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� � ����������Vol. 33, pp. 279�286, 2005
MATER�NALP5 ����������
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���1 Andrea Jurisicova2 Robert F. Casper 2
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� �MATER �maternal antigen that embryo require��NALP5 $�%�&'()&*+,-./01�� Mater ��� 2�3456+7589$:;01-<55� =5�>�$ 2%�?0*@ABCDED�F� GH� MATER A�%�IJK�L���0*+DEM-NOAPQRSA� =5TU$�01-� =N0� Mater ��� �34�%�5IJK�L��TUVWX,-NO&YZE� MATER A�%�IJK�L��&[METUDEM-\U]V^_DS� 8�12`5Mater ���abMater wild type �WT�2�345�%�IJK�L��5�cde]� e]fg �ROS�e]� hijkl�mnVopDS� Mater ����%�5IJK�L���cde]$ Mater WT Y�<�q&rsZS� �%�IJK�L��&[t-ROS e]$ Mater WT Y�< Mater ��� 0r9� hijkl�mn$ Mater ��� 0uMvw&1ZS� xy5z{s|� Mater ��� 0$� �%�5IJK�L��}~A(;& �DSz{� e]fgA��,-A� hijkl�A��DEM-S�e]fgV�W0��� %���V�SD%��A��QR-O��|R-� �S� MATER $:;��*�� '&��?�?&[ME���./01-A� �>x�5�%�&[ME��DEM-\U]AP�QR-�
����MATER� NALP5� �%�� IJK�L��� e]fg
Maternal antigen that embryos require �MATER�$� �34&[t-�*�� �premature ovarianfailure: POF� 5��W5��0*�QR� �%�A:;���V��,-S�&���./01-1�� MATER ./$H!*�QRS NALP pro-
tein family 5��0� �� NALP 5 O<� RE
M-2��3�� NR�05¡¢0� NALP protein family$� �£J¤¥4¦§¨©ª5¥h�i«¬&�DEM-NOA®|REM-4��9��MATER $�%�&'()&*+,-./0�
�¯��5°?Y�*+D� �? blastocyst�0^±QR-10�� ��� Mater mRNA $�340$²³��Y�*+D� �¯��0Gr´O�-A� =5µ$��*@OO<&��D� �>µ5 2�4%�?&$^±QR�9�-10�� Mater ��� 2�34s|¶�QRS�>�$·���,-<55� 2%�?0*@ABCDED�F �2-cell block�11�� 2%�?0�*�ABC,-+¸$� Mater � 5�%�
1 �������� ¶�¹�º»2 Samuel Lunenfeld Research Institute �SLRI�, Mt.Sinai Hospital,
J¼�J¶�¹�º»
279
63
� Mater ������������� ���Figure 1�� ���� �����������MATER ����������� �!��"�#$%�&'���� (� MATER )����*$+,�!��"-� MATER �./�0������*$�12345� 2��������678����3�9 �NALP protein family �:;)� <;*$�=�
��>>"��� MATER �?3�) (@��9A�: � BC� MATER )����DEF��G3� D�HIJKLMN�O�PQ8���R��10��S��� �TU�)� MATER )�8�V�
������� �*$3� �#$%�"��WX� ���Y�HIJKLMN*$�> �Z[3�
�����
�� ����� ��Dr. Larry Nelson �NICHD � NIH� Bethesda,USA���Mater +��\ B6A �C57BL�6J�A�J� F1 ]^_�`��� Samuel Lunenfeld ResearchInstitute animal facility �Toronto, Canada� �abc+,3� ����]^_cdefg�h� Ma-ter wild type �WT��Mater ���\]^_cij�k�3� lm�=n)� opqr0-st3DNA �High Pure PCR Template Preparation Kit,1�796�828, Roche, USA ck�� �� PCR �c� �u�� PCR primer )� Mater WT ) sense
� 5’-TCA TGT CCT TGG ATG GCA TG-3’�antisense � 5’-CCA CGT GCT TTC AAG ATTGC-3’c� Mater ���) sense� 5’-ACC GGT GGATGT GGA ATG TG-3’� antisense � 5’-CCA
