haploid round spermatids. Furthermore, Brdt interacts with Smarce1, a member of the SWI/SNF family. We have studied the genomic organization of smarce1 and identified that it has two splice variants expressed during spermatogenesis. The N terminus of Brdt is involved in the recognition of Smarce1 as well as in the reorganization of hyperacetylated round spermatid chromatin. Interestingly, the interaction between Smarce1 and Brdt increases dramatically upon histone hyperacetylation both in vitro and in vivo. Thus, our results indicate this interaction to be a vital step in the chromatin remodeling process during mammalian spermiogenesis.     

Spermiogenesis in three species of cicadas (Hemiptera: Cicadidae)

Spermiogenesis in three species of cicadas representing one cicadettine (Monomatapa matoposa Boulard) and two cicadines (Diceroprocta biconica [Walker] and Kongota punctigera [Walker]) was investigated by light and electron microscopy. Although spermiogenesis was occurring in the testis of adult males of all species, earlier spermiogenic stages were observed in D. biconica only. While spermiogenesis was similar to that described for other insects, some differences were noted. For example granular material did not assemble around the centriole to form a centriolar adjunct but did accumulate in the cytoplasm of early spermatids adjacent to a region of the nuclear membrane where nuclear pores were aggregated. In late spermatids this material accumulated anterior to the mitochondrial derivatives in a developing postero‐lateral nuclear groove. While this material has been named the ‘centriolar adjunct’ by previous authors, its formation away from the centriole raises questions about its true identity. Second, during acrosome maturation an ante‐acrosomal region of cytoplasm develops. Although present in later spermatids, this region is lost in spermatozoa. Interspecific variations in chromatin condensation patterns and the number of microtubule layers encircling the spermatid nucleus during spermiogenesis were noted.     

Spermiogenesis and structure of spermatozoa in the oribatid mite,Hafenrefferia gilvipes (C.L. Koch) (Acari,Oribatida)

The process of spermiogenesis and the structure of spermatozoa in the mite, Hafenrefferia gilvipes (Koch) were studied ultrastructurally. Spermiogenesis was divided into six stages. The spermatids at stage 1 have the usual structure. At stage 2 the structure of the mitochondria and their distribution in the spermatid start to change, leading to the formation of specific mitochondrial derivatives which are subsequently incorporated into the nucleus of the spermatozoon. Parallel to the transformation of mitochondria occurs a reorganization of the nuclear material. The fully formed spermatozoon has a tadpole-like shape, with the cell nucleus located in the distended part of the cell, and containing mitochondrial derivatives in its karyoplasm. Acrosome, flagellum and centrioles are absent. The participation of peripherally distributed microtubules, present in spermatids at stages 4 to 6, in the shaping of the spermatozoon has been suggested.     

Spermatogenesis and distinctive mature sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879)

The structures of differentiating male germ cells in the testis of the giant freshwater prawn, Macrobrachium rosenbergii, were studied by light and electron microscopy. Based on ultrastructural characteristics, the developing male germ cells are classified into 12 stages, including spermatogonia, six phases of primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis and metaphase), secondary spermatocyte, three stages of spermatids and mature sperm. During spermatogenesis, the differentiating germ cells have characteristics similar to those of other invertebrates, but they exhibit some unique characteristics during spermiogenesis. In particular, an early spermatid has a round nucleus with highly condensed heterochromatin, appearing as thick interconnecting cords throughout the nucleus. In contrast to most invertebrates and vertebrates, the chromatin begins to decondense in one-half of the nucleus at the mid spermatid stage. In the late spermatid, the chromatin becomes almost entirely decondensed with only a small crescent-shaped heterochromatin patch remaining at the anterior pole of the nucleus. Mature sperm possess an everted umbrella-shaped plate with a spike covering the anterior pole of the nucleus, whose chromatin is totally decondensed as only small traces of histones H3 and H2B remain. The acrosome appears at the ruffled border of the spike plate as small sac-like structures. Few mitochondria remain in the cytoplasm at the posterior pole.     

