David M. Phillips

Surface architecture of the mouse and hamster zona pellucida and oocyte.

The surface of the mouse and hamster zona pellucida and oocyte were examined with the scanning electron microscope. Both zonae and ova were studied before fertilization, at the time of fertilization, during development of the zygote, and following cleavage. No change was observed in surface of the mouse or hamster zona pellucida or oocyte. These observation are in contrast to previous studies which have described marked changes in surface of the mouse zona and oocyte at fertilization and during early development. It is suggested that previous workers may have confused preparative artifacts with developmental changes.

Mechanics of sperm entry in cycling hamsters

At various times after mating, fertilized eggs were flushed from the ampulla of the oviduct of estrus hamsters. Cumulus cells were removed with hyaluronidase before fixation. After fixation, the zonae pellucidae were removed mechanically. The initial association between the fertilizing spermatozoon and the oocyte surface appears to be between the tips of oocyte microvilli and the equatorial segment. As the sperm head is incorporated, it undergoes a pronounced bend such that the anterior tip is the last portion of the sperm to be incorporated. A small incorporation cone forms at the site where the sperm head was incorporated into the ooplasm. As the sperm tail is incorporated, it forms numerous bends which distort the oolemma into a row of small elevations. From these observations, we have been able to propose a model for the mechanics of sperm entry into the ovum in vivo.

Nuclear shaping during spermiogenesis in the whip scorpion.

The mature spermatozoan of the whip scorpion or vinegaron Mastigoproctus giganteus is a spherical cell within which the nucleus and flagellum are tightly coiled. Spermiogenesis in the whip scorpion starts out in a manner basically similar to spermiogenesis in species with conventional elongate sperm. As in many other animal species, the spermatid nucleus is surrounded by a manchette of microtubules and elongates in an axis parallel to the microtubules. Late in development, however, the nucleus and flagellum undergo unorthodox contortions in which the nucleus becomes spiralized and the flagellum is wound up into the body of the cell. No microtubules appear to be associated with the spermatid nucleus during the process of coiling. Pronounced changes in the configuration of chromatin occurs concomitantly with the coiling which suggests that chromatin alteration may be related to the changes in nuclear shape. Peculiar spermatozoa similar to the type found in Mastigoproctus have been described in spiders, whereas scorpions have more conventional sperm. Thus, whip scorpions may be more closely related to spiders than scorpions.

Morphological studies of Dalkon Shield tails removed from patients

Abstract Examination of the tails of Dalkon Shields removed from patients showed that approximately 34% of the tails had breaks or holes in the nylon sheath immediately below the double knot at the base of the Shield. The location of these holes is such that most of them would have been within the endometrial cavity. For control purposes, unused sterile Shields were removed from their pouches, and the tails were inspected for breaks in the sheath. Breaks were found in approximately 9% of these. Most of the holes were in the same location as those seen in tails removed from patients. The internal contents of the short terminal segments of Dalkon tails located beyond the double knot, from Shields removed from patients, were studied. Thirty-five (35) segments were evaluated by phase contrast microscopy and 10 were studied subsequently by transmission electron microscopy. Bacteria were found within the interfilamental spaces inside the sheath of 8 of the 10 tails. These observations suggest that bacteria which have ascended through the tail from the vagina could exit through these breaks in the sheath or from the terminal end of the tail directly into the endometrial cavity. The potential clinical implications of the data presented in this paper must be taken into consideration in the management of the non-pregnant asymptomatic wearer of a Dalkon Shield.

Mitochondrial disposition in mammalian spermatozoa

A modified technique for preparing surface replicas simplified the analysis of mitochondrial arrangement in the midpiece of spermatozoa from nine mammalian species. The plasma membrane was removed from epididymal sperm by detergent to expose the underlying mitochondria. Sperm were attached to poly- l -lysine-coated coverslips and critical-point dried. The disposition of mitochondria as revealed in platinum replicas of these preparations varied among species. Rabbit sperm possessed coils of mitochondria in a quadruple or quintuple helix. The mitochondrial helix was triple or quadruple in bull sperm, double or triple in mouse sperm, and single or double in rhesus monkey sperm. The outer faces of individual mitochondria appeared rectangular in Chinese hamster, diamond-shaped in rhesus monkey, S- or V-shaped in rat, and irregular in guinea pig and degu.

Mycoplasmal infection of lymphocyte cell cultures: Infection withM. salivarium

SummaryMany conclusions concerning cell culture mycoplasmas are based on data from studies in fibroblast cultures. Some conclusions may not be valid in other types of differentiated cell cultures.M. salivarium was isolated from 35 human lymphocyte cultures (HLC), 34 from the same laboratory. The organism grew to more than 108 colony forming units (CFU) per ml of lymphocyte suspensions and was readily detectable by microbiological culture, uridine phosphorylase, and uridine/uracil assays. Direct mycoplasmal assays on HLC by DNA fluorochrome staining and scanning electron microscopy (SEM) yielded artifacts that interfered with diagnosis. For DNA and SEM of HLC, inoculation into indicator cell cultures is recommended.M. salivarium infection of HLC did not produce any immediate difference in growth rates; however, infected cultures eventually died 14 to 29 passages after infection in contrast to uninfected controls. The same organism in 3T6 fibroblasts effected a 60% decrease in growth rate. AlthoughM. salivarium is a frequent isolate from the oral cavity, it is a rare cell culture isolate.M. salivarium was able to initiate growth over a wide pH range, grew as well in cell cultures as in cell-free media, and was resistant to 50 μg per ml of gentamycin, tylocine, kanamycin, and erythromycin. By C0t1/2 analysis,M. salivarium had a genomic molecular weight of 4.2×108 daltons.M. salivarium did not increase chromosome aberrations in one HLC. Some of these results have application to infection of HLC by other mycoplasmal species.