CGT GCT TTC AAG ATT GC-3’c� � PCR)� 95�C 5 c 1 vwxy� 95�C 30 z� 59�C 30z� 72�C 1 c 37vwxy�{|�3� 1.25�Agarose gel �}~��3� Ethidium bromide so-lution����� Mater WT) 396 bp, Mater ���)242 bp, Mater +�� )�������KLc��3������)� Samuel Lunenfeld Research In-stitute animal facility ���232�C�� ��652��� ����5 : 00�17 : 00����� �����Purina Mouse Chow� ��)���X���3� ������ij)� Mount Sinai HospitalAnimal Care Committee �����������u���� ��8�12��� Mater WT, Mater ��� \]^_������%%����y�K �pregnant mare’s se-rum gonadotoropin: PMSG� �NHPP, NIDDK, Dr.A. F. Parlow��`� 5 IU� 48 ¡��¢I��%%����y�K �human chorionic gonado-toropin: hCG� �Wyeth, Canada� 5 IUc�£¤¥3� 12 ¡���¦c§t3� ¨�� ©�ª�200 mlY�� 26G«¬��¦�®�c¯°3��±���²-³(����c´µ�3��� ©�ª�3� 0.5� bovine serum albumin �BSA;
Figure 1. Maternal lethal e#ect. The embryo of F1 Mater ��� becomesarrested during early stages of development. Therefore, there is
no o#-spring produced �F2 less mutation�.
¶·¸¹ º»¼´� �280
64
Sigma, Canada���� modified human tubal fluidmedium �mHTF; Irvine Scientific, USA� ������ �� 0.3 mg�ml hyaluronidase ����� ��� 1���� �������������� ��!"�#$%&'(�)'(*+�,�-�.� 0.5� BSA ��� mHTF �����/0�� )1'(�2345��)�67����12���� ��� fragmentation �Mater WT8Mater ������9)'(�� 0.5�
BSA ��� human tubal fluid medium �HTF ;Irvine Scientific� USA� ��:� 37�C� 5� CO2� ��� ;)<�0 <=�� 24 <=.� 48 <=.'(>9 fragmentation �?@�#� A9BCD�EB����� �������� ����a� F�GHIJK&LMNOPQDePsipher �DePsipherTM Kit� TA700 ; R&D Sys-
tems Inc., USA� 0.5 ml, HTF 890 ml, reaction bu#er
100 ml, stabilizer 10 ml �RS�#� 30�= 37�C�THU"VW!XH��� Y� 13,000 g # 1�=Z[�,�� �\9]� DePsipher ��8�#����� ;^�� Mater WT� Mater ��� 9)'(�� DePsipher ��:� 37�C� 5� CO2 �25�= ���� 0.5� BSA��� mHTF ��� 1 �/0��.� _�` ��a�#bc�de�� Y� deconvolution microscope �Olym-pus IX70; Applied Precision Inc.�USA��)'(�?@��� f98g� hijk�lmn J-aggre-gate 8oijk�lmn J-monomer �ApqpRITC �rhodamine isothiocyanate�8 FITC �fluore-scein isothiocyanate� rstW��`#uB���f9vw�x� yzF�GHIJK� J-aggregate{uB�pn{� &LM{|}��F�GHIJK�xfp{uB�p~`� J-aggregate 8 J-mo-nomer 9�S� Delta Vision Software �AppliedPrecision Inc.� USA� ��`#���� J-monomer�mn J-aggregate 9�S�F�GHIJK&LMNO8]~���b� ROS �Reactive oxygen species� NOPQDimethyl sulfoxide �DMSO; Sigma� Canada�
990 ml 8 2’,7’-dichlorodihydrofluorescein diacetate�H2DCFDA� �Molecular ProbesTM; Invitrogen�USA� 10 ml�RS�� fp �1mMH2DCFDA��j
