Late Spermatid Nuclei Research Articles

An Androgen Receptor in the Nuclei of Late Spermatids in Testes of Male Rats*

We recently identified and characterized, by RIA of bound testosterone (T), an androgen receptor in nuclei isolated from seminiferous tubules of the male rat. By use of similar methods, we now report the presence of a similar androgen receptor in salt extracts of late spermatids from stage 12 on, which were prepared by sonication of homogenized testes or mechanically dissected seminiferous tubules and isolated on a discontinuous sucrose density gradient. Purity of the spermatid preparation was approximately 99%, as established by light microscopy. The major component of a 0.4 M KC1 extract of spermatid nuclei migrated at 4.0S on a glycerol density gradient and coeluted on hydroxylapatite with a salt extract of ventral prostatic nuclei radioactively labeled with 5α-[3H]dihydrotestosterone. In both isolation systems, there were additional minor components in the spermatid macromolecular complex, as compared to the prostatic androgen receptor. A comparison was made between the bound T concentration in late s...

Spermatogenesis in the mouse. I. Autoradiographic studies of nuclear incorporation and loss of 3H-amino acids.

Autoradiographic and electron microscope methods were used to correlate changes in nucleoproteins with nuclear fine structure during spermatogenesis in the mouse. Testes were fixed at daily intervals after intratesticular injectionwith labeled amino acid. [3H]Arginine, lysine, valine, and proline were rapidly incorporated into primary spermatocyte nuclei, retained through subsequent spermatocyte divisions and through spermatid differentiation to step 12 of spermiogenesis, but were lost with spermatid differentiation beyond step 12. Arginine and lysine (not valine or proline) also were rapidly incorporated into certain elongated spermatid nuclei but differed strikingly in their distribution and fate. Nuclei of late step-12 through step-15 spermatids were initially labeled with arginine. This label was retained through subsequent spermatid differentiation and sperm maturation in the epididymis. By contrast, lysine was initially incorporated only into late step-12 and step-13 spermatid nuclei, and was retained only to early step 14 of spermiogenesis. Spermatid incorporation of lysine coincided with the initiation of chromatin condensation in late step-12 nuclei, and loss of lysine coincided with the completion of condensation in step-14 nuclei.

Fractionation of cricket testis nuclei on gradients of colloidal silica for study of basic protein changes during spermiogenesis

Nuclei isolated from testes of the house cricket were centrifuged in a gradient of colloidal silica with a density range of about 1.12 to 1.18 g/ml. Fractions were collected from the bottom to the top of the gradient, and the types of nuclei in them were classified by phase microscopy. The distribution of nuclear types in the gradient indicated relatively large increases in nuclear density during spermatogenesis, and that silica-gradient centrifugation can readily yield fractions enriched for nuclei of specific developmental stages needed to study basic protein changes during sperm development. Basic proteins could be extracted from nuclei spun through silica if they were washed with polyvinylpyrrolidone. The histones in different fractions of nuclei were analysed electrophoretically. Fractions of spermatocyte and early spermatid nuclei contained histones of the somatic types as their only basic proteins. Fractions with mixtures of mid-spermatid and earlier nuclei also yielded somatic histones primarily. Essentially pure samples of late spermatid nuclei were obtained. They lacked somatic histones. In one fraction of late nuclei, the spermatid-specific histones TH1 and TH2 were the major proteins present. In another, two additional histone-like components, not detected in previous studies, were also prominent.

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.

Formation of packaging units in cricket late spermatid nuclei as visualized by spreading techniques

The nuclear organization of a particular step of cricket late spermiogenesis was examined by an electron microscope study of spermatids dispersed by air-liquid surface tension. Cell spreading techniques facilitate a display of condensing spermatid nuclei sufficient to allow interpretation of chromatin packaging processes. Results indicate that nuclei of late developing cricket spermatids are integrated by multiple packaging units as the result of an orderly aggregation of individual chromatin fibers. Each packaging unit consists of a thick fasicle, formed by the alignment of smooth chromatin fibers, which frays out into tassels of looped fibers.

The effect of X-radiation on spermatogenesis and the fertility of Schistocerca gregaria (Forsk.)

ABSTRACT Normal spermatogenesis and the effects of X-radiation on the male locust germ cells have been studied with the aid of light and electron microscopy. Adult insects were irradiated with doses of 100–500 rad (i.e. 1–5 J kg−1) and subsequently examined, at intervals, up to 55 days later. In secondary spermatogonia all doses caused nuclear fragmentation and necrosis, and in primary spermatogonia and a few spermatocytes caused a delay in division. Spermatids and the majority of spermatocytes developed into spermatozoa which were passed from the follicles to the seminal vesicles. The supply of sperm was, subsequently, temporarily stopped. The follicles were eventually repopulated by the division of the primary spermatogonia. Radiation-induced abnormalities, e.g. the formation of supernumerary centrioles, flagella and definitive mitochondria, were most common in the cytoplasm. The Golgi bodies and acrosome seemed to be unaffected. Abnormal outgrowths of the late spermatid and sperm nuclei appeared to be caused by the presence of the numerous centrioles and flagella. The bright yellow coloration of the cuticle, which is characteristic of sexually mature males, was not fully developed in irradiated males. Breeding experiments showed a significant reduction, when compared to the controls, in the number and percentage hatchability of eggs obtained from females mated to irradiated males. The doses of radiation employed did not appear to affect the longevity of the adults.