Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm.

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin-phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.

Supranucleosomal organization of sea urchin sperm chromatin in regularly arranged 40 to 50 nm large granular subunits.

The organization of the chromatin of sperm nuclei of sea urchin (Parcentrotus lividus and Sphaerechinus granularis) was studied by electron microscopy using thin sections of fixed cells and spread preparations of chromatin obtained after various procedures involving incubation in low salt buffers. In fixed cells and in moderately dispersed preparations chromatin appeared in the form of fibrillar chains formed by closely apposed, distinctly sized granules that were larger than chromatin subunit structures so far described in other kinds of chromatin (mean diameters of particle units determined were 36 nm, in thin sections, and 47 nm, in spread preparations). Upon prolonged incubation in low salt buffer an increasing number of these large granular units was transformed into extended nucleosomal chains (nucleofilaments). Estimations indicated that the large granules contained 20 to 26 nucleosomes, i.e. 4.8 to 6.2 kb DNA, resulting in a linear DNA contraction ratio of 33 to 55, which is in a similar range as values reported for the supranucleosomal chromatin particle of the SV40 minichromosome but greater than contraction ratios of "superbead" structures. The unravelling of the large supranucleosomal granule into the extended nucleofilament chain in low salt buffers seemed to be a gradual process since intermediate stages of variable particles sizes and shapes were observed. This novel type of chromatin particle is considered to represent an example of a naturally occurring globular supranucleosomal packing unit of chromatin which is predominating, if not the only forms of supranucleosomal levels of chromatin organization and in relation to the special DNA content and histone composition of sea urchin sperm nucleosomes.

Isolation of myosin from starfish sperm heads.

Myosin was extracted and partially purified from the head portion of spermatozoa of the starfish, Asterias amurensis. The sperm myosin showed a specific Ca2+-activated ATPase [EC 3.6.1.3] activity of 0.2 mumoles Pi/min/mg at high ionic strength and pH 6.5. It resembled egg myosin in forming thick filaments, becoming attached to actin filaments. subunit composition, and serological properties.

Correlation between elemental composition and motility of human spermatozoa.

X-ray microanalysis of human sperm cells in donor semen having a range of motilities from 0% to 85% showed that elemental composition is not strongly correlated with spermatozoal motility. Only copper in the midpiece was positively correlated with motility when high- and low-fertility groups were compared. Aging of cells in semen caused large changes in subcellular elemental concentrations as motility decreased, notably with uptake of zinc, but these changes were not reflected in the range of motilities in the clinical samples. Electrolyte balance as measured by sodium to potassium ratios also appeared not to be correlated with motility. Subcellular elemental distribution is not a major factor in determining sperm motility in normal human semen.

PIP: A possible correlation between the elemental composition of human spermatozoa and sperm motility was investigated. X-ray microanalysis did not reveal any strong correlation between sperm motilities of C-85% and elemental composition. However, when high- and low-fertility samples were compared, a significant (p less than .025) correlation was found between copper in the midpiece region and motility. As cells aged and motility decreased, there were considerable changes in subcellular elemental concentrations, particularly with regard to zinc uptake. However, these changes were not correlated with the range of motilities. No apparent correlation was found between electrolyte balance, as determined by sodium to potassium ratios, and motility. It is concluded that the subcellular distribution of elements in semen is not a determining factor in sperm motility.

Histochemical studies on the mucosubstances in the ovotestis of a hermaphrodite land pulmonate Semperula maculata in seasonal breeding-aestivation cycle.

With a view to augment the cytochemical localization characterization and, probable functional significance of mucosubstances in the ovotestis in the reproductive physiology of land pulmonates, the mucosubstances were investigated in the ovotestis of a land snail, Semperula maculata (Stylommatophora) in the seasonal breeding-aestivation cycle. Widely accepted, recent and well-established histochemical techniques were emoloyed. The results showed that the cells in the germinal epithelium of the follicular wall, were endowed with a capacity of synthesis of neutral muco-substances. They also contained glycogen. The nurse cells contained neutral mucosubstances and glycogen in them, while maturing ovum contained hyaluronic acid and protein. Among all the male germ cell stages, only matured sperms showed sialoglycoprotein in their heads and glycogen in the tails. The seasonal variations in the mucosubstances showed that the synthesis of the mucosubstances in the male germ cells of the ovotestis was initiated at the end of the breeding season and attained a maximum in the post-breeding season. Most strikingly the presence of the sperm in the follicular cavity was a permanent feature throughout the seasonal breeding-aestivation cycle of the snails. The synthesis of the mucosubstances in the ovum and the nurse cells was initiated somewhat earlier in the post-breeding season and attained a peak prior to the active breeding season. The importance of the mucosubstances synthesised by the different cells of the ovotestis, is discussed with reference to the reproductive physiology of the snails.

