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PubMed2000

Poor outcome with round spermatid injection in azoospermic patients with maturation arrest.

OBJECTIVE: To compare the outcome of intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI), both obtained by testicular sperm extraction (TESE), and to compare the results of fresh versus frozen ROSI. DESIGN: Retrospective study. SETTING: An IVF unit at a university hospitalPatient(s): Eighteen infertile couples with nonobstructive azoospermia. INTERVENTION(S): TESE with ROSI or ICSI of mature spermatozoa into metaphase II oocytes was performed. The resulting embryos were transferred to female partners. The spare round spermatids were frozen. MAIN OUTCOME MEASURE(S): Fertilization and cleavage rates, embryo quality, and clinical pregnancy rates. RESULT(S): Seventeen ROSI cycles and six ICSI cycles were compared. Fertilization rate following ROSI (44.9%) was significantly lower than with ICSI (69%). A significantly higher rate of cleavage arrest occurred following ROSI (40.8%) as compared to ICSI (8.2%). The morphology of embryos resulting from ROSI was significantly poorer. No pregnancies were achieved following ROSI as compared to a 50% clinical pregnancy rate in the ICSI group. The fertilization and cleavage rates following ROSI with fresh versus frozen-thawed spermatids were comparable. CONCLUSION(S): In azoospermic patients with maturation arrest at the stage of round spermatids the efficiency of ROSI appears to be extremely poor. The role of ROSI in the treatment of nonobstructive azoospermia should be reevaluated.

Adult↗
PubMed1996

Beneficial effects of electrical stimulation before round spermatid nuclei injections into rabbit oocytes on fertilization and subsequent embryonic development.

OBJECTIVE: To evaluate the effects of electrical stimulation of rabbit oocytes before round spermatid nuclear injection procedure on oocyte activation and fertilization. DESIGN: The ratio of activated oocytes to the number of successfully injected oocytes and the proportion of offspring to the number of activated oocytes after round spermatid nuclear injections into oocytes stimulated via mechanical stimulation (group A) or a combination of electrical and mechanical stimulation (group B) was compared. INTERVENTIONS: Round spermatid nuclei were isolated from mature male rabbits and microinjected into the oocytes of groups A and B. Injected oocytes were cultured for 24 hours. The embryos developed from groups A and B were transferred to synchronized recipient does. RESULTS: Embryos that developed normally through implantation in groups A and B were carried successfully through complete gestation in the recipient does. The ratio of the activated oocytes to the number of successfully injected oocytes and the proportion of offspring to the number of activated oocytes were significantly higher in group B. CONCLUSION: Electrical stimulation of oocytes before ooplasmic spermatid nuclear injections and ET procedures has beneficial effects on oocytes activation, fertilization, and subsequent embryonic development.

Animals↗
PubMed1996

Fertilization and embryo cleavage after intracytoplasmic spermatid injection in an obstructive azoospermic patient with defective spermiogenesis.

OBJECTIVE: To achieve fertilization and cleavage by spermatids without tails from testicular biopsy. DESIGN: Clinical trial. SETTING: Reproductive unit of a university teaching hospital. PATIENT: A patient of obstructive azoospermia with defective spermiogenesis. INTERVENTION: Testicular biopsy after scrotal exploration and spermatid injection into the cytoplasm of oocyte. MAIN OUTCOME MEASURES: Fertilization and cleavage. RESULTS: Four of 13 spermatid injections achieved normal fertilization and 2 of them cleaved. CONCLUSION: Intracytoplasmic spermatid injection may be a possible treatment for patients of defective spermiogenesis who have no viable spermatozoa available.

Adult↗
PubMed1996

Spermatid micronucleus analysis of aging effects in hamsters.

Spermatid micronuclei (MN) from Armenian hamsters in different age groups were compared with regard to frequencies and kinetochore status (presence or absence) as determined with immunofluorescent staining. Six thousand cells analyzed from each of fifteen young animals (3 months) revealed a group mean frequency of 0.45 MN/1000 spermatids; kinetochore staining was uniformly negative. Six thousand cells scored from each of fifteen older animals (2 years) revealed a group mean frequency of 1.00 MN/1000 spermatids. Most of the MN in these animals were negative for kinetochore staining, although a significant representation of MN with positive kinetochore staining was also observed. The results indicate that frequencies of spermatid MN increase with advancing age, and suggest that the increase is due to significant elevations in both chromosome breakage and chromosome loss.

