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PubMed1992

Purification and characterization of the rat spermatid basic nuclear protein TP4.

Following elongation of spermatids in mammals, the histones are replaced by a set of basic nuclear transition proteins; in the rat there are four, named TP1-TP4. Of these, TP1 and TP2 are well characterized. Here we report the purification to homogeneity of TP4 from rat spermatids. It is a low molecular mass (about 13-20 kDa) basic protein with arginine and lysine constituting 24 mol % and histidine 2.2 mol %. Its 25 N-terminal amino acids were sequenced, and no sequence homologies with any known protein were found. Polyclonal antibodies raised against it in rabbit did not cross-react with other transition proteins, protamines, or histones. The presence of TP4 during sperm development was monitored by cell separation studies. No TP4 was detected in round spermatids, and along with TP1 and TP2, it is present in step 13-15 spermatids and its amount decreased in steps 16-19. Trace amounts of TP4 were also detected in epididymal sperm. A possible role for TP4 in spermatid and sperm chromatin structure is discussed.

Amino Acid Sequence↗
PubMed1982

[The indirect roles of microtubules in the nuclear morphogenesis of spermatids].

The depolymerization of the microtubules of the spermatid manchette was effective for 4 to 5 h in rat, starting 30 min after one intratesticular injection of colcemid in vivo. The manchette built up again later was normal in old elongating spermatids but very oddly shaped in young elongating spermatids in which the nuclear shape was abnormal and the relations between the nucleus and the endoplasmic reticulum aberrant, ie destruction of the apparatus for nucleocytoplasmic exchange. From 24 to 96 h after the injection, a progressive shifting of the cellular associations indicated that young spermatids elongating at the time of injection did not differentiate further. This could result from the lack of two roles not played by the new manchette: the guiding effect of the intranuclear chromatin and the role of skeleton for the cytoplasmic apparatus devoted to nucleocytoplasmic exchange. Step 14 to 16 spermatids were not modified by the removal of the manchette due to correct reformation of a typical manchette 4 to 5 h later. The associations of the microtubules and the nuclei and the speculative roles of the microtubules during spermiogenesis have been discussed.

Animals↗
PubMedDate not supplied

[From spermatid to spermatozoa : what changes are necessary for development?].

The male gamete modifications during spermiogenesis were summarized in view to answer the question : "Is it a risk for a baby conceived by fertilization with a spermatid to be different of a baby conceived with a spermatozoon?" Cytoplasmic changes in spermiogenesis mainly concern the Golgi apparatus (acrosome vesicle), centrosomal material (flagelium), mitochondria (periaxonemal ring) and ooplasm volume (drastic reduction). These changes are not necessary in case of microfertilization (ICSI) and their absence could be without consequences on post fertilization development. Nuclear changes affect chromatin with the substitution of histones by protamines to insure sperm ADN stability across male and female genital tracts. Spermatid gene transduction brings new proteins which are related to fertilization (acrosomal enzymes, flagellum proteins, protamines...) and seem no longer useful after gametes fusion. There is no report of genomic imprinting occurring during spermiogenesis in mammals. Moreover, the important sperm changes during epididymal maturation (formation of disulfure bonds, acquisition of motility and of molecules for oocyte recognition, methylation of certain genes...) were found not necessary for embryo development in case of ICSI with epididymal or testicular spermatozoa. Normal and fertile animals (mice and rabbits) were born from ICSI with spermatids originated from normal males; an animal model with sterile males delivering apparently normal spermatids is lacking. Although the risk for defectuous babies from fertilization with spermatids is highly subjective, caution is needed and requires team competence, complete informed consent by the patients and exhaustive analysis of the methods used and results obtained. Moreover the evident risk to "reproduce" the father infertility must be compared with the psychosocial injury induced by using the alternative option of donor insemination.

Animals↗
PubMedDate not supplied

Spermatids from mice after cryptorchid and reversal operations can initiate normal embryo development.