ki�8�#�`�� ;^�� Mater WT, Mater��� 9)'(�� 0.5� BSA ��� HTF 1ml jki� 1 ml����� ��:�� 37�C� 5� CO2� 15�= ���� deconvolution microscope �FITC rstW����#�p���� DeltaVision Software �bcmnf8*e# ROS NO�Q�����c� �st��HPQ;^�� Mater WT, Mater ��� 9)'(��HTF�0.5� BSA 988 ml H2O 11.4 ml����� ��:� 37�C� 5� CO2 � 90�= ���� �{��mn 15�� ��: monochlorobi-mane � 1 ml���#)'(F�GHIJK�9�st��H��i��� deconvolution microscope� CFP rstW��`#�p���� DeltaVision Software �bcmnf8*e#�st��H��Q������ ��������x���������� 2�=�9u�x
c2uQ �Fisher’s exact test��`# p0.05��e# ¡�¢+8£Q���
� �
�� ��� fragmentation �)'(9'(> fragmentation Dx� ;)<�0<=�xMater WT 4.151.92�� Mater ��� 1.060.76�� 24<= �.x Mater WT 8.126.53��Mater ��� 2.431.62�� 48 <= �.x MaterWT 64.606.49�� Mater ��� 68.769.18��¢+� ¤�=��x¥p~¦e� �Figure 2���� �������� ����a� F�GHIJK&LMNO)'(F�GHIJK9&LMNOx� MaterWT �x 0.740.05� Mater ��� �x 0.950.10�¢+� Mater ���9)'(3§�xMater WT *+� ¡¨��©�� �p0.02� �Figure 3��ª�� Mater WT �x J-aggregate x)'(9«¬®�#`�9��� Mater ��� �x)'(3§¯®�#`� �Figure 4��b� ROS �Reactive oxygen species� NOPQ)'(�°n ROS NOx� Mater WT �x74.9923.39�105� Mater ��� �x 110.147.91�105 �¢+� ¡�x~¦e�{ Mater ��� �xMater WT *+�¨`±²¢e� �Figure 5��
MATER 9)'(�°n³´ 281
65
0
0.2
0.4
0.6
0.8
1.0
1.2
Mitochondrial membrane potential
in oocyte
WT -/-
P<0.02
Mater WT ����������� ROS �������� Mater ��� ��������������� �Figure 6��c� ����� !"#��$%&'����� (�� Mater WT�166.30�9.25�107� Mater ��� � 113.59�1.98�107
�)*� Mater WT��Mater ��� +*,-.$�(/01� �p�0.01� �Figure 7��
� �
MATER�NALP5�� NALP protein family23�#���4$567'89�)'� NALP pro-
tein family �:;<=>?� Pyrine domain
�PYD-�, NACHT domain� @�ABC=� Leuci-ne-rich repeats �LRRs-� �DEFGH/-1� :IJKCLMNOPQ�C�R�ST$UV1��'�W�XYZ��'4��9�� [\L�Nalp]S^�_`� Nalp 12, Nalp alpha, Nalp beta, Nalp
gamma, Mater�Nalp 5, Nalp delta, Nalp epsilon,Nalp zeta, Nalp eta, Nalp theta � Chromosome 7Aa$bc1� Nalp iota, Nalp 10� Chromosome7F a$bc1�%*� Mater�Nalp5 �d� Nalp
]S^Wef�gh$ij1�bc1��'2��kl 2�4m�[\L�no/pq7'W� rstu��#vN/��1�w5xy �premature ovar-ian failure: POF�WF'13��16��� [\LMATER��� POF z{�[\L�#v�G|$561�
Figure 2. Spontaneous cytoplasmic fragmentation �FR�rate in oocytes. There was no statistical
significant di#erence in spontaneous cyto-
plasmic fragmentation rate between Mater WT
and Mater ��� oocytes. In WT, 0hr: n232,24hr: n84, 48hr: n73. In ���, 0hr: n461,24hr: n89, 48hr: n60.
Figure 3. Mitochondrial membrane potential in oocytes.
There was a significant di#erence between
Mater WT �n19� and Mater ��� �n20� inoocyte mitochondrial membrane potential
�p�0.02�.
Figure 4. Images of oocytes stained with DePsipher.
J-aggregates �red� were localized to the outerregion of the Mater WT oocyte. However,
J-aggregates were distributed all over the
oocyte in Mater ���.
