John H. D. Bryan

A naphthol yellow S and erythrosin B staining procedure for use in studies of the acrosome reaction of rabbit spermatozoa.

Rabbit spermatozoa suspended in Krebs-Ringer-phosphate containing 0.25% glucose were smeared on polylysine-coated slides and dried in air at room temperature for 30 min at room temperature, blotted, rinsed in 1.0% aqueous acetic acid for 10-15 sec, drained and stained for 7 min in a mixture of equal parts of aqueous naphthol yellow S and erythrosin B (final concentration of each dye 0.1% w/v) at pH 4.6-5.0 (pH adjusted with acetic acid). Stained slides were well rinsed in distilled water adjusted to pH 4.6-5.0 with acetic acid, blotted, allowed to dry completely, rinsed in xylene and mounted in synthetic resin. Acrosomal caps were stained cherry-red (apical ridge) to pink (dorsal and ventral aspects); postnuclear caps stained pale pink; nuclei were either unstained or stained a very faint yellowish-pink. The mid-piece and flagellum were stained different shades of pink. The procedure is simple, rapid, and gives highly reproducible results. When present, acrosomes are easily detected regardless of the density of the smear.

An eosin-fast green-naphthol yellow mixture for differential staining of cytologic components in mammalian spermatozoa.

Air-dried smears of saline suspensions of mammalian spermatozoa were stained for 10-60 min at room temperature in a mixture of eosin Y, fast green FCF, and naphthol yellow S (0.1% w/v, each dye) in 1.0% aqueous acetic acid. They were then blotted, rinsed in 1.0% acetic acid until no more dye was removed (0.5-1.5 min), blotted and allowed to dry completely, rinsed in xylene and mounted in synthetic resin. In all species examined except the rat, acrosomes were stained greenish blue to bluish green or blue depending on their thickness; in the rat, they displayed more affinity for eosin and were reddish. In all species, spermatozoan nuclei were strongly stained by naphthol yellow. In intact sperm heads, postnuclear cap had a yellowish green appearance. Midpieces of rodent spermatozoa, especially those of younger gametes, were stained bright red while those of ejaculated bull and rabbit spermatozoa were stained blue-green. Cytoplasmic droplets associated with rodent spermatozoa were consistently stained a dark...

Brain development in hydrocephalic-polydactyl, a recessive pleiotropic mutant in the mouse.

Animals homozygous for the recessive, pleiotropic, mutation hpy (hydrocephalic-polydactyl) progressively lag behind their wild-type littermates in increase in body weight and brain dry weight over the period from 1–40 days post-partum; many homozygotes die within the first 14 days after birth. Light microscope observations of serial sections of brains revealed a mild to severe dilation of the entire ventricular system and damaged ependyma. Ciliated ependymal cells appeared reduced in number and destruction of ependymal cells over wide areas of the ventricular surfaces was observed. Preliminary scanning electron microscope studies confirmed the light microscope observations and revealed large numbers of erythrocytes and phagocytes associated with the ependymal surface. Neither the histological studies nor experiments involving intracerebral injections of tracer dyes demonstrated obstruction or stenosis of the aqueduct of Sylvius. Individual neurons appeared to be present in normal numbers and to be developing normally and at the same rate as in wild-type animals.

Spermatogenesis revisited: I. On the presence of multinucleate spermatogenic cells in the seminiferous epithelium of the mouse

SummaryThis paper reports the results of detailed studies of the morphology of living and of specially fixed and stained spermatogenic cells of the mouse. Particular emphasis was given to studies of multinucleate cells. Evidence is presented to show that such cells may he found in all stages of spermatogenesis from type A spermatogonia onward. Spermatogenic cells containing odd numbers of nuclei appeared to be as frequent as those containing even numbers. After considering various lines of evidence bearing on the probably origin (s), fate and biological significance of multinucleate cells it was concluded that: (1) multinucleate cells do existin vivo; (2) they may be generated either through the failure of cytokinesis, the coalescence of conjoined sister cells, or both; (3) multinucleate cells containing odd numbers of nuclei are generated from even-numbered parents, by addition or substraction of nuclei, through an auxiliary mechanism involving cell fusions and/or a pinching-off process; (4) multinucleates undergo normal meiotic development and give rise to apparently normal spermatozoa; and (5) the multinucleate condition is another cytological indication of the biological importance of synchronous differentiation in gametogenesis.

