Mitotic arrest of female germ cells during prenatal oogenesis. A colcemid-like, non-apoptotic cell death

Abstract

The sequence of events and a possible reason for germ cell death during oogenesis in the prenatal ovary were studied in rat and mouse embryos. ED 14-22 rat and ED 14-16 mouse embryos were studied using semithin sections for light microscopy and serial ultrathin sections for electron microscopy. In addition, the rat material was 3H-thymidine labelled for historadioautography and cytospin preparations of freshly obtained gonads were immunohistochemically analysed. During the transition from the proliferating oogonial stage to the meiotic prophase about 16% of the postmitotic oocytes do not pass the initial meiotic checkpoint on ED 18/19 in the rat (ED 15/16 in the mouse). These germ cells first show structural signs of mitosis; the diploid number of 'super-condensed' chromosomes are globally formed and are concentrated in the center of the cell. Although the germ cells show all morphological signs of living cells they never have mitotic spindles; the micro-tubulus-organisation-centres (MTOCs) are found peripherally and become concentrated, forming a single centrosomal body (acentriolar MTOC) as detected by immunohistochemistry for the centrosomal protein MPM2 and gamma-tubulin. EM studies show 25 nm tubule-like profiles within the MTOC bodies. The centrioles frequently lie separate from the MTOC material or are not present at all; the germ cells are apparently arrested in a prophase- or metaphase-like stage when they have reached the postmitotic G2/preleptotenal transition and are unable to enter meiosis. Forty-eight to 72 h after the first mitotically arrested germ cells are found, degeneration is seen in these germ cells. This second event, the germ cell death proper, shows neither criteria of apoptosis (cell shrinkage, marginal condensation of chromatin, DNA fragmentation) nor signs of necrosis (cell swelling, pycnosis, inflammation). Both arrested pro- and metaphase-like stages are found with signs of cell death and phagocytosis. The morphological signs of phagocytosis are found in neighbouring pregranulosa cells. The final heterocytotic bodies contain the remnants of the centrosomal (MTOC) material and DAPI-positive DNA material. The pregranulosa cells are mitotically silent during the phase when mitotic arrest and germ cell degeneration is found. The results suggest the presence of a hypothetical 'anti-spindle' factor, which under normal conditions is necessary for induction of meiotic prophase. The structural events of 'arrested mitosis' is reminiscent of those induced by the antimitotic, tubule-degrading drug colcemid. This type of arrest may inhibit meiosis of more than 33% prenatal germ cells and induce their cell death.

Quantification of healthy follicles in the neonatal and adult mouse ovary: evidence for maintenance of primordial follicle supply

“…In the superficial cortex of day 7 and day 12 ovaries, we found two mitotic cells and occasional PCNA-positive cells, but their identity could not be confirmed as proliferating germ cells. Such mitotic figures may represent oogonia arrested in mitosis as suggested by Wartenburg et al (2001) and McClellan et al (2003). Despite a thorough search for mitoses and PCNA-positive cells among primordial follicles at later ages, we found no mitotic figures and only rare examples of PCNA-labelled cells up to day 25 but none in older ovaries.…”

Analysis of programmed cell death in mouse fetal oocytes

“…The process of oocyte death remains, however, incompletely characterized and alternative forms of PCD for embryonic germ cells remain possible (Wartenberg et al 2001). Furthermore, the causes and the molecular mechanisms underlying oocyte PCD are little understood (for reviews, see , Tilly 2001, De Felici et al 2005.…”

The bridge-partitioning complex is absent in a distinct type of defective germ cell division in the gonads of the golden hamster

“…Several cytologic features of the arrested germ cells suggest that mitotic and early meiotic events occur in parallel within these cells (Wartenberg et al 2001). These are, on the one hand, a rapid and strong condensation of the chromosomes and the complete breakdown of the nuclear membrane, which are both indicative of mitosis.…”

“…Some features, on the other hand, resemble events that characterize meiotic prophase: the centrosomal apparatus is unable to nucleate regular microtubules and to organize the metaphase spindle. This is supposed to imply the continuous action of some inhibitory, possibly colcemid-like influence on the microtubule apparatus of the cells (Wartenberg et al 2001). In addition, the centrioles, if present, are abnormally localized centrally within the group of chromosomes, and the normal pericentriolar material (MTOCs) is lacking.…”

“…In both the ovary and the testis, these germ cell divisions are irreversibly arrested in a state marked by strongly condensed chromosomes and a disturbed centriolar spindle apparatus. The cells finally degenerate (Baker 1963;Miething 1998;Wartenberg et al 2001) and thus represent one factor contributing to the loss of germ cells characteristic of gametogenesis Morita and Tilly 1999).…”