Figure 5. ROS activity in oocytes. ROS activity in
Mater ��� oocytes �n45� was higher than thatin Mater WT oocytes �n28�, although therewas no statistical di#erence.
}~�� ���2� Y282
66
����������� �1�� MATER �������������� Mater ��� ���������������� Mater ��� �����!"��#�$%�&'� (�)*+,-�� �.�� �����/ Mater WT ����0���10�1� Mater ��� ���234�� �����56�78�/��� 29�����1:;<=�> ��&�11�� Mater +�� ��� ?(��@��� F1 AB� Mater ���� ������� 14�� �1� F1� Mater ��� ����Mater WT ����?(��@� Mater +�� �CDE"?/F���?G��/� H�������I���JK���1:;<=�>� 4L?4��M&�� H ? F2 NO�P�$��=�Figure 1��H�Q=�� H <�� MATER ���������RS8TU��V� ��� 2� �9�� MATER 1�!���/22W3X� Y
��9��Z[�.��\]3 �&�� <��MATER ��9��^_`�abc��!��10�� YH�� de�Mater ���� Mater WT ����^_`�abc.�?�f8�H��Qg�MATER ��9�^_`�abc�Z[�.�?h�8�H�?�i���<X� �9��^_`�abcj,�k��6F�� Y��lm?\]�� H�no�p��DePsipher�q�r�,��st�,uv �5, 5’, 6,6’-tetrachloro-1, 1’, 3, 3’-tetraethylbenzimidazolyl
carbocyanine iodide� �� 6��cw_xy��z{��|�^_`�abc�lm�}~?��8���p�� �19��21�� DePsipher �� �%&�lm?����^_`�abc��_b�������< ��� �u�� �J-aggregate� ?�8�� �g� �u��?�8�^_`�abc�j,���H�?�8� 6�� �lm?}~�^_`�abc������< X� 9�����u�� �J-monomer� ?�8�� H���� DePsi-pher �^_`�abc������< &�1�^_`�abc�-����8�������>��u�����i��lm?}~�^_`�abc?�8���3 �� Figure 3 ���Q=��Mater WTQ�/Mater ��� �^_`�abcj,1 ���� �9���� ^_`�abc�B�j,+�v}!"� �B��������# � Z�� �����$z{�H 3�¡�� ��'21��22�� 2� Mater ��� ���9��JK�8��^_`�abcj,1$%� ���H�1%¢£�Q��32�&g�� H ��MATER 1^_`�abcj,?�¤�@�.�?&8�H�?�¥8�� <�� Mater WT��j,��^_`�abc1�9��¦§�¨©������ Mater ��� ���9�ª«�¬©��� H �� Mater WT�^_`�abcj,1 � ��1� Mater ��� �^_`�abc?j,�8� .�1�¤��H�?�8�2/ &��^_`�abc�9���Z[�j,�v�®�&��¯�&g��1� H�j,�v�DNA+°'�� q�&±?²³� 9�´?(µ8�23��H�¢£�Z[� ROS ¶·�¸¹��� &º»��3 &2g�1Mater ����Mater WTQ�/¼�����g�� �g� Mater ��� ��9��
Figure 6. Images of oocytes stained with H2DCFDA.
ROS activity were more concentrated in the
centre of the oocyte in Mater WT, but they
were spread around the cytoplasm in Mater
���.
Figure 7. Glutathione level in oocytes. Glutathione
levels in Mater ��� oocytes �n�46� were sig-nificantly lower than in WT oocyte �n�34��p0.05�.
