Identifying a new locus that regulates the development of rete ovarian cysts in MRL/MpJ mice.

Abstract

MRL/MpJ (MRL) is a mouse model for autoimmune disease and develops ovarian cysts with age. The ovarian cysts originate from the rete ovarii, which is considered to be the remnant of fetal mesonephric tubules. In a previous study, we analyzed the genetic background of ovarian cysts by using backcross progenies between MRL and C57BL/6N (B6) mice. By interval mapping, suggestive linkages were detected on several chromosomes (Chrs), and a significant linkage on Chr 14 was designated as MRL Rete Ovarian Cyst (mroc). In the present study, which evaluated 113 F2 intercross progenies, a significant linkage appeared on Chr 6 at the marker position D6Mit188 (likelihood ratio statistic = 18.5). In particular, the peak regions of Chrs 6 and 14, which contain major causative loci by backcross analysis, showed close reverse interaction. From these results, a locus on Chr 6 was identified as mroc2, the second major locus associated with ovarian cyst formation in MRL mice.

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Title
Identifying a new locus that regulates the development of rete
ovarian cysts in MRL/MpJ mice
Author(s)
Lee, Shin-Hyo; Ichii, Osamu; Otsuka, Saori; Hashimoto,
Yoshiharu; Namiki, Yuka; Kon, Yasuhiro
Citation Japanese Journal of Veterinary Research, 59(2&3): 79-88
Issue Date2011-08
Doc URLhttp://hdl.handle.net/2115/47148
Right
Typebulletin (article)
Additional
Information
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

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Japanese Journal of Veterinary Research 59(2&3): 79-88, 2011
FULL PAPER
Identifying a new locus that regulates the
development of rete ovarian cysts in MRL/MpJ
mice
Abstract
MRL/MpJ (MRL) is a mouse model for autoimmune disease and develops ovarian cysts with
age. The ovarian cysts originate from the rete ovarii, which is considered to be the remnant
of fetal mesonephric tubules. In a previous study, we analyzed the genetic background of
ovarian cysts by using backcross progenies between MRL and C57BL/6N (B6) mice. By
interval mapping, suggestive linkages were detected on several chromosomes (Chrs), and a
significant linkage on Chr 14 was designated as MRL Rete Ovarian Cyst (mroc).
In the present study, which evaluated 113 F2 intercross progenies, a significant linkage
appeared on Chr 6 at the marker position D6Mit188 (likelihood ratio statistic ＝ 18.5). In
particular, the peak regions of Chrs 6 and 14, which contain major causative loci by
backcross analysis, showed close reverse interaction. From these results, a locus on Chr 6
was identified as mroc2, the second major locus associated with ovarian cyst formation in
MRL mice.
Key words: cystic rete ovarii, MRL/MpJ mice, mroc2, QTL analysis, rete ovarii
Shin-Hyo Lee1), Osamu Ichii1), Saori Otsuka1),
Yoshiharu Hashimoto2), Yuka Namiki2), and Yasuhiro Kon1,＊)
1) Laboratory of Anatomy, Department of Biomedical Sciences, Graduate School of Veterinary Medicine,
Hokkaido University, Sapporo 060-0818, Japan
2) Office for Faculty Development and Teaching Enriched Veterinary Medicine, Graduate School of
Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Received for publication, June 2, 2011; accepted, June 21, 2011
cystic changes, cause economic loss by affecting
the fertility of farm animals such as cattle,
sheep, and swine.16)
　　The rete ovarii is homologous to the male
rete testis. It is considered to be the remnant
of mesonephric tubules and is composed of
mesenchymal cells that have migrated from the
mesonephros to the embryonic gonad.6,24-26) While
Introduction
　　In humans and animals, ovarian cysts are
major causes of female reproductive dysfunctions.
They are derived from several components of
the ovary, such as ovarian surface epithelium,
follicles, corpus luteum, and rete ovarii.2,16) In
veterinary medicine, ovarian disorders, including
*Corresponding author: Yasuhiro Kon, Laboratory of Anatomy, Department of Biomedical Sciences, Graduate
School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Phone and Fax: +81-11-706-5189. E-mail: y-kon@vetmed.hokudai.ac.jp

