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The induction of sex-chromosomal nondisjunction and diploid spermatids following x-irradiation of pre-spermatid stages in the northern vole Microtus oeconomus.

Chromosome nondisjunction seems to be one of the most important mutagenic effects occurring in man and makes an enormous contribution to human foetal wastage. As yet, little or no information is available on which environmental factors are important in inducing nondisjunction and accordingly we have investigated the effect of X-irradiation on inducing nondisjunction in male germ cells of an experimental mammal, the Northern vole-Microtus oeconomus. Using a staining technique based upon the presence of heterochromatin we have scored the number of sex chromosomes in early spermatids in both irradiated and unirradiated animals. A significant increase in nondisjunction, following treatment, was found with all doses between 25 and 200 R. However, variations in nondisjunction induction at various time intervals following irradiation suggest variations in cell stage sensitivity. More surprising was the large induction of diploid gametes which also demonstrated a significant induction with all irradiation doses. From the distribution of sex chromosomes we conclude that both nondisjunction and diploid gamete induction occur at both meiotic divisions. At present it is not possible to conclude whether the radiation response is linear and to define the cell-stage sensitivity with precision. The reasons for this appear to be variations in sensitivity between animals and also that there is a clear overlap between the duration of the early spermatid stage analyzed (4 days) and the interval between sampling times.

Animals

Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl-, ethyl-, propyl-, and isopropyl methanesulfonate.

In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.

1-Propanol

[Morphological studies on abnormal human spermatids and spermatozoa (author's transl)].

Malformed spermatids and spermatozoa in the testes, epididymes, and ejaculates of 29 apparently healthy men and in 171 patients suffering from different andrological diseases were investigated with the electron microscope. The following types of malformations are described: 1) Malformations of the acrosome in contact with the nucleus of the spermatid: Vesicular inclusions in the acrosome vesicle, asymmetric thickenings of the acrosome cap, enfolding of the acrosomal lamina into the spermatid's nucleus, partial separation of the acrosome from the nucleus, apical knob-like thickenings of the acrosome. 2) Malformations of the acrosome independent from the spermatid's nucleus: the acrosomal material is contained in small vesicles, large vacuoles, in ring-like structures, in globular or shell-like laminar structures. The nucleus of such spermatids lacks an acrosome. 3) Malformations of the nucleus: inclusions of vesicles or membranes, excessive packages of membranes, gigantic nuclear vacuoles, lack of condensation of the caryoplasm. 4) Multinucleate spermatids: two or more nuclei within the pericaryon of one spermatid are joined by an acrosome. 5) Duplicate acrosome anlagen. 6) Malformations of the tail: disorganization of the axonema, a lack or excessive numbers of tubules and fibres, doublets of the axial filaments, destruction of the neck region, a lack of the mitochondrial sheath, and enlargement of the fibrous sheath. Malformed germ cells were observed to occur in varying amounts both in apparently healthy men and in those who were apparently infertile. This indicates that germ cell malformations are no absolute indicator of male infertility. However, certain distinct types of malformations (such as round-headed spermatozoa) are definitely associated with infertility, but only if all spermatozoa in the ejaculate of a patient have the same defect.

Acrosome

The lipid composition of isolated rat spermatids and spermatocytes.

The lipids composition of enriched fractions of spermatids and spermatocytes, isolated from rat testicular tissue, has been investigated. More than 20% of the total fatty acids of spermatids but only 10% of those of spermatocytes, isolated from testes of mature rats, was 4,7,10,13,16-docosapentaenoic acid. Spermatocyte-enriched fractions isolated from testes of immature rats had fatty acid compositions similar to those isolated from testes of mature rats. On the other hand, spermatids isolated from immature rats had a level of docosapentaenoic acid which was intermediate between the level found in spermatocytes and that of spermatids from mature rats. Major phospholipid classes and the triacylglycerols of spermatids contained much more of the docosapentaenoic acid than the corresponding lipid types from spermatocytes. Differences in content of total phospholipids, individual classes of phospholipids and triacylglycerols among spermatocytes, spermatids and late spermatids were also observed.

