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Cell cycle analysis of fetal germ cells during sex differentiation in mice.

Spiller C, Wilhelm D, Koopman P - Biol. Cell (2009)

Bottom Line: Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling.Lastly, in XX germ cells we observed a down-regulation of genes involved in both G1- and G2-phases of the cell cycle consistent with their entry into meiosis.The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics and Development, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

ABSTRACT

Background information: Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex-specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G1/G0 arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours.

Results: In order to unravel the mechanisms utilized by germ cells to achieve and maintain the correct cell cycle states, we analysed the expression of a large number of cell cycle genes in purified germ cells across the crucial time of sex differentiation. Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling. A robust mechanism for apoptosis and checkpoint control was observed in XY germ cells, characterized by p53 and Atm (ataxia telangiectasia mutated) expression. Additionally, a member of the retinoblastoma family and p21 were identified, linking these factors to XY germ cell G1/G0 arrest. Lastly, in XX germ cells we observed a down-regulation of genes involved in both G1- and G2-phases of the cell cycle consistent with their entry into meiosis.

Conclusion: The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.

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Related in: MedlinePlus

Validation of significantly up- and down-regulated transcripts identified from 12.5 versus 14.5 dpc germ cell populationsqPCR analysis from purified populations normalized to 18S RNA, n=3. (A) Increased fold expression of Tnfs5ip, Rbl2 and Camk2g and decreased fold expression of E2f6, Gas2 and Cks1b in XY 14.5 dpc populations relative to XY 12.5 dpc populations. (B) Increased fold expression of Tnfs5ip, Camk2a and Camk2g and decreased fold expression of Rbl1, Dst and E2f6 in XX 14.5 dpc populations relative to XX 12.5 dpc populations. Gc, germ cell; Rel., relative.
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Figure 3: Validation of significantly up- and down-regulated transcripts identified from 12.5 versus 14.5 dpc germ cell populationsqPCR analysis from purified populations normalized to 18S RNA, n=3. (A) Increased fold expression of Tnfs5ip, Rbl2 and Camk2g and decreased fold expression of E2f6, Gas2 and Cks1b in XY 14.5 dpc populations relative to XY 12.5 dpc populations. (B) Increased fold expression of Tnfs5ip, Camk2a and Camk2g and decreased fold expression of Rbl1, Dst and E2f6 in XX 14.5 dpc populations relative to XX 12.5 dpc populations. Gc, germ cell; Rel., relative.

Mentions: Using cDNA generated from the isolated germ cell populations, we profiled the expression level of 112 cell cycle-related genes for each time point (Figure 2A). Membrane hybridization represented three individual germ cell isolations for each sex and time point (Figure 2B). Using these populations we were able to make four comparisons: XX versus XY germ cells at 12.5 and 14.5 dpc and 12.5 versus 14.5 dpc for XX and XY germ cells. Because microarray analysis is generally known to underestimate fold changes in gene expression (Dallas et al., 2005), we focused on fold differences greater than 1.3. This threshold has also been used in other microarray studies (McHale et al., 2009). Using these criteria, gene expression changes across the germ cell populations revealed both up- and down-regulation of genes across all phases of the cell cycle (Table 1). From each comparison we next validated a selection of genes using qPCR [quantitative real-time RT–PCR (reverse transcription–PCR)] (Figure 3). Six out of nine genes analysed displayed a greater fold change than that detected by the array (Table 2), most likely reflecting the sensitivity of qPCR analysis relative to the microarray analysis (Dallas et al., 2005).


Cell cycle analysis of fetal germ cells during sex differentiation in mice.

Spiller C, Wilhelm D, Koopman P - Biol. Cell (2009)

Validation of significantly up- and down-regulated transcripts identified from 12.5 versus 14.5 dpc germ cell populationsqPCR analysis from purified populations normalized to 18S RNA, n=3. (A) Increased fold expression of Tnfs5ip, Rbl2 and Camk2g and decreased fold expression of E2f6, Gas2 and Cks1b in XY 14.5 dpc populations relative to XY 12.5 dpc populations. (B) Increased fold expression of Tnfs5ip, Camk2a and Camk2g and decreased fold expression of Rbl1, Dst and E2f6 in XX 14.5 dpc populations relative to XX 12.5 dpc populations. Gc, germ cell; Rel., relative.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2722160&req=5

Figure 3: Validation of significantly up- and down-regulated transcripts identified from 12.5 versus 14.5 dpc germ cell populationsqPCR analysis from purified populations normalized to 18S RNA, n=3. (A) Increased fold expression of Tnfs5ip, Rbl2 and Camk2g and decreased fold expression of E2f6, Gas2 and Cks1b in XY 14.5 dpc populations relative to XY 12.5 dpc populations. (B) Increased fold expression of Tnfs5ip, Camk2a and Camk2g and decreased fold expression of Rbl1, Dst and E2f6 in XX 14.5 dpc populations relative to XX 12.5 dpc populations. Gc, germ cell; Rel., relative.
Mentions: Using cDNA generated from the isolated germ cell populations, we profiled the expression level of 112 cell cycle-related genes for each time point (Figure 2A). Membrane hybridization represented three individual germ cell isolations for each sex and time point (Figure 2B). Using these populations we were able to make four comparisons: XX versus XY germ cells at 12.5 and 14.5 dpc and 12.5 versus 14.5 dpc for XX and XY germ cells. Because microarray analysis is generally known to underestimate fold changes in gene expression (Dallas et al., 2005), we focused on fold differences greater than 1.3. This threshold has also been used in other microarray studies (McHale et al., 2009). Using these criteria, gene expression changes across the germ cell populations revealed both up- and down-regulation of genes across all phases of the cell cycle (Table 1). From each comparison we next validated a selection of genes using qPCR [quantitative real-time RT–PCR (reverse transcription–PCR)] (Figure 3). Six out of nine genes analysed displayed a greater fold change than that detected by the array (Table 2), most likely reflecting the sensitivity of qPCR analysis relative to the microarray analysis (Dallas et al., 2005).

Bottom Line: Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling.Lastly, in XX germ cells we observed a down-regulation of genes involved in both G1- and G2-phases of the cell cycle consistent with their entry into meiosis.The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics and Development, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

ABSTRACT

Background information: Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex-specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G1/G0 arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours.

Results: In order to unravel the mechanisms utilized by germ cells to achieve and maintain the correct cell cycle states, we analysed the expression of a large number of cell cycle genes in purified germ cells across the crucial time of sex differentiation. Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling. A robust mechanism for apoptosis and checkpoint control was observed in XY germ cells, characterized by p53 and Atm (ataxia telangiectasia mutated) expression. Additionally, a member of the retinoblastoma family and p21 were identified, linking these factors to XY germ cell G1/G0 arrest. Lastly, in XX germ cells we observed a down-regulation of genes involved in both G1- and G2-phases of the cell cycle consistent with their entry into meiosis.

Conclusion: The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.

Show MeSH
Related in: MedlinePlus