Aging↗
PubMed1989

Relationship between chromosome content and nuclear diameter in early spermatids of Drosophila melanogaster.

We have studied, using light microscopy, the relationship between chromosome content and nuclear diameter in early spermatids of males carrying different combinations of wild-type and compound chromosomes in Drosophila melanogaster. By using these genotypes we have been able to observe spermatid nuclei bearing various numbers of chromosomes ranging from only one sex chromosome and no major autosomes to almost twice the normal chromosome complement. We have found that variations in the chromosome content are accompanied by increasing the variance in early spermatid nuclear diameter; the more gametic classes produced, the higher the variance of nuclear diameters. These results indicate that measuring nuclear diameters in early spermatids represents a useful way to estimate the levels of meiotic non-disjunction and thereby to improve the characterization of lethal or male sterile mutants in which analysis of meiotic chromosome non-disjunction cannot be achieved by conventional genetic methods.

Animals↗
PubMed1992

Ultrastructure of the spermatid and spermatozoon of Macracanthorhynchus hirudinaceus.

The ultrastructure of the spermatid and spermatozoon of Macracanthorhynchus hirudinaceus (Archiacanthocephala) was studied by means of transmission electron microscopy. The flagellum and nucleus in the spermatid gradually expanded simultaneously. The karyoplasma of the spermatid transformed into dense inclusions and a multibarrel structure, which were also found in the spermatozoan body. The multibarrel structure was located close to the flagellum and consisted of many irregular microtubes. The flagellum of the developing spermatozoon was observed in a concavity of the spermatid nucleus. The microtubule arrangement of the flagellum was "9 + 2". No mitochondria or acrosome were observed in spermatozoa.

Acanthocephala↗
PubMed2003

Microtubule-associated epithelial protein E-MAP-115 is localized in the spermatid manchette.

A microtubule-associated protein E-MAP-115 has been originally isolated and characterized from HeLa cells. Because of its predominant expression in cultured cells of epithelial origin, it has been suggested to be involved in the regulation of cell polarization. The present immunocytochemical, Northern blot and in situ hybridization analysis of E-MAP-115 in the mouse and rat seminiferous epithelium indicates its distinct association with the spermatid manchette, a unique microtubular structure which appears in the cytoplasm of spermatids at step 8 when nuclear polarization and elongation starts. At steps 15-16 when manchette has been disassembled, immunoreactivity for E-MAP-115 disappeared. At immunoelectron microscopical level, E-MAP-15 was associated with the microtubules of the manchette. In the Western and Northern blot analysis, a distinct stage-dependent expression of a single E-MAP-115 polypeptide and two mRNA species (3.4 and 2.4 kb) could be identified. MTEST 60, a spermatid-specific transcript, showed a 100% homology over region of 68-193 bp of E-MAP-115 sequence. The reported specific localization of E-MAP-115 to the spermatid manchette strongly supports its role as a regulator of cell polarization. This, in turn, supports the hypotheses concerning the dynamic function of the manchette during spermiogenesis.

Animals↗
PubMed2005

Polar nuclear localization of H1T2, a histone H1 variant, required for spermatid elongation and DNA condensation during spermiogenesis.

Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell involving replacement of the somatic histones by protamines packing the DNA into the condensed spermatid nucleus and elimination of the cytoplasm during the elongation phase. We describe H1T2, an histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis. In round and elongating spermatids, H1T2 specifically localizes to a chromatin domain at the apical pole, revealing a polarity in the spermatid nucleus. Inactivation by homologous recombination shows that H1T2 is critical for spermiogenesis as male H1t2(-/-) mice have greatly reduced fertility. Analysis of spermiogenesis in H1t2 mutant mice shows delayed nuclear condensation and aberrant elongation. As a result, mutant spermatids are characterized by the presence of residual cytoplasm, acrosome detachment, and fragmented DNA. Hence, H1T2 is a protein required for proper cell restructuring and DNA condensation during the elongation phase of spermiogenesis.