Aging

The effects of temperature and glucose on protein biosynthesis by immature (round) spermatids from rat testes.

A method is described for the preparation of highly purified fractions (greater than 80% pure) of immature spermatids (round, steps 1--8) from rat testes by centrifugal elutriation in sufficient yields for biochemical studies when four rat testes are used. Electron microscopy established the identity of the cells and demonstrated that the cell membrane is intact. Some cells develop nuclear and cytoplasmic vacuoles during the 2 h required for preparation. Immature spermatids prepared by this method use glucose with an increase in oxygen consumption, lactate production, and protein synthesis over control levels (no glucose). The testicular cell suspension from which spermatids are separated, like whole testis and spermatids themselves, show higher incorporation of amino acids into TCA-precipitable material at 34 degrees C than at 38 degrees C and in the presence of glucose. A subcellular system prepared from immature spermatids with excess ATP shows greater incorporation of amino acids into TCA-precipitable material at 34 degrees C than at 38 degrees C. This difference does not result from increased breakdown of protein. It is concluded that body temperature (38 degrees C) inhibits some aspect(s) of protein synthesis in addition to previously reported effects on amino acid transport and production of ATP (Means and Hall. 1969. Endocrinology. 84:285--297.).

Animals

Unscheduled DNA synthesis induced in mouse spermatids after combined treatment with methyl methanesulfonate and X-rays.

Unscheduled DNA synthesis (UDS), which is considered to be DNA repair, has been studied in early- to mid-spermatid stages of the mouse after combined treatments with X-rays and methyl methanesulfonate (MMS). UDS in spermatids was detected by giving testicular injections of [methyl-3H]thymidine ([3H]dThd) and making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes. X-rays and MMS are each able to induce UDS in mouse spermatids. However, there was a statistically significant reduction in the amount of UDS observed when X-ray exposures of from 200 to 600 R were given 4 h before an i.p. injection of 75 mg/kg of MMS and concurrent testicular injections of [3H]dThd. This reduction in UDS is more than can be explained by the completion of repair of X-ray-induced DNA lesions. We suggest that the reduction in UDS is the result of an X-ray-produced impairment of a least a part of the repair mechanism involved in correcting MMS-induced DNA lesions. When the time interval between a 600-R X-ray exposure and MMS treatment was between 3 and 20 h (latest time interval s;udied) there was a statistically significant reduction of UDS in the spermatids. No significant decrease in UDS response occurred when the time interval between radiation exposure and MMS treatment was less than approximately 3 h.

Animals

The packaging unit: a basic structural feature for the condensation of late cricket spermatid nuclei.

The alignment, folding and packaging of cricket chromatin was examined during late spermiogenesis by an electron-microscope study of nuclei dispersed by air--liquid surface tension forces after detergent treatment. Late developing spermatid genomes arrange themselves in multiple packaging units in a stepwise process which includes: (1) a loss of the beaded repeating structure of chromatin as nucleoprotein fibres become smooth and gradually assume a uniform diameter; (2) a side-by--side alignment of structurally modified chromatin fibres; and (3) a regular folding into packaging units. Alignment and folding of chromatin fibres are presumably mediated by intermolecular bonds easily disrupted by spreading forces. In very late spermatids, interfibre binding forces are difficult to overrride by spreading alone, indicating a stronger cross-linking of increasingly coalescent packaging units. 'Unit to unit' coalescence stabilizes the nuclear structure, first limiting and afterwards denying penetration of phosphotungstic acid, as displayed in thin sections of extremely cricket spermatid nuclei. Binding of phosphotungstate by nuclear basic proteins can be facilitated by limited protein solubilization after disulphide reduction of unfixed cricket tests with sodium dodecyl sulphate and dithiothreitol. Results of this study permit the proposal of model experiments useful for clarifying the organization of highly condensed spermatid genomes and for evaluating the structure of genome segments in systems wherein changes of chromatin-associated protein occur.

Animals

Spermiogenesis in Nautilus pompilius. II. Sertoli cell-spermatid junctional complexes.