[Physico-chemical properties of deoxyribonucleoprotamine from sturgeon sperm heads]. / Nekotorye fiziko-khimicheskie svoistva dezoksiribonukleoprotamina iz golovok spermiev osetra

Deoxyribonucleoprotamine (DNPn) from sonicated nuclei of sturgeon sperm heads was studied by means of ring dichroism. A derivative analysis of DNA and DNPn melting curves in 1 mM Tris. HCl pH 8.0 revealed the fraction of protein-free DNA being about 30% and suggested the preferable binding of protamine molecules with AT-rich DNA regions. The latter is also confirmed by the data on ring dichroism of protein-poor soluble DNAPn fraction in 0,14 M NaCl. Ring dichroism of DNA and DNPn in 1 mM Tris coinsides at the wavelength of 310-240 nm at concentrations of 500-50 mkg/ml. Dilution of DNPn to 5 mkg-ml resulted in the decrease of the ellipticity at 275 nm and produced no effect at 260-210 nm. The effect observed is suggested to be due to a partial transition of DNA in DNPn into C-form under the dilution as a result of a higher molecule hydration and a destruction of some hydrogen bonds between guanidine residues of arginine and oxygen of phosphate groups, stabilyzing DNA in the B-form. Ring dichroism spectrum of protamine, calculated by the subtraction of DNA spectrum from DNPn spectrum at the region of 240-210 nm coinsides with that of free protamine and indicates the absence of an ordered structure in protamine molecules in DNPn.

Ethanol extraction of basic proteins from ejaculated human spermatozoa.

A method for the extraction of basic proteins from human ejaculated spermatozoa has been developed. It relies on the previously unreported observation that such basic protein is soluble in a solution containing 60% (v/v) ethanol. This unconventional method yields a high percentage of arginine-rich basic protein which is then able to be characterized on the basis of its amino acid composition. This method also allows comparisons to be made between single ejaculates by the banding pattern each displays when subjected to polyacrylamide gel electrophoresis.

An optical model for fluorescence of mammalian sperm in flow cytometry.

When flat sperm heads that have been stained to fluorescence are examined in a flow cytometer, unexpectedly, skewed pulse height distributions are obtained despite the apparent homogeneity of the samples. This anomaly has been ascribed to an optical artifact that arises when the cells are oriented in flow. We have extended our model for fluorescent scattering to spheroids and here explore some aspects for oblate spheroids which serve to model sperm heads. Although computational limitations have restricted these studies to oblate spheroids about 1.5 micrometer in diameter and an eccentricity of 0.1, the results clearly show effects of particle size, shape, optical properties and particularly of orientation on the differential scattering cross-sections. This plethora of information contained in the fluorescent signals may suggest further experiments.

Localization of actin in the sperm head of the plains mouse, Pseudomys australis.

Actin was localized in the sperm head of the plains mouse, Pseudomys australis, using NBD-phallacidin, a fluorescent-labeled phallotoxin. It was found to be present in the two ventral hooks in sperm from the testis, epididymis, and vas deferens. Faint fluorescence was observed in other regions of the sperm head, whereas autofluorescence occurred in the midpiece of the tail in some preparations. The material in the ventral hooks was also found to be birefringent, which is consistent with it showing preferential orientation.

Deoxyribonucleic acid (DNA) content of morphologically different sperm types from normal and subfertile human males.