We investigated fertilization and embryo development following intracytoplasmic injection of round spermatids collected from mice after cryptorchid and reversal operations. The rates of oocyte activation, blastocyst formation, and development to live offspring were compared after intracytoplasmic injection of round spermatids from intact, cryptorchid males (control) or males that had undergone the reversal operation. The incidence of normal fertilization and embryo development was about the same when the oocytes were injected with spermatids from intact cryptorchid males or males after reversal operation. The rates of the development of two-cell embryos to term were also about the same, regardless of the origin of spermatids injected. Round spermatids from both cryptorchid and previously cryptorchid testes can initiate normal embryo development when injected into mature oocytes.

Animals↗
PubMed1998

Intracellular Ca2+ homeostasis in rat round spermatids.

Intracellular calcium, [Ca2+]i, can regulate meiotic progression of mammalian oocytes. However, the role of [Ca2+]i in the regulation of the spermatogenic process and its cellular homeostatic mechanisms in spermatogenic cells has not been elucidated. Using intracellular fluorescent probes for Ca2+ and immunodetection of plasma membrane (PM) Ca(2+)-ATPases, we report that: a) rat round spermatids maintain [Ca2+]i levels of 60 +/- 5 nM (SEM), as estimated with fluo-3 in single cells or fura-2 in cells in suspension; b) these cells regulate [Ca2+]i by actively extruding it using a PM Ca(2+)-ATPase; c) rat spermatids also actively transport Ca2+ by sarco-endoplasmic reticulum type ATPases (SERCA); d) rat spermatids possess non-mitochondrial intracellular Ca2+i stores insensitive to thapsigargin but releasable by ionomycin; and e) rat spermatids do not activate Ca2+ entry mechanisms by the release of Ca2+ from SERCA-regulated stores. These results demonstrate that rat round spermatids can generate modulated intracellular Ca2+ signals upon activation of Ca2+ channels or Ca2+ release from intracellular stores.

Adenosine Triphosphatases↗
PubMed2000

Prognostic factors for successful testicle spermatid recover.

Men with non-obstructive azoospermia exhibit different histopathologic syndromes in the testicle biopsy, varying from aplasia, Sertoli cell only syndrome, maturation arrest and hypoplasia. The genetic basis of these syndromes is discussed. We present the diagnostic testicle biopsies performed on 160 consecutive non-obstructive azoospermic patients, and these results were correlated with the findings after multiple bilateral treatment testicle biopsy. Each syndrome had to be reevaluated as for the presence of at least one focus of spermatogenesis up to the primary spermatocyte, round spermatid, elongating spermatid, elongated spermatid, or spermatozoa stages. The clinical outcome using donor sperm-IVF, spermatid or sperm intracytoplasmic injection is thereafter presented. A new prognosis based on the findings of this large clinical series coupled to results obtained with Y chromosome molecular screening is offered. Alternative treatments to donor sperm for men without spermatids are discussed.

Biopsy↗
PubMed2003

HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis.

Chromatin remodeling is a major event that occurs during mammalian spermiogenesis, the process of spermatid maturation into spermatozoa. Nuclear condensation during spermiogenesis is accomplished by replacing somatic histones (linker histone H1 and core histones) and the testis-specific linker histone, H1t, with transition proteins and protamines. It has long been thought that H1t is the only testis-specific linker histone, and that all linker histones are replaced by transition proteins, and subsequently by protamines during spermiogenesis. Here, we report the identification and characterization of a spermatid-specific linker histone H1-like protein (termed HILS1) in the mouse and human. Both mouse and human HILS1 genes are located in intron 8 of the alpha-sarcoglycan genes. HILS1 is highly expressed in nuclei of elongating and elongated spermatids (steps 9-15). HILS1 displays several biochemical properties that are similar to those of linker histones, including the abilities to bind reconstituted mononucleosomes, produce a chromatosome stop during micrococcal nuclease digestion, and aggregate chromatin. Because HILS1 is expressed in late spermatids that do not contain core histones, HILS1 may participate in spermatid nuclear condensation through a mechanism distinct from that of linker histones. Because HILS1 also belongs to the large winged helix/forkhead protein superfamily, HILS1 may also regulate gene transcription, DNA repair, and/or other chromosome processes during mammalian spermiogenesis.

