Meiotic prophase progression and germ cell elimination in fetal and neonatal mouse ovaries.

摘要

In most mammalian species, all oogonia cease mitotic proliferation and enter meiosis in fetal ovaries. Furthermore, more than half of the maximum number of germ cells is eliminated from ovaries by neonatal life, thus limiting the oocyte reserve for reproduction. The cause or mechanism of this female germ cell loss remains largely unknown. A major loss occurs in the oocytes which reach the pachytene stage of meiotic prophase, suggesting that oocytes with meiotic or recombination errors may be eliminated by a checkpoint mechanism. It remains to be determined whether oocytes are eliminated by apoptosis and if so in which pathway. The purpose of my study is to investigate a mechanism of oocyte loss in the mouse ovary during meiotic prophase. We used an Msh5 null mutant mouse strain, in which all oocytes are eliminated by neonatal life. Msh5 encodes a protein required for meiotic chromosome synapsis.;Meiotic progression was studied by GCNA1 and SC (synaptonemal complex) or SC and gammaH2AX double immunolabeling of chromosome spread preparations. We found that meiosis in MT was blocked at zygotene-pachytene transition. No normal pachytene was observed in MT.;The role of apoptosis in elimination of oocytes during meiotic prophase was investigated by analyzing the cleavage of various caspases (caspase 2, 3, 6, 7, 9) as well as PARP1 by western blot using the lysate of whole ovaries. The activation of initiator caspase 9 increased from 17.5 to 18.5 dpc and decreased by 19.5 dpc. Caspase 2L activation also increased in a similar pattern but at much lower levels. The activation of effector caspase 3 or 6 remained at low levels. The activation of caspase 7 also was low but increased slightly at 19.5 dpc. The cleavage of PARP1 was high at all investigated stages. There were not major differences in the average level of activation between WT and MT. By immunolabeling of ovarian sections we observed that cleaved caspases and PARP1 were localized in oocytes but also in cells negative for GCNA1.;These results suggest that a mitochondrial pathway of apoptosis may play a role in the elimination of oocytes during meiotic prophase, involving activation of caspase 9 and cleavage of PARP1. However further studies are necessary for identification of an effector caspase.;Msh5 heterozygous mutant mice were crossed and ovaries were isolated from female progeny at 14.5 -- 22.5 days postcoitum (dpc). We studied the loss of germ cells in Msh5 -/- (MT) females comparing to the Msh5 +/+ (WT) and Msh5 (+/-) (HT) females by immunolabeling of ovarian sections for GCNA1 or MVH (both germ cell markers) or by counting GCNA1 positive germ cells in cell suspension preparations. Our results showed a continuous loss of GCNA1 positive cells in both MT and WT although the loss in MT was constantly larger than in the WT. A significant difference between WT and MT was found at 19.5 dpc.