Anita M. Mandl

The ultrastructure of oogonia and oocytes in the foetal and neonatal rat

Ovaries from eighty foetal and neonatal rats (aged 16·0 days post coitum to 4 days post partum) were examined under the electron microscope. All the normal developmental stages (oogonia and oocytes in the leptotene, zygotene, pachytene, diplotene and dictyate phases of meiotic prophase) were identified. Patterns of degeneration (‘atretic divisions’, ‘Z’ cells and atresia at the diplotene phase), whose histological appearance and incidence have been recorded by Beaumont & Mandl (1962), were also recognized. The nuclei of oocytes at the leptotene phase contain single electron dense threads which become aligned in parallel pairs at the following phase (zygotene). A third finer fibril half-way between them appears at pachytene (tripartite ribbon). The longitudinal segments of threads, visible in ultra-thin sections, become shorter, presumably due to coiling. Nuclei at the diplotene phase contain single threads essentially similar to those seen at leptotene. As the oocyte enters the dictyate or resting phase, electron dense threads disappear from the nucleus. These observations suggest that the threads represent chromosomal ‘cores’. Nucleolus-like components persist throughout meiotic prophase, and at the diplotene phase regain the more complex form typical of oogonia. The cytoplasmic organelles become more numerous and complex as the oocyte approaches the dictyate phase. ‘Atretic divisions’ in oogonia are characterized by the persistence of long segments of nuclear membrane and the appearance of vesicles enveloped by a double membrane resembling the nuclear envelope. The dense masses of ‘chromatin’ (mitotic chromosomes) are more rounded than in normal cells at metaphase, and tend to coalesce. Spindle fibres have not been observed. Cytoplasmic organelles tend to increase in number and complexity. ‘Z’ cells (cells degenerating largely at the pachytene phase) show heavy ‘chromatin’ condensation around the tripartite ribbons. The major cytoplasmic changes consist in swelling of the endoplasmic reticulum, vacuolation of mitochondria and increase in incidence of multilamellar bodies. Atretic oocytes at the diplotene phase differ markedly from ‘Z’ cells in that ‘chromatin’ condensation around electron dense threads (single) is markedly less prominent. Cytoplasmic changes are similar to those of ‘Z’ cells, but also include a rise in the incidence of multivesicular and other complex bodies. All three types of degenerating cells are removed from the ovary by the phagocytic activity of neighbouring somatic cells.

THE RADIOSENSITIVITY OF GERM CELLS

The radiosensitivity of germ cells depends on (a) the criterion by which it is assessed, and (b) the developmental stage at the time of irradiation. For any given parameter and cell‐type, the final effect exerted by ionizing radiations depends on the genetic make‐up and age of the organism; type of irradiation; method of irradiation (acute, fractionated, chronic at different dose rates); oxygen tension; and temperature. Variations between different developmental stages may be due to inherent changes in oxygenation, metabolic activity, chromosomal configuration; and other factors, as yet to be determined, are probably also involved. The yield of mutations observed after irradiation of different developmental stages may be at least partly determined by the selective elimination, during subsequent development, of cells sensitive to immediate or delayed cell‐death.

Pre-Ovulatory Changes in the Oocyte of the Adult Rat

The sequence of nuclear changes in the oocyte of the adult rat has been investigated, with particular emphasis on early pre-ovulatory stages. The condition of the chromosomes during the so-called dictyate phase is discussed, and an attempt made to determine the time at which diakinesis occurs. The relationship between the onset of the successive stages of the maturation division and the diurnal rhythm of light and darkness (‘natural’ and artificial) was determined. The first clear-cut change (condensation of chromatin) occurs at about 5 p. m. under ‘natural’ light conditions, and within the first 1 h of the onset of darkness under artificial illumination (12 h light: 12 h darkness). Ovulation occurs some 9 to 10 h later. An approximate estimate was made of the duration of each stage of the division up to second metaphase. The variability in the onset of the various stages of the division was greater between individual rats kept under ‘natural’ light conditions than between those kept in light-controlled rooms.

The Effect of Cysteamine on the Survival of Spermatogonia after X-irradiation

SummaryThe reduction in the number of spermatogonia and resting pre-spermatocytes following exposure of the scrotum of adult rats to 230–460 r was partially inhibited by pre-treatment with 30 mg cysteamine.