Amino Acid Sequence↗
PubMed1997

Germ cell mutagenicity of three metabolites of 1,3-butadiene in the rat: induction of spermatid micronuclei by butadiene mono-, di-, and diolepoxides in vivo.

Three metabolites of the industrial chemical 1,3-butadiene (BD), namely butadiene monoepoxide (BMO, 3,4-epoxy-1-butene), diepoxide (DEB, 1,2;3,4-diepoxybutane), and diolepoxide (DE, 3,4- epoxybutane-1,2-diol) were studied for germ cell mutagenicity using the rat spermatid micronucleus (MN) test. All three epoxides increased slightly, but significantly, the frequency of spermatid MN. The most sensitive stage to the action of BMO and DEB was preleptotene (meiotic S phase) harvested at 18-day time intervals after treatment. The dose-response for BMO followed a second order curve at this time interval, with maximum MN induction at the dose of 186 mumol/kg and lower induction of higher doses. Late stages of the meiotic prophase (late pachytene-diplotene-diakinesis) also showed some sensitivity to the three epoxides. Stem cell spermatogonia were affected by DEB as observed by a slight induction of spermatid micronuclei 50 days after treatment. No clear cytotoxic effects were observed by measuring testicular weight or cell numbers of seminiferous epithelial stage 1 18 days after the treatments. DEB at the dose 387 mumol/kg caused a slight inhibition of spermatogonial DNA synthesis in stage I and a delay of meiotic DNA replication observed in stage XII 72 hr after treatment. Since BMO is able to induce spermatid MN in the rat, the present results, together with previous data, indicate that rat bone marrow MN results that are negative for both BD and BMO cannot directly predict mutagenicity in male germ cells. The results also emphasize that tissue; species, and strain-specific differences in metabolism have to be taken into account when the genetic risks of human butadiene exposure are evaluated. The results support the conclusion that 1,3-butadiene is a germ cell mutagen-possibly also in humans.

Animals↗
PubMed1996

Actin and major sperm protein in spermatids and spermatozoa of the parasitic nematode Heligmosomoides polygyrus.

Nematode spermatozoa are amoeboid cells. In Caenorhabditis elegans and Ascaris suum, previous studies have reported that sperm motility does not involve actin, but, instead, requires a specific cytoskeletal protein, namely major-sperm-protein (MSP). In Heligmosomoides polygyrus, a species with large and elongate spermatids and spermatozoa, cell organelles are easily identified even with light microscopy. Electrophoresis of Heligmosomoides sperm proteins indicates that the main protein band has a molecular weight of about 15 kDa, as MSP in other nematodes, and is specifically labelled by an anti-MSP antibody raised against C. elegans MSP. A minor band at 43 kDa was specifically labelled by an anti-actin antibody. Reaction of anti-actin and anti-MSP antibodies is specific to, and restricted to, their respective targets. Actin and MSP localisation, studied by indirect immunofluorescence in male germ cells of Heligmosomoides polygyrus, are similar: spermatids show rows of dots, corresponding to the fibrous bodies, around an unlabelled central longitudinal core; spermatozoa are labelled strictly in an anterior crescent-shaped cap, at the opposite pole to the nucleus, which contains fibres of the MSP cytoskeleton. Phalloidin labelling shows that F-actin is present in spermatids, but absent in spermatozoa. Tropomyosin shows a distinct pattern in spermatids, but is located in the MSP and actin-containing cap in spermatozoa. It is hypothesized that actin plays a role in the shaping of the cell and in the arrangement of its organelles during nematode spermiogenesis, when MSP is present, in an inactive state, in the fibrous bodies. The concentration of actin and tropomyosin in the anterior cap is not compatible with previous theories about the MSP cytoskeleton, which is supposed to act in the absence of actin.