Amino Acid Sequence↗
PubMed2006

Transcriptional regulation by Modulo integrates meiosis and spermatid differentiation in male germ line.

Transcriptional activation in early spermatocytes involves hundreds of genes, many of which are required for meiosis and spermatid differentiation. A number of the meiotic-arrest genes have been identified as general regulators of transcription; however, the gene-specific transcription factors have remained elusive. To identify such factors, we purified the protein that specifically binds to the promoter of spermatid-differentiation gene Sdic and identified it as Modulo, the Drosophila homologue of nucleolin. Analysis of gene-expression patterns in the male sterile modulo mutant indicates that Modulo supports high expression of the meiotic-arrest genes and is essential for transcription of spermatid-differentiation genes. Expression of Modulo itself is under the control of meiotic-arrest genes and requires the DAZ/DAZL homologue Boule that is involved in the control of G(2)/M transition. Thus, regulatory interactions among Modulo, Boule, and the meiotic-arrest genes integrate meiosis and spermatid differentiation in the male germ line.

Amino Acid Sequence↗
PubMed1995

Premature translation of protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice.

Translational control is a major form of regulating gene expression during gametogenesis and early development in many organisms. We sought to determine whether the translational repression of the protamine 1 (Prm1) mRNA is necessary for normal spermatid differentiation in mice. To accomplish this we generated transgenic animals that carry a Prm1 transgene lacking its normal 3' untranslated region. Premature translation of Prm1 mRNA caused precocious condensation of spermatid nuclear DNA, abnormal head morphogenesis, and incomplete processing of Prm2 protein. Premature accumulation of Prm1 within syncytial spermatids in mice hemizygous for the transgene caused dominant male sterility, which in some cases was accompanied by a complete arrest in spermatid differentiation. These results demonstrate that correct temporal synthesis of Prm1 is necessary for the transition from nucleohistones to nucleoprotamines.

Animals↗
PubMed1998

Two novel odorant receptor families expressed in spermatids undergo 5'-splicing.

We report the identification of two novel families of odorant receptor (OdR)-like proteins, termed spermatid chemoreceptors (SCRs), in rat spermatids of the testis. The full-length genomic clones encode seven transmembrane domain receptors that share 35-40% identity with certain OdRs and are among the most divergent members of the OdR superfamily based on phylogenetic analysis. RNase protection assays and in situ hybridization studies confirmed the expression of SCRs in spermatids, the post-meiotic, differentiating cell population in the testis. SCR transcripts were undetectable in the prepubertal testis but were readily identified in spermatids of sexually maturing and mature testis. Rapid amplification of cDNA end-polymerase chain reaction and genomic clone sequencing led to the discovery that SCRs are spliced upstream of their presumptive starting methionines. 5'-Splicing of OdRs may regulate the expression of functional chemoreceptors.

Alternative Splicing↗
PubMed2003

Cloning and developmental analysis of murid spermatid-specific thioredoxin-2 (SPTRX-2), a novel sperm fibrous sheath protein and autoantigen.

Thioredoxins compose a growing family of proteins that participate in different cellular processes via redox-mediated reactions. We report here the cloning, developmental expression, and location of murid Sptrx-2. Mouse and rat SPTRX-2 proteins display a high homology to their human ortholog in the thioredoxin and NDP kinase domains, and the coding genes are located at syntenic positions. Northern blotting and in situ hybridization confirmed the testis-specific expression of murine Sptrx-2 mRNA, mostly in round spermatids. Immunohistochemical analysis of the 19 steps of rat spermiogenesis showed that SPTRX-2 expression becomes prominent in the cytoplasmic lobe of step 15-18 spermatids and diminishes in step 19 just before spermiation. However, in the spermatid tail, SPTRX-2 immunoreactivity increased from step 15 to 19 and was confined to the principal piece. By immunogold electron microscopy, SPTRX-2 was first detected scattered throughout the cytoplasm of the axoneme in step 14-15 spermatids, but began to be incorporated by step 16 into the fibrous sheath (FS). During steps 17-18, the labeling increased over the ribs and columns of the assembled FS. It peaked in step 19 and remained in the FS of epididymal spermatozoa. Immunoblots of isolated FS obtained from spermatozoa confirmed that SPTRX-2 is an integral component of the FS and a post-obstruction autoantigen in vasectomized rats. Our data indicate that SPTRX-2 incorporation into the FS lags well behind FS assembly, suggesting it is required during the final stages of sperm tail maturation in the testis and/or epididymis, where extensive disulfide bonding of FS proteins occurs.