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QTL analysis of ovarian cysts in MRL mice
80
　　In the present study, we analyzed F2
intercross progenies to obtain more detailed
information about candidate QTLs related to the
development of ovarian cysts in MRL mice.
Comprehensive and accurate QTL analysis using
both backcross and F2 progenies is fundamental
for identifying the genes responsible for the
development of rete ovarian cysts. These results
would provide novel genetic information about
the pathogenesis of ovarian disease in human
and veterinary reproductive medicine.
Materials and Methods
Animals: Mouse MRL and B6 strains were
purchased from Japan SLC Inc. (Hamamatsu,
Japan), and F1 progenies from these were
generated in our laboratory. A total of 113 F2
progenies were generated by an MRL × B6 cross
between F1 mice of both sexes. Some F1
progenies were mated with MRL mice to produce
213 MRL × [MRL × B6] F1 backcross progenies
as previously described.14) These animals were
maintained in specific pathogen-free conditions
with a room temperature of 22°C ± 4°C, relative
humidity of 55% ± 20%, and a 12-h light and
dark cycle in the animal facility of the Graduate
School of Veterinary
University. The mice were allowed free access to
water and rodent food in pellet form. The mice
were housed in an animal facility approved by
the Association for Assessment and Accreditation
of Laboratory Animal Care, and the project was
approved by the Animal Experiment Committee
of the Graduate School of Veterinary Medicine,
Hokkaido University.
Medicine, Hokkaido
Phenotyping: The phenotyping of ovarian cystic
change was performed according to our previous
study.14) Briefly, ovaries of F2 progenies were
collected at 8 months of age, fixed with Bouin’s
solution overnight, processed through graded
alcohol, and embedded in paraffin by a routine
procedure. Serial sections 3-μm-thick by 30 μm
the rete system is considered to play a central
role in ovary development, the rete ovarii in
adults is generally a nonfunctional vestigial
tissue.4,5) However, some authors have hypothesized
that the rete ovarii has a secretory role, which is
supported by observations of eosinophilic and
periodic acid-Schiff-positive secretion, columnar
epithelium, and wide lumina of rete tubules in
several species.27) On the other hand, the rete
ovarii is reportedly associated with pathological
processes via lumen dilation and hyperplastic
and/or hypertrophic epithelial
mice.3,15,23)
　　MRL/MpJ (MRL) mice are the representative
model for autoimmune diseases, including dermatitis,
vasculitis, arthritis, and glomerulonephritis.1,10,22)
In addition to these autoimmune-mediated
inflammatory characteristics, this strain shows
interesting phenotypes such as active regeneration,
metaphase-specific apoptosis in spermatogenesis,
heat-shock resistance in spermatocytes, and
the appearance of oocytes in testes.17,20,21)
Furthermore, we identified a new phenotype:
spontaneous development
originating from the rete ovarii in aged female
MRL mice.11) This finding suggests that MRL
mice could be spontaneous models of ovarian
cysts and provides fundamental information
about female reproductive dysfunction.
　　In a previous study, to determine the
susceptibility loci of
originating from the rete ovarii, we performed
quantitative trait loci (QTL) analysis using
backcross progenies with C57BL/6N (B6) mice.14)
The marker positions D3Mit182-244, D4Mit248,
D6Mit316, and D11Mit212 show suggestive
linkages to the development of ovarian cysts. The
locus at the D14Mit37 marker position on
chromosome (Chr) 14, which showed significant
linkage (likelihood ratio statistic [LRS] ＝ 13.5),
was designated as MRL Rete Ovarian Cyst
(mroc). Furthermore, these findings suggest that
the development of rete ovarian cysts in MRL
mice is regulated by multiple loci on their
autosomes.
changes in
of ovarian cysts
MRL ovarian cysts