Actins↗
PubMed1980

Evolution of the endoplasmic reticulum in the Sertoli cell cytoplasm encapsulating the heads of late spermatids in the rat.

Throught stage VII and early stage VIII of the cycle of the seminiferous epithelium, the heads of the late spermatids, located in a juxtaluminal position, are embedded in apical processes of Sertoli cells. These processes contain cisternae of endoplasmic reticulum (ER) of two main types, i.e., flattened and tubular, which communicate with each other to form a continuous system. Throughout the long stage VII of the cycle, these two types of cisternae undergo marked changes. In early stage VII, the flattened cisternae, developing from the subsurface cisternae which compose the "junctional specialization," form concentric sheets at the periphery and in the middle of each apical process. The less conspicuous tubular cisternae form a continuous network which is present in the bridge connecting the Sertoli cell body to the apical process, and extends along the dorsal and ventral aspects of the spermatid's head to end up as cup-shaped flattened cisternae capping the bulbs of the tubulobulbar complexes described by Russell and Clermont ('76). In mid stage VII, the flattened cisternae start to regress, while the tubular cisternae become more abundant. In late stage VII, only fragments of the flattened cisternae are present, while the tubular cisternae form a profuse and elaborate network throughout the apical process. In the following stage VIII, the tubular cisternae disperse and only remnants of ER are present at the time of the release of the spermatid into the tubular lumen. These transformations of ER cisternae suggest a complex alteration in the relationship between Sertoli cells and late spermatids prior to their release as spermatozoa.

Animals↗
PubMed1980

Acrosomal and nuclear morphogenesis in ram spermatids: an experimental study of hypophysectomized and testosterone-supplemented animals.

An ultrastructural study of spermiogenesis was carried out in adult hypophysectomized rams supplemented with testosterone at a dose which induced a normal or excessive (1 to 3) concentration of the steroid within the rete testis fluid (Monet-Kuntz et al., '76). Most of the spermatids from 15- or 20-day treated animals displayed a normal nuclear appearance but possessed acrosomes with morphological abnormalities. The process of acrosome formation as well as its binding to the nucleus was severely impaired in young spermatids, whereas only morphological changes of the acrosomes were seen in old spermatids. The suggestion is made that acrosome development is under the control of endocrine-dependent cellular events occurring before the beginning of spermiogenesis, possibly via Sertoli cell/germ cell interactions. The spermatids from hypophysectomized rams supplemented with testosterone for 40 days were normal in appearance but reduced in number. The Sertoli cell ultrastructure differed for the two durations of treatment.

Acrosome↗
PubMed1987

Distribution of actin in spermatids and adjacent Sertoli cell regions of the rat.

In this study, we describe the distribution of actin filaments in and around spermatids that are mechanically dissociated, in the presence and absence of exogenous trypsin, from the seminiferous epithelium of the rat. NBD-phallacidin and subfragment 1 of the myosin molecule (S-1) are used as probes for filamentous actin at the light and electron microscopic levels, respectively. The fluorescence associated with spermatids mechanically dissociated in the absence of trypsin is due to actin both in the spermatogenic cells themselves and in attached Sertoli cell ectoplasmic specializations. Fluorescence generated by labelled actin in ectoplasmic specializations occurs in linear tracts that follow the outer contour of spermatid heads. The residual fluorescence seen when trypsin is used to detach Sertoli cell fragments is diffuse and due to f-actin in the subacrosomal space. Electron microscopic data agree with the fluorescence results. This study conclusively demonstrates that Sertoli cell ectoplasmic specializations remain attached to spermatids mechanically dissociated from the seminiferous epithelium. This observation may be useful when attempting to isolate ectoplasmic specializations for biochemical analyses.

Actins↗
PubMed1990

Immunocytochemical localization of proteins utilized in the formation of outer dense fibers and fibrous sheath in rat spermatids: an electron microscope study.