Endogenous HPRT activity in a cryptic strain of mycoplasma and its effect on cellular resistance to selective media in infected cell lines

Abstract A cryptic strain of mycoplasma, which contains a high level of endogenous hypoxanthine phosphoribosyltransferase (HPRT) activity, was isolated from a subclone of mouse cell mutant A9 that had been maintained for over 100 cell generations in 8-azaguanine medium. HPRT-deficient cells deliberately infected with the mycoplasma acquire an apparent wild-type level of HPRT when assayed in cell-free extracts, even when cultured in medium with 8-azaguanine or 6-thioguanine. However, both normal and HPRT-deficient cells become extremely sensitive to HAT medium after infection. As a consequence, HPRT-deficient mutants contaminated with this mycoplasma will not yield HAT-resistant colonies when hybridized with HPRT-positive cells. Biochemical analysis suggests that the resistance of the mycoplasma to purine analogues may be due to elevated substrate binding constants of the mycoplasma HPRT, which render it non-functional at the physiological concentrations of the substrates. The mycoplasma-coded HPRT has characteristic electrophoretic mobility, substrate specificity and thermolability, which together distinguish it from animal HPRT allozymes in man, mouse, rat, Chinese hamster, Syrian hamster and chicken. This mycoplasma strain can grow only in association with animal cells, fails to incorporate exogenous thymidine, bands with a density of 1.217 g/ml in sucrose gradients, and passes through 0.22 μm membrane filters. Growth medium of animal cells deliberately infected with the mycoplasma can reach a maximum titer of 108 infectious particles per ml within a few days. The mycoplasma was positively identified by scanning electron microscopy and typed as Mycoplasma hyorhinis with a monospecific antiserum.

Nucleolus-like bodies in micronuclei of cultured Xenopus cells.

Two of the 36 chromosomes in Xenopus laevis are known to carry nucleolar organizer loci. Partitioning of the chromosomes of cultured, early-passage Xenopus cells among variable numbers of micronuclei could be induced by extended colcemid treatment. A large, obvious nucleolus occurred in a maximum of 4 micronuclei per colcemid-induced tetraploid cell. The large, deeply-stained nucleoli incorporated [3H]uridine and appeared by electron microscopy to have typical nucleolar morphology with fibrillar and granular areas disposed in nucleolonema. In situ hybridization to radioactive ribosomal RNA (rRNA) resulted in heavy labelling of nucleoli in no more than 4 micronuclei per cell. The other micronuclei generally contained small bodies (blobs) which stained for RNA and protein as well as with ammoniacal silver. In the electron microscope, these appeared as round, dense bodies resembling nucleoli segregated by actinomycin D treatment. Nucleoplasmic RNA synthesis occurred in all micronuclei regardless of whether they contained definitive nucleoli. These observations suggest that micronuclei which formed large, typical, RNA-synthesizing nucleoli contained nucleolar organizer chromosomes, while the other micronuclei, which contained nucleolus-like “blobs” probably lacked nucleolar organizer loci. It is possible that the nucleolus-like bodies may have been aggregates of previously synthesized nucleolar RNA and protein trapped in micronuclei after mitosis.

Difference in the Manner of Association of Acrosome‐Intact and Acrosome‐Reacted Hamster Spermatozoa with Egg Microvilli as revealed by Scanning Electron Microscopy

Scanning electron microscopy was employed to examine the manner of association between in vitro capacitated spermatozoa and zona‐free eggs of the hamster. Spermatozoa with intact acrosomes, which were unable to fuse with eggs, were seen in general associated with egg microvilli in the region of the acrosomal cap. Acrosome‐reacting spermatozoa were seen associated with egg microvilli with the dissociating acrosomal caps. Acrosome‐reacted spermatozoa, which were able to fuse with eggs, generally associated with egg microvilli by the equatorial segment and the anterior portion of the postacrosomal region. It is inferred that the completion of the acrosome reaction signals changes in the plasma membrane over the equatorial segment of the acrosome and the anterior area of the postacrosomal region which give it a greater affinity to and fusibility with the oolemma.

Detection of Mycoplasma Contamination of Cell Cultures by Electron Microscopy

Since mycoplasma infections of cell cultures are a frequent and serious problem, it behooves the cell culturist to find reliable and reasonably easy methods for the detection of mycoplasma contamination. In this chapter we will discuss the electron microscope as a tool for the detection of mycoplasma infections of cultured cells. We will describe how mycoplasma infections appear in the scanning and transmission electron microscope and will discuss the advantages and disadvantages of electron microscopy in terms of reliability, sensitivity and practicality. The reader is referred to other chapters of this book for thorough discussions of various methods of detection and for discussions of the particular ways which mycoplasma alter the physiology, chemistry and morphology of cell cultures. It is often necessary to employ more than one technique to detect mycoplasma or to be reasonably certain that a culture is mycoplasma free. Each method has its advantages and disadvantages. Different techniques for detecting mycoplasma, therefore, often complement each other.