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Shin-Hyo Lee et al.
81
whole-genome scanning, 6 F2 mice of high trait
in ascending order of rete ovarii circumference
(＞ 7,000 μm) were tested by chi-square (χ2)
analysis to identify the causative chromosomes.
To identify the QTLs of ovarian cysts, the data of
the trait score and genotyping were analyzed by
MapManager QTXb20. The permutation tests
were performed in 1-cM steps for 10,000
permutations to determine the statistically
suggestive, significant, and very significant
levels. Final results of interval mapping were
considered to be suggestive for an LRS of 9.2,
significant for an LRS of 17.2, and highly
significant for an LRS of 28.8.
　　Among whole-genomic-locus-coded microsatellite
markers, the interactive locus throughout the
genome was analyzed by χ2 statistics by
MapManager QTXb20.
phenotype values of the interactive locus were
investigated in detail and compared with those of
the significant locus on Chr 6 by F2 QTL analysis.
The genotype and
Results
Histological features and distribution of ovarian
cyst sizes in B6 and MRL progenies
　　Fig. 1 shows the histology and appearance of
dilated rete ovarii in MRL, B6, F1, and F2
progenies. From 113 F2 progenies, 31 mice
showed cystic changes in the rete ovarii
according to our histological criteria. However,
the circumference of rete ovarii varied among
these mice (Fig. 1a and b). The circumferences of
rete ovarii in MRL and B6 mice and their
progenies were compared as shown in Fig. 1c.
The average values of F2 progenies tended to be
smaller than those of backcross progenies and
larger than those of F1 progenies (Fig. 1c). In
Fig. 1d, the individual traits of F2 progenies (n
＝ 113) were arranged by the circumference of
rete ovarii and compared with those of backcross
progenies (n ＝ 213). The trait distribution curve
of F2 progenies was sharper than that of backcross
progenies, and the maximum circumferences of
intervals with bilateral ovaries were cut and
stained with hematoxylin and eosin by standard
procedures. Only the section containing the
biggest cystic rete ovarii was chosen for phenotype
evaluation, photographed with a digital camera,
and measured by ImageJ (NIH, http://www.nih.
gov/ij/). Because we had already confirmed that
the circumference of the rete ovarii reflected the
severity of ovarian cysts,14) these values were
used as a quantitative trait in QTL analysis.
When calculating the rete ovarii circumference in
all mice, the F2 progenies with both larger
values than the average B6 value and flattened
epithelia were considered to show cystic changes.
Statistical analysis: The phenotype values of
MRL, B6, and their progenies were evaluated by
the nonparametric Kruskal-Wallis test (Schéffe’s
method) (Fig. 1c).
Genotyping: Genomic DNA was extracted from
the spleen with a standard protocol and applied
to a genome-wide scan analysis based on
polymerase chain reaction (PCR), using 98
polymorphic microsatellite markers (Table 1). For
0.2- to 40.0-cM intervals (average ＝ 15.1 cM),
primer pairs of microsatellite markers were
selected from the database at Mouse Genome
Informatics (http://www.informatics.jax.org/).
　　PCR was carried out on a PCR thermal
cycler (iCycler; Bio-Rad, Madison, WI, USA) with
a cycling sequence of 94°C for 2 min (1 cycle)
followed by 40 cycles consisting of denaturation
at 94°C for 40 s, primer annealing at 58°C for
30 s, and extension at 72°C for 30 s. Taq DNA
polymerase and buffers were purchased from
Promega (Madison, WI, USA). The amplified
samples were electrophoresed in 2% NuSieve 3:1
Agarose Gel (Cambrex Bio Science Rockland Inc.,
Rockland, ME, USA) or Agarose 3:1 (AMRESCO
Inc., Solon, OH, USA), stained with ethidium
bromide, and photographed under an ultraviolet
lamp.
Genomic linkage and statistical analysis: Before