Affinity purified antibodies prepared against proteins isolated from fibrous sheath (FS) and outer dense fibers (ODF) were utilized in an immunocytochemical study of spermatids at various steps of spermiogenesis. This study, using the immunogold technique, was performed on sections of Epon or Lowicryl embedded tissues examined with the electron microscope. In the case of FS antibodies there was a selective immunoreactivity of the FS itself from step 10 onwards, but no reactivity over the plasma membrane associated FS anlagen. In addition there was a diffuse immunoreaction over the cytoplasmic matrix from step 9 until step 18 of spermiogenesis but no reactivity over the various types of dense bodies (e.g., granulated bodies, reticulated body, etc.) seen in the cytoplasm of these spermatids. In the case of ODF antibodies the ODF were immunolabeled throughout their development from step 11 onward. In addition to a diffuse immunoreactivity of the cytoplasmic matrix of spermatids from step 9 until step 18 of spermiogenesis, there was an immunolabeling of "granulated bodies." These bodies appeared in relation to ER cisternae during steps 10-14, increased in number and size during steps 15-17 and decreased in number thereafter leaving only a few coarsely granulated bodies in the residual cytoplasm which detached from late step 19 spermatids. No other cytoplasmic structures were labeled with the ODF antibody-gold complexes. Thus the granulated bodies appeared to serve as a transitory storage site for some proteins destined to form ODF, a major cytoskeletal element of the tail of rat spermatozoa.

Acrosome↗
PubMed2005

Ectopic expression of KitD814Y in spermatids of transgenic mice, interferes with sperm morphogenesis.

Kit is a receptor tyrosine kinase that plays a fundamental role during the development of germ cells. Additionally, a truncated product, tr-kit, expressed in haploid spermatids and mature spermatozoa can induce parthenogenetic activation when microinjected into mouse eggs, through the activation of PLCgamma-1. In this work, we induced ectopic expression of a mutated Kit protein, Kit(D814Y) during germ cell development. The in vivo expression of this mutant in spermatids produced malformations in mature spermatozoa, and in the most severe cases, sterility. Ultrastructural analysis indicated that condensing spermatids in the transgenic mouse presented a mislocalization of the manchette; a structure that has a crucial role during the elongation steps of spermiogenesis. This morphogenetic phenotype was accompanied by an increased phosphorylation of PLCgamma-1 in spermatogenic cells. Interestingly, we also found that, in wild-type testis, PLCgamma-1 is specifically phosphorylated in condensing spermatids, coincident with the timing of expression of tr-kit in spermiogenesis. We propose that alterations of PLCgamma-1 activity artificially promoted by ectopic Kit(D814Y) expression are related to the abnormalities of spermiogenesis. Our observations suggest that PLCgamma-1 activity could be involved in the shaping of spermatozoa.

Animals↗
PubMedDate not supplied

Cloning and characterization of rat spermatid protein SSP411: a thioredoxin-like protein.

In an attempt to identify new sperm-specific genes that are involved in sperm maturation, fertilization, and embryo development, such as the mammalian ortholog of the sperm-supplied protein gene, spe-11, in Caenorhabditis elegans, we cloned and characterized a new spermatid-specific protein gene, ssp411, from adult rat testes. The ssp411 cDNA shared >85% sequence identity with an unnamed human protein, FLJ21347, and an uncharacterized mouse testicular protein called transcript increased in spermiogenesis 78 (TISP78). A 2.8-kb ssp411 mRNA was expressed in a testis-specific and age-dependent manner; the mRNA was evident at 28 days and remained at high levels throughout adulthood. An SSP411 protein of molecular weight 88 000 was detected in testicular extracts by Western blot analysis. Ssp411 mRNA and SSP411 protein, as analyzed by in situ hybridization and immunohistochemistry, were both expressed in a stage-dependent fashion during the cycle of the seminiferous epithelium. The ssp411 mRNA was predominantly localized to round and elongated spermatids, with maximal expression at stages VII-XII. The SSP411 protein was mainly observed in elongated spermatids and reached its highest levels during stages V-VI. A conserved thioredoxin-like domain was detected in the N-terminal region of SSP411 and its orthologs. An analysis of the predicted 3-dimensional structural modeling and folding pattern further suggested that SSP411 is identifiable as a member of thioredoxin family. In summary, we have identified a new rat spermatid protein gene, ssp411, and its orthologs in human and mouse and demonstrated that SSP411 might belong to a testis-specific thioredoxin family. This suggests that SSP411 may play a role in sperm maturation, fertilization, and/or embryo development, as has been shown in thioredoxin family.