A "ball and socket-like" junction between branches of the Sertoli cells and the developing spermatids is described. A cytoplasmic extension of the Sertoli cell fits into a pocket in the spermatid and has a constricted neck region. Frequently there are large multivesiculate bodies in the Sertoli cell extension and small vesicles frequently appear in the spermatid cytoplasm, in the area of the "ball and socket-like" junction. This suggests the possibility that there may be communication of materials between the two cells. The possible function of the junctions is discussed and it is concluded that they more likely have a role in nutrition than in coordination.

Animals

Location of glycogen in spermatids and spermatozoa of the shipworm, Bankia australis (Teredinidae, Bivalvia, Mollusca).

The periodic acid-thiocarbohydrazide-silver proteinate technique and alpha-amylase digestion were used to locate glycogen in the spermatids and sperms of the bivalve Bankia australis. Glycogen was found in the middle piece and around the acrosome in spermatozoa, apparently randomly scattered throughout the cytoplasm of young spermatids, and in a cytoplasmic bead in old spermatids.

Acrosome

The behaviour of centrioles and the formation of the flagellum in rooster and drake spermatids.

Developmental changes in the formation of the centrioles and flagellum during spermiogenesis in the rooster and drake were studied. Changes in the length and thickness of the wall of the centrioles were observed from an early stage of spermatid development. Before the proximal centriole is attached to the nucleus microtubules were observed near the centrioles joined to them. At this stage of spermatid development changes on the nuclear membrane were observed at a place where the proximal centriole is attached to the nucleus. At the later stage of spermatid differentiation three to five dense extensions in the space of the nuclear invagination and dense bodies or granules near the distal centriole were present. The anterior part of the newly formed flagellum is covered by a cytoplasmic membrane displaying extension which is approximately 1.3 mum long. Slight differences between the two species were observed.

Animals

Morphology of the spermatid and spermatozoon of Amblyomma hebraeum Koch (Acarina; Ixodidae).

Spermatids and spermatozoa of Amblyomma hebraeum were examined by scanning and transmission electron microscopy as well as by freeze-etch and freeze-fracture techniques. The external surface of the spermatid is smooth except for a rim of two parallel grooves near the anterior apex. This rim probably marks the level at which a cap detaches, permitting extrusion of the inner cord during spermatozoon maturation. Both ends of the spermatid are closed, the anterior part being slightly thicker than the posterior. The spermatozoon is clavate anteriorly and tail-like posteriorly. The tip of the anterior part is a hemispherical structures carrying numerous bulbous expansions. Apart from the hemispherical structure, the entire spermatozoon surface is covered with parallel longitudinal cellular processes. Waves of constrictions on the tail-like part of the spermatozoon and undulations of the cellular processes are considered to be involved in sperm motility.

Animals

Mutagenic effect of thioTEPA in laboratory mice. V. Influence of female genotype on realization of dominant lethal mutations induced in the spermatids of males.

Hybrid male mice (F1 males derived from the cross DBA/2 x AKR) were injected intraperitoneally with thio-TEPA at a 2 mg/kg dose. The mutagenic effect was determined from the frequency of dominant lethal mutations induced in spermatids of males. The males were mated during the second and the third week after the injection to females of the strains C57BL/6, CBA/lac and to the F1 hybrid females derived from the cross CBA x C57BL/6 differing in the frequency of spontaneous and induced aberrations in bone marrow cells. In the course of investigation of pregnant C57BL/6 females F1 (CBA x C57BL/6) and CBA/Lac females the frequency of dominant lethals induced in late spermatids was established to be 63.4, 54.0 and 37.9% respectively. The differences were interpreted as the evidence of different rates of recovery of a certain proportion of premutational lesions of the chromosomes of pserms in fertilized egg cells of female mice. No significant differences between the groups of females were observed in case of the treatment of early spermatids. It is establihsed that the F1 hybrids derived from the cross DBA/2 x AKR are characterized by a high mutability.

Animals

Development and validation of a deep learning model based on cascade mask regional convolutional neural network to noninvasively and accurately identify human round spermatids.