The population of germ cells in immature male rats following irradiation at birth

Male rats were irradiated with 19 r on the day of birth, and killed at intervals ranging from 5 to 18 days. Estimates were made of the absolute and relative numbers of germ cells at different stages of spermatogenesis in 64 irradiated and 61 untreated specimens. In the normal rat, the calculated population of germ cells increased from about 160000 at 5 days to 30 million at 18 days. Only negligible numbers of primordial germ cells (gonocytes and transitional cells) persisted beyond the age of 10 days. Small numbers of spermatogonia type A appeared at 5 days (15000) and their population rose to about 1 million at 12 days, and 2 million at 18 days (7 % of all germ cells). Intermediate spermatogonia first occurred in appreciable numbers (23000 to 55000) at 8 or 9 days, when the population of type-A spermatogonia was 360000. The subsequent rise in the population of intermediate spermatogonia was more rapid than that of type A (4 million at 18 days). Spermatogonia type B and primary spermatocytes appeared at 9 to 10 days, and their numbers rose more steeply still (6.5 and 16 million at 18 days, respectively). Irradiation at birth exerted no rapid effect on the cytological appearance of primordial germ cells. Transformation from gonocytes to transitional cells appeared to proceed normally and the estimated total population of germ cells at 5 days was no smaller than in the controls. Subsequently, however, many of the transitional cells failed to divide: they enlarged to form giant cells, acquired bizarre nuclear outlines, and persisted for unusually long periods. Some degenerated at mitotic prophase or metaphase, while a few seemed to die at interphase, without entering division. The calculated total population of germ cells in irradiated rats rose from 160000 at 5 days to 9.4 million at 18 days. Small numbers of spermatogonia type A, presumably derived from such primordial germ cells as were able to complete mitosis, appeared some 2 to 3 days later than in controls. The number of type-A spermatogonia in 7-day-old irradiated rats was 44000, cf. 215000 in controls; the difference became less pronounced with time, and by the age of 18 days, the population of 1.9 million was comparable to that estimated for the controls. Small numbers of intermediate spermatogonia appeared on the 9th (8000) and 10th day (35000), when the population of type-A spermatogonia was about 110000 and 260000 respectively. By the 18th day, intermediate spermatogonia numbered 2 million. The populations of type-B spermatogonia and primary spermatocytes rose from 11000 to 13000 at 10 days to 1.6 and 3.4 million, respectively, at 18 days. The difference in the absolute and relative numbers of germ cells between normal and irradiated testes widened progressively with advance in the developmental stage of the germ cells. Analysis of the results indicates that in the reduced population of spermatogonia type A after irradiation, the pattern of spermatogonial mitoses is modified so as to favour the formation of more type-A, in preference to intermediate, spermatogonia.

The ultrastructure of male germ cells in rats X-irradiated at birth

A histological and ultrastructural study was made of the testes of rats, aged 1 to 15 days, which had received partial-body X-irradiation (ca. 100 r) on the day of birth. Nineteen serially sectioned testes were subjected to a quantitative analysis including counts of germ cells and measurements of nuclear volume. The type, position and general form of germ cells, together with the incidence of various organelles, was assessed semi-quantitatively from 533 electron micrographs of irradiated (370) and normal (163) tissues. Exposure to 100 r at birth has no marked effect on the total population of germ cells during the first 5 days. The transformation of gonocytes into transitional cells is not affected. Subsequently, the treated testes differ from those of coeval controls by the almost complete absence of germinal mitoses and, consequently, of spermatogonia type A. Many of the transitional cells grow into irregularly-shaped giant cells, usually with a highly lobed nucleus and enlarged nucleoli. Although some cells show condensation of chromatin which may indicate the onset of mitotic prophase, the majority appear to degenerate at interphase. By 10 days, the testes contain only a fraction of the original population of germ cells. No significant changes are detectable in the ultrastructure of germ cells up to 4 days after irradiation. The treatment only induces the sporadic appearance of two minor features (pinocytotic vesicles containing electron-dense material; peripheral granular bodies with some internal organization) which are normally absent from corresponding cells in the controls. Electron micrographs of giant transitional cells confirmed the cytological observations of irregularity of nuclear outline and nucleolar enlargement. The nucleoli are frequently irregular in shape and contain material of two distinct electron densities. In general, the organelles of giant transitional cells are normal and healthy in appearance. With advancing age, an increasing proportion of germ cells show degenerative changes leading to pyknosis. Dead cells appear to be engulfed by neighbouring somatic cells. In both normal and irradiated testes, the simple form of Golgi apparatus in the gonocyte changes to a more complex type in the transitional cells. The persistence of giant cells is associated with a tendency for the Golgi apparatus to revert to the simple type, or to disperse into a number of small simple units. The most striking feature is the relatively high incidence of centrioles, suggesting that radiation-induced mitotic inhibition and gigantism may be accompanied by endomitosis. Irradiation does not markedly affect the appearance and incidence of A- and B-bodies (Franchi & Mandl 1964). These organelles tend, however, to persist over a longer period in irradiated than in untreated young rats.

The effect of β-mercaptoethylamine on the sensitivity of oocytes to X-irradiation

The rapid decrease in the population of primordial oocytes which normally occurs after exposure of the ovaries of the rat to X -irradiation has been partially prevented by administering β-mereaptoethylamine about ½ h before irradiation.