The immotile cilia syndrome

When homozygous the recessive, pleiotropic, mutationhpy (hydrocephalic-polydactyl) produces post-natal hydrocephalus, complete sterility in males, and reduced reproductive performance in females. Because the fertility problems and the development of hydrocephalus could arise as consequences of defective flagella and ciliary axonemes, this mutant type might serve as a useful animal model for the immotile cilia syndrome. Ultrastructural defects seen in axonemes of flagella, and of cilia from the trachea, oviduct, and ependyma included: a deficiency of inner dynein arms (the most frequent defect); an absence of one or both central-pair tubules; extra central tubules; a displacement of one outer doublet and/or the central-pair tubules. Some axonemes showed more than one of these defects. The frequency of dynein-deficient axonemes in all three tissues was similar (about 35%) and fell within the range reported for human patients with the immotile cilia syndrome. On this basis, this mutant type might be considered as a useful animal model for such studies. There were no indications of situs inversus, nor was there a marked increase in respiratory problems. Sohpy/hpy mice do not exhibit all of the clinical symptons characteristic of the human condition.

Abnormal cilia in a male-sterile mutant mouse

In mice homozygous for the mutation hydrocephalic-polydactyl (hpy) ciliary axonemes from tracheal, oviducal, and ependymal lining cells showed a variety of abnormalities. Defects included: a deficiency of inner dynein arms, extra central tubules, a displacement of one outer doublet and/or the central tubules, and double axonemes. More than one kind of defect was seen in some axonemes. None of the types of defects observed in mutants were encountered in equivalent samples from non-mutant littermates. Except for the most common defect, the deficiency in dynein arms, which occured to about the same extent (approximately 34%) in all three tissues, there were marked variations in frequency among the tissue types with respect to the other defects. In general, defects such as central tubule anomalies, displaced tubules, and double axonemes occured with the highest frequencies in axonemes from tracheal epithelial cells and with the lowest frequencies in samples of oviducal epithelium. Fused cilia were seen only in ependymal cell samples. Some of the defects encountered were common to sperm flagella axonemes while others appeared restricted to somatic tissues, suggesting, perhaps, each tissue type may exert its own modulating influence on the expression of the mutant gene.

Cytochemical localization of non-specific esterase and acid phosphatase in spermatozoa of the mouse (Mus musculus)

SummaryThis paper describes methods for the cytochemical demonstration of non-specific esterase and acid phosphatase activities in spermatozoa of the mouse. The acetate and phosphate esters of 1-naphthol were used as substrates, and hexazonium pararosanilin was used as coupler in both techniques. Specificity was assured by the use of appropriate positive and negative controls. Best results were obtained with unfixed smears which had been stored at room temperature for a few days prior to use. Chemical fixation, especially with formol-calcium solutions, is not recommended for use with rodent spermatozoa. According to our investigations the murine acrosome contains very low levels of non-specific esterase and acid phosphatase which are not amenable to detection by the “standard” methods employing short incubations and/or with material fixed in a formalin-containing fixative.

Non-specific esterase activity in bovine acrosomes

SynopsisFixed and unfixed smears of bovine spermatozoa were stained for esterase activity at pH 6.1–6.2 using 1-naphthyl acetate as substrate and hexazonium pararosaniline as coupler. Best results were obtained with unfixed smears prepared from suspensions of spermatozoa washed free of seminal plasma. The most intense staining was obtained when smears were incubated at 4°C for 40 hr with replacement of the incubating medium every 12 hr. No staining was detected in spermatozoa when smears were incubated in medium lacking substrate or when heat-inactivated smears were incubated in complete medium. Enzyme activity was restricted to the acrosomal cap; no final reaction product was detected in or on the post-nuclear cap, the mid-piece or the remainder of the flagellum. Enzyme activity was distributed quite uniformly throughout the acrosome with no indication of the existence of localized regions with high activity. The use of fixatives, especially those containing formalin, is not recommended for esterase studies of bovine spermatozoa.