20 male patients (G2), affected by idiopathic oligozoospermia, and 10 normal fertile males (G1) were investigated. The DNA content of 50 Feulgen stained sperm heads per subject was determined, using single cell photometry. In a second step area, circumference, length, and width of the same heads were measured, using a semi-automatic image analysis system. Considering only morphologically normal heads, the mean DNA content is only slightly smaller in G2 than in G1. On the contrary, the DNA variation is strongly higher in G2 than in G1. Unfortunately, morphologically defect heads show an increased DNA variation, too. Thus, determining the DNA variation of a semen, containing many morphologically defect heads, a combined determination of the DNA content and head morphology is necessary. This can only be achieved by single cell cytophotometry, which is therefore superior to flow cytophotometry as regards this problem. Many heads with a strongly abnormal DNA content do not show any morphological abnormalities. These subcellular changes can only be detected by the determination of the DNA content.

Localization and distribution of actin in mammalian sperm heads.

Actin was identified in boar and mole spermatozoa by utilizing indirect immunofluorescence, immunoelectron microscopy, and SDS-PAGE, followed by blot and screening with an anti-actin monoclonal antibody. Actin was detected in two places in the sperm head: the equatorial segment of the acrosome and the postacrosomal region. The protein was present in a nonfilamentous form and was localized under the plasma membrane. A small amount of actin was also detected in the sperm tail. The function of actin in the sperm head is discussed.

Nuclear zinc in human epididymal and ejaculated spermatozoa.

Sperm nuclear zinc content (expressed as zinc/phosphorus and zinc/sulphur ratios) was determined with X-ray microanalysis in individual, air-dried, epididymal spermatozoa from elderly men, and in ejaculated spermatozoa from healthy donors. Ejaculated sperm nuclei contained (also after treatment with sodium dodecyl sulphate) more zinc than epididymal spermatozoa. The results indicate that the human spermatozoon accumulates zinc from the prostatic fluid upon ejaculation.

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei.

Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts. Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days. The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.

Electrophoretic patterns of total, nuclear, and flagellar proteins from ejaculated human spermatozoa.

By means of selective solubilization methods and slab gel electrophoresis, reproducible patterns of 19, 37, and 56 protein bands were found to be associated with nuclear, "flagellar," and total human spermatozoa, respectively. Forty protein bands were found between the molecular weight of 12,400 to 160,000 daltons. Twelve bands were associated with values lower than 12,400 daltons. The nuclear major bands were located in a low molecular weight zone, while "flagellar" ones were located in a high molecular weight zone. None of these bands represents degradation products since a) in the solubilized samples neither acrosin, chymotrypsin, nor trypsin activities were present, b) in the presence of two protease inhibitors the same electrophoretic patterns were observed, and c) labelled globins added during sample manipulation were quantitatively recovered without degradation.

Investigation of fading and recovery of fluorescence intensity at 73.5 K.

Fading and recovery of Af-feulgen stained sperm heads are investigated at 73.5 K. The fast fluorescence signals are measured and stored by two coupled transient recorders. 100% recovery was reached after a dark time of 3 s. This shows that the photodecomposition is mainly caused by change of the probability density of energy level and not by destruction of the chromophore groups. The recovery effect allows measurement of the fluorescence intensity of the same sample more than 50 times. Therefore the unaffected spectrum can be measured directly, provided that between the short-term measurements at constant wavelength an appropriate dark phase has been put into operation.

Isolation, structure and protein composition of the perforatorium of rat spermatozoa.

Heads of spermatozoa were sonically separated from tails and treated in 1 N NaOH until the perforatoria were partially detached from the nucleus. Their complete detachment was then assured by repeatedly passing the suspension through a 22-gauge needle. The perforatoria were then separated from nuclei on sucrose gradients and the purity of the fraction was verified by electron microscopy. The isolated perforatoria were denatured and used to raise antibodies or run on polycrylamide gels. Such gels revealed many polypeptide bands, six of which were most prominent (Mr approximately 13,000, 13,400, 16,000, 33,000, 35,000, and 43,000). Of these, the 16,000 Mr polypeptide was major. Anti-perforatorium serum reacted with the perforatoria of fixed spermatozoa, with a substance found between the plasmalemma and the outer acrosomal membrane of the acrosomal head cap and with the inner component of the ventral spur, but not with the postacrosomal dense lamina. This observation indicated that the perforatorium and dense lamina, although structurally continuous to form the perinuclear theca, are biochemically distinct. On Western blots, the anti-perforatorium serum reacted with the prominent polypeptides of the perforatorium and cross-reacted with some less prominent polypeptides of the fibrous sheath (FS) and outer dense fibers (ODF), most notably with a 16,000 Mr polypeptide found in both. Likewise anti-FS or anti-ODF serum cross-reacted with some major and minor polypeptides of the perforatorium, again most notably with a major 16,000 Mr polypeptide. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. These results demonstrate that the perforatorium is composed of several polypeptides, is immunologically distinct from the postacrosomal dense lamina, may be immunologically similar to a substance found between the plasmalemma and outer acrosomal membrane and to a substance found on the inner aspect of the ventral spur, has antigenic determinants in common with the FS and ODF, and may share a 16,000 Mr polypeptide with these two cytoskeletal structures of the flagellum.