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QTL analysis of ovarian cysts in MRL mice
82
(32.53 cM) on Chr 6 showed the most significant
linkage to cystic changes in rete ovarii (Table 2).
　　In QTL analysis, the highest linkage above
the significant level (LRS ＞ 17.2) was detected
only in Chr 6, and suggestive linkages (9.2 ＜
LRS ＜ 17.2) were not found in any chromosomes
(Fig. 2a). Linkage details are shown in Fig. 2b.
The loci at the D6Mit188 (32.53 cM) marker
position showed the highest linkage (LRS ＝
the rete ovarii of F2 progenies were lower than
those of backcross progenies.
Genetic analysis of ovarian cysts in F2 progenies
　　To clarify the relationships between genotypes
of F2 progenies and each chromosome locus, the
χ2 test was performed using 6 selected progenies
showing large rete ovarii circumferences in
ascending order. The marker position D6Mit188
Table 1. List of the microsatellite markers used in the genotyping of F2 progenies. For positions of the
microsatellite markers, refer to Mouse Genome Informatics.
Marker
D1Mit64
D1Mit123
D1Mit303
D1Mit134
D1Mit191
D1Mit107
D1Mit403
cM
3.67
17.67
31.79
41.24
52.66
70.19
81.03
D2Mit369
D2Mit107
D2Mit148
24.51
65.13
100.49
D3Mit130
D3Mit182
D3Mit244
D3Mit103
D3Mit288
D3Mit320
D3Mit129
2.55
21.73
35.01
46.83
52.94
66.75
80.49
D4Mit235
D4Mit178
D4Mit301
D4Mit145
D4Mit12
D4Mit251
D4Mit127
D4Mit42
3.57
34.92
42.14
43.30
57.76
69.05
80.52
82.64
D5Mit352
D5Mit201
D5Mit101
18.40
39.55
80.16
D6Mit166
D6Mit74
D6Mit33
D6Mit316
D6Mit243
D6Mit188
1.99
23.70
24.42
27.41
32.27
32.53
Marker
D6Mit323
D6Mit105
D6Mit10
D6Mit194
D6Mit374
cM
37.75
49.71
52.75
62.90
64.60
D7Mit178
D7Mit82
D7Mit105
2.02
32.76
79.29
D8Mit224
D8Mit226
D8Mit205
D8Mit343
D8Mit50
D8Mit248
D8Mit242
D8Mit211
D8Mit200
D8Mit56
19.38
23.05
28.85
39.33
43.51
44.99
50.07
54.51
61.37
76.14
D9Mit91
D9Mit181
D9Mit76
D9Mit18
20.74
46.58
50.18
71.49
D10Mit166
D10Mit42
D10Mit271
2.06
39.72
72.31
D11Mit62
D11Mit230
D11Mit212
D11Mit199
D11Mit48
5.78
16.15
54.34
65.48
82.96
D12Mit185
D12Mit136
D12Mit158
9.85
13.00
38.14
Marker
D13Mit17
D13Mit13
D13Mit260
cM
7.73
30.06
63.73
D14Mit10
D14Mit133
D14Mit141
D14Mit142
D14Mit37
D14Mit193
D14Mit265
D14Mit266
6.41
16.80
24.28
27.68
33.21
37.26
50.90
64.86
D15Mit111
D15Mit71
D15Mit245
13.02
37.80
48.21
D16Mit131
D16Mit140
D16Mit70
D16Mit106
3.41
40.30
48.81
57.68
D17Mit198
D17Mit119
D17Mit221
14.59
38.15
58.77
D18Mit177
D18Mit51
D18Mit186
21.39
34.41
45.63
D19Mit68
D19Mit80
D19Mit91
D19Mit33
3.38
18.24
40.53
51.76
DXMit166
DXMit130
DXMit186
28.26
55.45
76.75