Amino Acid Sequence↗
PubMed1962

Observations on the fine structure of the developing spermatid in the domestic chicken.

The kinetic apparatus, the acrosome and associated structures, and the manchette of the spermatid of the domestic chicken have been studied with the electron microscope. The basic structural features of the two centrioles do not change during spermiogenesis, but there is a change in orientation and length. The proximal centriole is situated in a groove at the edge of the nucleus and oriented normal to the long axis of the nucleus and at right angles to the elongate distal centriole. The tail filaments appear to originate from the distal centriole. The plasma membrane is invaginated along the tail filaments. A dense structure which appears at the deep reflection of the plasma membrane is identified as the ring. The fine structure of the ring has no resemblance to that of a centriole and there is no evidence that it is derived from or related to the centrioles. The tail of the spermatid contains nine peripheral pairs and one central pair of tubular filaments. The two members of each pair of peripheral filaments differ in density and in shape: one is dense and circular, and the other is light and semilunar in cross-section. The dense filaments have processes. A manchette consisting of fine tubules appears in the cytoplasm of the older spermatid along the nucleus, neck region, and proximal segment of the tail. The acrosome is spherical in young spermatids and becomes crescentic and, finally, U-shaped as spermiogenesis proceeds. A dense granule is observed in the cytoplasm between acrosome and nucleus. This granule later becomes a dense rod which is interpreted as the perforatorium.

Animals↗
PubMed1958

The spermatid nucleus in two species of grasshopper.

The nuclear changes accompanying spermatid elongation have been studied in two species of grasshopper, Dissosteira carolina and Melanoplus femur-rubrum. Testes were fixed in 1 per cent buffered OsO(4), imbedded in butyl methacrylate, and examined as thin sections in the electron microscope. In both species nuclear changes during spermatid development involve (1) an early period, during which the nuclear contents are predominately fibrous; (2) a middle period, characterized by the lateral association of the nuclear fibers to form plates or lamellae which are oriented longitudinally in the major axis of the elongated nucleus; and (3) a late period, involving coalescence of the lamellae into a crystalline body which eventually becomes so dense that all resolvable detail is lost. The fibers seen in the early spermatid nucleus are about 150 A in diameter and so are similar to fibers described from other types of nuclei. The thickness of the lamellae varies from about 150 A when first formed to 70 A during the later stages. The lack of evident chromosomal boundaries in the spermatid nucleus makes it difficult to relate either the fibers or lamellae to more familiar aspects of chromosome structure. We see no apparent reason to consider that the fiber alignment described here is related to conventional chromosome pairing.

Animals↗
PubMed1960

Spermatogenesis in animals as revealed by electron microscopy. VIII. Relation between the nutritive cells and the developing spermatids in a pond snail, Cipangopaludina malleata Reeve.

This paper deals with spermatogenesis in Cipangopaludina malleata Reeve, with special regard to the relation between the nutritive cells and the developing spermatids. The nutritive cell gives rise to numerous, slender or broad, elongate pseudopodia which extend from its surface toward the seminiferous lumen. They are characteristically provided with rows of circular, oval, and elongate profiles identical in form and position with the profiles of the endoplasmic reticulum. As the elongate pseudopodia increase in number, they become more slender and more closely packed until they coalesce into a continuous sheet circumferentially disposed around the nucleus and the full length of the middle piece of the typical spermatid. Thus the mantle of the typical spermatozoon of the pond snail is formed by a thin fold of the cytoplasm of the nutritive cells. This wrapping appears to contain 16 to 18 elements of the smooth surfaced endoplasmic reticulum, which run parallel and helically (50 to 100 mmicro apart). It is suggested that these constitute a conductor system for nutritional supply from the nutritive cells to the developing typical spermatids. The mantle is assumed to be a transient structure which disappears when the sperms are detached. The atypical spermatids develop while lodged in deep indentations of the surface of the nutritive cells.