INTRODUCTION: The difficulty of identifying human round spermatids (hRSs) has impeded applications of the human round spermatid injection (ROSI) technique. RSs can be accurately screened through flow cytometric analysis utilizing the Hoechst fluorescence profile reflecting DNA, but this method is not suitable for isolating hRSs due to the toxicity associated with Hoechst staining. OBJECTIVE: To evaluate the capacity of a deep learning model grounded in a cascade mask region-based convolutional neural network (R-CNN) for the noninvasive and accurate identification of hRSs. METHODS: In this study, we presented the development and validation of a deep learning model for identifying hRSs through the analysis of 3457 optical light microscope images of sorted hRSs obtained via flow cytometric analysis. The model's accuracy and specificity were evaluated by calculating the mean average precision (mAP). Furthermore, a double-blind experiment was conducted to access the reliability of the proposed model in accurately identifying hRSs. It detected the expression of protamine (PRM1) and/or peanut lectin (PNA), which are established markers for RSs. RESULTS: Our deep learning-based model demonstrated a high precision, achieving a mAP of over 0.80 for isolating hRSs in test datasets. The expression of PRM1 and/or PNA was observed in all cells noninvasively selected by our AI model during an independent double-blind test. This phenomenon confirmed the accuracy and effectiveness of the proposed model. The model's capability for noninvasive and accurate isolation of hRSs among spermatogenic cells highlighted its robustness and generalizability for clinical applications. CONCLUSION: The deep learning AI model based on a cascade R-CNN has the ability to accurately identify hRSs among spermatogenic cells. The application of this noninvasive method, which requires no additional procedures in clinical practice, is able to facilitate the widespread implementation of ROSI technique. Therefore, it can provide patients with spermatogenic arrest the opportunity to become biological fathers.

Humans

A further type of transient cytoplasmic organelle in human spermatids.

Osmiophilic granules with surrounding vesicles resembling flower-like structures occur transiently during the differentiation of human spermatids. These organelles are incorporated into the residual bodies when mature spermatids are released from the germinal epithelium.

Cytoplasmic Granules

"Cytogenetic" studies of spermatids of mice carrying Cattanach's translocation by flow cytometry.

The DNA content of spermatids of mice carrying Cattanach's translocation has been measured with high precision by flow cytometry. The observation that the two peaks of DNA content in the haploid region of the DNA histograms represent X- and Y-bearing spermatids was tested and confirmed. Using flow cytometry, the difference in DNA content between the X and Y chromosomes in these mice was measured to be 5.2 +/- 0.1% of the total haploid genome as compared to 3.4 +/- 0.1% in normal mice. These results demonstrated the precision of flow cytometry for cytogenetic studies. Additional information on spermatogenesis in mice bearing Cattanach's translocation was obtained and showed a gradual loss of cells during spermatogenesis in those bearing the balanced form of the translocation.

Animals

Ultrastructural characteristics of idiopathic spermatidic arrest.

Previous studies on some cases of idiopathic spermatidic arrest showed an anomaly in glucose metabolism, in particular decreased utilization of glycogen at stage 3 of spermatogenesis, when condensation of the spermatid head commences. Histological patterns seen on light microscopy confirmed the reduced utilization of glycogen at stage 3 of spermatogenesis. Electron microscopy revealed an arrest at stage 3 of spermiogenesis and, at the cytoplasm level of the Sertoli cell, a modification in the smooth endoplasmic reticulum characterized by the presence of dilated cisternae. From a morphological viewpoint, the equivalent of a biochemical modification which characterizes this maturation disorder in the germinal epithelium is hypothesized.

Humans

Ultrastructural studies on the differentiation of spermatids in the domestic fowl.

Four phases of spermatid maturation are recognized on the basis of nuclear morphology. The formation of the acrosome, perforatorium and axial filament complex are described in relation to these phases. The functional significance of the microtubular systems in nuclear elongation and spermatid curvature are discussed. A membranous structure, hitherto unidentified, and referred to in this study as the 'membrane body' is compared with the chromatoid body. Morphological evidence suggests that this structure, together with the annulate lamellae, removes excessive nuclear material.

Animals