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Screening of genes involved in chromosome segregation during meiosis I: towards
the identification of genes responsible for infertility in humans
Hiroshi Kogo1, Hiroe Kowa-Sugiyama 1, Kouji Yamada1,2, Hasbaira Bolor1, Makiko
Tsutsumi1, Tamae Ohye1, Hidehito Inagaki1, Mariko Taniguchi1,3, Tatsushi Toda3, and
Hiroki Kurahashi1*
Supplementary information
Supplementary Results
Supplementary Table 1, 2
Supplementary Figure legends
Supplementary Figure 1
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SUPPLEMENTARY RESULTS
Isolation of the meiotic cells
To isolate differentiating male meiotic cells, we first performed discontinuous
Percoll density gradient centrifugation of testis cells from 15 day old mice
(Supplementary Figure 1a). The cell fractions were characterized by morphological
analysis and by the expression of cell lineage- and spermatogenic stage-specific genes.
Morphological observations using Nomarski interference contrast microscopy revealed
that spermatogonia were highly enriched in the lower cell fractions, whereas pachytene
spermatocytes were most abundant in the upper cell fractions (data not shown).
Consistent with these observations, semi-quantitative RT-PCR analysis revealed
that the expression of Oct4, a marker for spermatogonia, was prominent in the lower
cell fractions, whereas that of Spo11, Sycp1, and Dmc1, which initiates in
leptotene/zygotene cells and peaks in pachytene spermatocytes, was most intense in
each case in the upper fractions (Supplementary Figure 1b). Although low levels of
Sertoli cells inevitably contaminated most of the cell fractions as demonstrated by the
presence of Wt1 expression, the levels of contamination did not significantly differ
between fractions (Supplementary Figure 1b).
In addition to density separation, we used testicular cells undergoing the first
round of spermatogenesis in developing testes at specific ages to more efficiently purify
each cell fraction. We eventually isolated spermatogonia from cell fraction 2 of 8 day
old testis, leptotene/zygotene spermatocytes from fraction 5 of 12 day old testis, and
pachytene spermatocytes from the fraction 8 of 15 day old testis. Morphological
observations were used to confirm that all of the fractions comprised more than 90% of
the target cells (data not shown).
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Supplementary Table 1. PCR primers used for RT-PCR
Genes
Primers
Length of PCR Product
Oct4
AGCTGCTGAAGCAGAAGAGG
GGTTCTCATTGTTGTCGGCT
Spo11
GACAACTTCTGCAGCAGGATG
TGGTCCACGTTCCCTGCTGT
Sycp1
206bp
GCCAGTTCACCGGTACAG
GCACATCCTGAATGCCTCG
Actb
746bp
TGACAGCTCAACTTCCAGGAACAGGCG
CTTGGCTGCGACATAATCAAGTAGCTCC
Wt1
514bp
CTGATTACTGTGGAACTCCAGAA
GACCTTTATCTCATATGCATTCAG
Dmc1
198bp
609bp
CTGTGCTATGTTGCTCTAGACTTC
TCTTTACGGATGTCAACGTCACAC
3
230bp
Supplementary Figure Legends
Supplementary Figure 1
Isolation of spermatogenic cells by Percoll density gradient centrifugation. (a)
Schematic representation of a discontinuous Percoll gradient showing the position of
each cell fraction. The percentage of Percoll and its density are indicated on the left. (b)
Semi-quantitative RT-PCR analysis of lineage or stage-specific genes. Each gene name
and the number of PCR cycles are shown on the right, while each sample fraction is
indicated at the top. P, positive controls.
Supplementary Figure 2
Expression pattern plots for representative genes that are expressed specifically in
spermatogonia, spermatocytes or Sertoli cells. Red lines indicate six genes specific for
spermatogonia (Oct4, EpCAM, Itgb1, Itga6, Cd9, Cdh1), green lines indicate eight
genes for spermatocytes (Msh4, Sycp1, Dmc1, Smc1b, Sycp3, Stag3, Spo11, Fkbp6), and
blue lines indicate six genes for Sertoli cells (Wt1, Gata4, Gata1, Sox9, Fshr, Shbg).
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