Dynamic aspects of spermiogenic chromatin condensation patterning by phase separation during the histone-to-protamine transition in charalean algae and relation to bryophytes

During early-to-middle spermiogenesis in multicellular, internally fertilizing charalean green algae (Chara fibrosa, Chara vulgaris, Chara tomentosa, Nitella missouriensis), patterning of chromatin/nucleoplasm in developing spermatid nuclei changes from granules → fibers → contorted lamellae → condensed chromatin. Cytochemical, immunocytochemical, electrophoretic studies on C. vulgaris and C. tomentosa spermatids (Kwiatkowska, Poplonska) and amino acid analysis of protamines in Chara corallina sperm (Reynolds, Wolfe), indicate that more positively charged protamines replace histones directly during spermiogenesis, not indirectly through other intermediate transitional proteins as in internally fertilizing neogastropods and sharks with more ordered spermatid lamellae. We hypothesize that such lamellar-mediated patterning is due to liquid–liquid phase separation by spinodal decomposition. This is a spontaneous thermodynamic process that involves diffusive instability of a lamellar chromatin network, a dominant pattern repeat distance and bicontinuity of chromatin/nucleoplasm phases. C. vulgaris sperm show contorted lamellae in the posterior region, whereas C. corallina sperm display contorted peripheral lamellae and interior fibrils. Among internally fertilizing liverworts, which may have evolved from Zygnematales, mid-spermatid nuclei lack lamellae. Instead they display self-coiled chromatin rods in Blasia pusilla, contain short chromatin tubules in Haplomitrium hookeri resembling those in internally fertilizing mosses and a hornwort and indirectly replace histones with protamines in Marchantia polymorpha.     

Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This "elementary" fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30-40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called "immature chromatin," which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.     

Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specificity and link to chromatin remodeling

In the course of mammalian spermiogenesis, a unique chromatin remodeling process takes place within elongating and condensing spermatid nuclei. The histone-to-protamine exchange results in efficient packaging and increased stability of the paternal genome. Although not fully understood, this change in chromatin architecture must require a global but transient appearance of endogenous DNA strand breaks because most of the DNA supercoiling is eliminated in the mature sperm. To establish the extent of DNA strand breakage and the stage specificity at which these breaks are created and repaired, we performed a sensitive terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay to detect in situ DNA strand breaks on both mice and human testis cross sections. In the mouse, we established that DNA strand breaks are indeed detected in the whole population of elongating spermatids between stages IX and XI of the seminiferous epithelium cycle perfectly coincident with the chromatin remodeling as revealed by histone H4 hyperacetylation. Similarly, TUNEL analyses performed on human testis sections revealed an elevated and global increase in the levels of DNA strand breaks present in nuclei of round-shaped spermatids also coincident with chromatin remodeling. The demonstration of the global character of the transient DNA strand breaks in mammalian spermiogenesis suggests that deleterious consequences on genetic integrity of the male gamete may arise from any disturbance in the process. In addition, this investigation may shed some light on the origin of the low success rate that has been encountered so far with intracytoplasmic injection procedures making use of round spermatids in humans.     

Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This “elementary” fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30–40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called “immature chromatin,” which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.     

Spermiogenesis in the Nile tilapia Oreochromis niloticus with notes on a unique pattern of nuclear chromatin condensation

Spermiogenesis in the Nile tilapia, Oreochromis niloticus, was observed ultrastructurally. The process of spermatid differentiation can be divided into six distinct stages based mainly on changes in the nucleus of spermatids. During the latter half of the process, nuclear chromatin condenses progressively to form many dense globules, which ultimately adhere tightly to pack the head of mature spermatozoa. During chromatin condensation the nucleus diminishes in size, and part of the nuclear envelope and nucleoplasm forms a vesicular structure that is finally discarded from the cells together with an associated thin layer of cytoplasm. The spermatozoon comprises a roundish head, a relatively small midpiece, and a relatively short flagellum consisting of the usual 9+2 axoneme. No acrosomal structure is developed during spermiogenesis.