Animals↗
PubMed2004

Cytoplasmic dynein-dynactin complex is required for spermatid growth but not axoneme assembly in Drosophila.

Spermatids derived from a single gonial cell remain interconnected within a cyst and elongate by synchronized growth inside the testis in Drosophila. Cylindrical spectrin-rich elongation cones form at their distal ends during the growth. The mechanism underlying this process is poorly understood. We found that developing sperm tails were abnormally coiled at the growing ends inside the cysts in the Drosophila Dynein light chain 1 (ddlc1) hemizygous mutant testis. A quantitative assay showed that average number of elongation cones was reduced, they were increasingly deformed, and average cyst lengths were shortened in ddlc1 hemizygous testes. These phenotypes were further enhanced by additional partial reduction of Dhc64C and Glued and rescued by Myc-PIN/LC8 expression in the gonial cells in ddlc1 backgrounds. Furthermore, DDLC1, DHC, and GLUED were enriched at the distal ends of growing spermatids. Finally, ultrastructure analysis of ddlc1 testes revealed abnormally formed interspermatid membrane, but the 9 + 2 microtubule organization, the radial spoke structures, and the Dynein arms of the axoneme were normal. Together, these findings suggest that axoneme assembly and spermatid growth involve independent mechanisms in Drosophila and DDLC1 interacts with the Dynein-Dynactin complex at the distal ends of spermatids to maintain the spectrin cytoskeleton assembly and cell growth.

Alleles↗
PubMed2000

Genetic analysis of a Y-chromosome region that induces triplosterile phenotypes and is essential for spermatid individualization in Drosophila melanogaster.

The heterochromatic Y chromosome of Drosophila melanogaster contains approximately 40 Mb of DNA but has only six loci mutable to male sterility. Region h1-h9 on YL, which carries the kl-3 and kl-5 loci, induces male sterility when present in three copies. We show that three separate segments within the region are responsible for the triplosterility and have an additive effect on male fertility. The triplosterile males displayed pleiotropic defects, beginning at early postmeiotic stages. However, the triplosterility was unaffected by kl-3 or kl-5 alleles. These data suggest that region h1-h9 is complex and may contain novel functions in addition to those of the previously identified kl-3 and kl-5 loci. The kl-3 and kl-5 mutations as well as deficiencies within region h1-h9 result in loss of the spermatid axonemal outer dynein arms. Examination using fluorescent probes showed that males deficient for h1-h3 or h4-h9 displayed a postmeiotic lesion with disrupted individualization complexes scattered along the spermatid bundle. In contrast, the kl-3 and kl-5 mutations had no effect on spermatid individualization despite the defect in the axonemes. These results demonstrate that region h1-h9 carries genetically separable functions: one required for spermatid individualization and the other essential for assembling the axonemal dynein arms.

Animals↗
PubMed1998

Ooplasmic injection of elongating spermatids for the treatment of non-obstructive azoospermia.

We applied the technique of ooplasmic elongating spermatid injection to the treatment of non-obstructive azoospermia. Mature oocytes were injected with elongating spermatids isolated from testicular biopsy material obtained from 13 non-obstructed azoospermic men. Seventy-three oocytes were successfully injected with elongating spermatids and were then cultured for 36 h. At 13 h post-injection 68 oocytes were found to be activated and 52 of them were fertilized. Forty-one 2- to 4-cell stage embryos developed from normally fertilized oocytes were transferred. At least two embryos were transferred to each female partner. Two pregnancies were achieved. Elongating spermatid injection may have a role in the treatment of non-obstructive azoospermia.

Adult↗