Chromatin organization in nuclei of sea urchin embryos. Comparison with the chromatin organization of the sperm.

The chromatin in sea urchin embryo nuclei and that in sperm heads are both organized in nucleosomes but show marked differences when analyzed by endonuclease digestion. Sperm chromatin DNA appears to be totally organized in nucleosomes that are highly resistant to nuclease hydrolysis. The kinetics of formation of acid-soluble oligonucleotides is slow and concerns only about 50% of the total DNA. In contrast, the DNA of embryo chromatin does not appear to be totally organized in nucleosomes since 5 to 10% is rapidly and preferentially hydrolysed into acid-soluble oligonucleotides without any appreciable fragmentation of the remaining parts. Futher digestion causes the formation of the usual pattern of DNA bands, as detected by gel electrophoresis. The length of the DNA segment associated with the embryo nucleosomes appears to be shorter than that of the DNA segment associated with the sperm nucleosomes. The kinetics of formation of acid-soluble oligonucleotides upon digestion of embryo chromatin is much faster than that of sperm chromatin and concerns almost all the chromatin DNA.

Studies on a protamine (galline) from fowl sperm. 4. Degradation of galline by trypsin-like protease of fowl sperm heads.

Galline, a protamine of domestic fowl, was obtained by two preparation procedures from the semen of a strain of White Plymouth Rock and submitted to fractionation by column chromatography on Bio-Gel CM-30. In the first procedure the specimen prepared from sperm heads was purified by the use of distilled water and dilute acetic acid and fractionated into almost eight fractions (G-I-G-VIII) in the same way as the specimen from a strain of New Hampshire (1,2). No difference could be found between galline specimens from the two different strains based on the amino acid and terminal analyses of each fraction. The specimen of galline from sperm heads purified with 1% citric acid (the second procedure) was composed of only one component, which was isolated as a single peak. The smaller fractions, G-I-G-VII, were found to be derived from G-VIII by the action of trypsin-like protease contained in the extract of sperm heads with 1% citric acid. This enzyme seems to originate in the acrosome of fowl spermatozoa. Consequently, it is concluded that intact galline is composed of only one molecular species and its total amino acid sequence is represented by the completed formula of G-VIII as shown in the preceding paper (4).

The chromosomal distribution of human satellite III DNA during meiosis.

In human meiotic cells DNA satellite II is located in the same sites as in mitotic cells, but in prophase the areas are less condensed. This differs from the situation in Plethodon where the sites of heavy satellite DNA are condensed throughout meiotic prophase (MacGregor and Kezer, 1971). The difference may be ascribed to the fact that Plethodon heavy satellite is pericentrically located, whereas few if any of the human satellite sites are actually at centromeres. A second difference is found in sperm, where the Plethodon satellite is located at a single site in the rear of the nucleus, while the satellite regions in mad do not have a common or constant orientation, suggesting that the respective satellites may well be functionally different.

A difference in dry mass between the heads of X- and Y-bearing human spermatozoa.

An integrating microinterferometer was used to measure the dry mass of sperm heads. The dry mass was found to be proportional to DNA content, and thus provides a useful method of estimating sperm DNA content. Using this technique we have confirmed that human spermatozoa which show none and one quinacrine-fluorescent spot are X- and Y-bearing respectively. However, the measurements suggest that many of the spermatozoa with two quinacrine-fluorescent spots are not YY-bearing, as previously thought, but might be incompletely condensed Y-bearing spermatozoa.