Amino Acid Sequence↗
PubMed1990

Extrusion of the residual body in spermatids of Ancylostoma caninum (Nematoda, Strongyloidea).

Transmission electron microscopy reveals that the spermatocytes of the hookworm Ancylostoma caninum contain an abundance of Golgi complexes, ribosomes, specialized membranous organelles, and long strands of smooth endoplasmic reticulum. These organelles remain abundant until the early spermatid stage of sperm development, when they reach their maximum abundance and maturity and the production of new ones ceases. Golgi complexes, ribosomes, and excess SER, which are not functional after this stage, become segregated and confined to the posterior portion of the spermatid in a polar lobe. Later, the polar lobe together with excess cytoplasmic matrix is bound by a membrane and dissociated from the spermatid as a residual body. The spermatid is then devoid of Golgi complexes and ribosomes. Formation of residual bodies as sperm cells mature may be considered a type of cell excretion.

Ancylostoma↗
PubMed2002

Rescue of infertile transgenic rat lines by intracytoplasmic injection of cryopreserved round spermatids.

Transgenic male rats carrying human alpha-lactalbumin with thymidine kinase gene (line name; LAC3) were found to be infertile due to expression of the transgene in the testes. Furthermore, it was not possible to maintain the line even by the use of intracytoplasmic sperm injection (ICSI). Therefore, round spermatids prepared from the LAC3 rats were microinjected into strontium-activated oocytes using a Piezo-driven micromanipulator. Of 263 oocytes microinjected with LAC3 spermatids, 244 (92.8%) survived the injection and 96 (39.3%) developed to the 2-cell stage. Three viable offspring were born after transfer (1.4%, 3/219), and two offspring carried the LAC3 transgene. In the control experiment using spermatids of Wistar rats, similar proportions of post-injection survival (91.3%, 241/264), cleavage (40.2%, 97/241), and development into offspring (0.5%, 1/206) were obtained. Thus, this paper reports not only the first rat offspring derived from round spermatid injection but also the practical application of the microinsemination technique to the rescue of transgenes of infertile transgenic male rats.

Animals↗
PubMed1991

Distribution of filamentous actin in and around spermatids and in spermatozoa of Australian conilurine rodents.

The distribution of filamentous actin around the maturing sperm head and in spermatozoa of four species of Australian conilurine rodents was investigated at the light and electron microscopic levels. Similar results were obtained for all the species studied. Mechanically isolated spermatids had NBD-phallacidin-positive longitudinal bands of fluorescence over the dorsolateral surface and, in late spermatids, bands of bright fluorescence passed perpendicularly from the dorsal convex to ventral concave surface. TEM observations indicated that these regions corresponded to filaments of ectoplasmic specializations and granular filamentous material around the tubulobulbar complexes, respectively. In testicular and cauda spermatozoa NBD-phallacidin fluorescent material was present in the two ventral processes that extended from the upper concave surface of the sperm head; also fainter material occurred along the concave border and as a dorsocaudal spur. Its distribution was identical for testicular and cauda spermatozoa. TEM of late spermatids showed that in the ventral process closest to the apical hook there were between 170 and 245 filaments, which attached to the inner surface of the postacrosomal dense lamina; in the more caudal ventral process about 70 filaments occurred. No filaments were, however, visible in the mature spermatozoon but, after immunocytochemical labelling for actin, deposition of gold particles was evident over ventral processes of both late spermatids and cauda spermatozoa. Within the female tract these ventral processes made contact with the zona matrix and were taken into the egg cytoplasm unchanged in morphology. The possible functional significance of the filamentous actin in these structures is discussed.

Acrosome↗