MATER ��9��Z[�.� 283
67
�� ��������� ������� �������������� ����� �!"��#�$� 2#�%&����'(�)'�*+� �#�&� ROS ����� MaterWT � Mater ��� &��,'� -*.� Mater WT&� ROS ��#�� /�&01�+��2�� Mater ��� &��#�34&56��7+� 6����89���&)'�� (�:-���� �#��;<07=>&?@A'��BC��7'�1D��,7�EFGHI����J���K������L�+M�NO;<PQA'�R�)'23��24���ST���� �#��UV'EFGHI�W�Mater ����U7�Mater WT �.D�8��X��7+� Y��� Mater ���&�Mater WT �.D���PQ��X��7'�Z[��'��ST&� 8�12\��]^_<`��+�� 6�a��#� fragmentation ��� Mater WT �Mater ��� &b�ca�,1�+d1a� 6�\�&��#��e�?@���7'd�fg��'� �1�� �#���������e<�hA'�� Mater ��� ]^_��#���������������U.� ���� ����L��ij�� �a�EFGHI��L��X��7'+k�� ��<PQA'���XA'6��fg��'� li���1a� Mater ��� ]^_��#���������m�� �������#��;��X)'7�#�$�����'�noa�'� ip�+�q�� Mater ��� ]^_�!"�� 2#�%&��<�rA'6���a��7'11��� 6� 2-cell block ���es���&)'� �ST&�� Mater ��� �#��;��X��7'+k� ��� 2#�%&�rA'(<�A'6�� MATER ��#����������EFGHI��L<07=>&?@A'e<��� *+� �������!"t<�X�u'6�<fg�+�vw� ]^_ Mater x#y�z{i|}~���07x#y���&$%��� 6�x#y�e������7'25�� ��� MATER�NALP5 ��a��#,e���vez����� ������&�J��'������J�(��R��)A'�noa�'�
� �
��<�o'�)+.� ��f� ���<�.*�+�]�����������(��G� *¡¢£�,a���+¤�0¥¢¦ST§�¨�©ª7+�*A� *+� ST�«<)o�7+¬R� ��,'�����®¯_<7+¬R*�+ SLRIDr. Casper�Brown Lab �°,�©ª7+�*A��ST�:�� 25th Great Lakes MammalianDevelopment Meeting �2005- 3±; Toronto, Can-ada�� ² 57³.���+¤��´�µ¶·´�2005- 4±; ¸/��U7��0�+�
����
1� Tong ZB, Nelson LM. A mouse gene encodingan oocyte antigen associated with autoimmune
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1999; 140: 3720�3726.2� Hamatani T, Falco G, Carter MG, Akutsu H,Stagg CA, Sharov AA, Dudekula DB, VanBu-
ren V and Ko MSH. Age-associated alteration
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Hum Mol Genet 2004; 13: 2263�2278.3� Dade S� Callebaut I, Paillisson A, Bontoux M,Dalbies-Tran R, Monget P. In silico identific-
ation and structural features of six new genes
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MATER �./0$%�12 285
69
Abstract
Role of MATER�NALP5 in Oocyte Mitochondria
Chiharu Tsuda1�2, Juichiro Saito1, Asako Taniuchi1�2, Marie Ino1,
Bunpei Ishizuka1, Andrea Jurisicova2, and Robert F. Casper2
Maternal antigen that embryo require �MATER� was identified as a possible factor inducing prematureovarian failure in mice. MATER, also known as NALP 5, belongs to the NALP protein family. Despite the
known domain structure of this protein, nothing is known about its function. In contrast to other NALP
family members, MATER is exclusively expressed by germ cells during oogenesis as well as by early cleaving
embryos. Female mice lacking functional Mater gene are infertile due to early embryonic arrest at 2-cell
stage, followed by cytoplasmic fragmentation. Recent studies suggest that MATER localizes to mitochon-
dria and nuclear envelope of oocytes. Therefore, we hypothesize that MATER functions as an anti-apoptotic
protein, and that disruption of Mater gene leads to altered mitochondrial function and accelerated ovarian
aging. Spontaneous cytoplasmic fragmentation of ovulated oocytes was not di#erent between Materwildtype and ���. Analysis of cellular parameters in ovulated oocytes lacking Mater revealed a significantincrease in mitochondrial membrane potential accompanied by increased accumulation of reactive oxygen
species �ROS� and decreased glutathione content. These results demonstrate that Mater decrease mitochon-drial membrane potential and ROS production. Furthermore, it is suggested that oocyte quality is impaired
in the Mater null females, leading to failure of early embryo development via alteration of mitochondrial
activity.
1 Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kawasaki, Japan
2 Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, University of Toronto, Toronto, Ontario, Canada
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