Page 42 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
42
Sch. of Med., the Univ. of Tokyo, Tokyo, Japan; 2) Dept. of Biom. Chem., Grad. Sch. of Med., the Univ. of Tokyo, Tokyo,
Japan; 3) JSPS Research Fellow.
In mammals, the reproductive processes are carried on under low-oxygen conditions. In case embryos are subjected to high-
oxygen environments, excessive reactive oxygen species (ROS) are produced, causing embryo injury. We have focused on
Sirtuins, a family of phylogenetically conserved NAD+-dependent protein deacetylases/ADP-ribosyltransferases, are implicated
in diverse biological processes, such as aging and metabolism, as molecules involved in stress defense mechanisms coupled with
cellular metabolic status. We, firstly, have characterized the role of sirtuins in mouse preimplantation development under in vitro
conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage.
Among them, the expression of Sirt3, which localizes to mitochondria and is involved in energy metabolism, was upregulated by
H
2
O
2
-
induced oxidative stress, indicating an important role for Sirt3 in mitochondrial functions, which is a crucial factor in
determining the developmental potential of embryos. Indeed, siRNA-induced knockdown and genetic deletion of
Sirt3
resulted in
increased intracellular ROS originated from mitochondria and decreased blastocyst formation. The antioxidant N-acetyl-L-
cysteine and low oxygen conditions rescued these adverse effects. We also found that ROS-induced upregulation of p53 was
responsible for this developmental arrest. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-
term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in
vitro aerobic conditions and that the effect was long lasting. Furthermore, microinjection of Sirt3 mRNA could improve the
developmental outcome of preimplantation embryos exposed to H
2
O
2
.
Thus, Sirt3 protects preimplantation embryos against
oxidative stresses under aerobic conditions through regulating mitochondrial functions. Recently, we are pursuing the possibility
that Sirt3 may improve the outcome of assisted reproductive technologies (ART), because of previous findings that ROS is
critical for ageing of germ cells. Since ART is widely used for the treatment of infertility in clinical practice, many issues such as
the low birth rate remain to be solved. We have obtained some preliminary evidences for a negative relation between Sirt3 levels
and aging-associated loss of egg quality; thus Sirt3 may work advantageously to improve the ART efficiency.
24
The mouse Y-encoded transcription factor ZFY promotes the second meiotic division and is essential for sperm tail
development and head remodelling. Nadege Vernet
,
Fanny Decarpentrie, Shantha Mahadevaiah, Paul Burgoyne. Stem cell
biology and developmental genetics, NIMR, Medical Research Council, UK, London.
Human ZFY and mouse
Zfy1
and
Zfy2
encode zinc finger transcription factors and were identified in the 1980s as promising
candidates for the mammalian Y-encoded testis determinant. The first functions were ascribed to these genes in 2010 and 2011
when they were shown to have important meiotic quality control functions in sex chromosomally aberrant mice;
thus
Zfy1
and
Zfy2
trigger the apoptotic elimination of XYY spermatocytes at the pachytene stage, and
Zfy2
the apoptotic
elimination of spermatocytes with a univalent X chromosome at the first meiotic metaphase.
Zfy1
and
Zfy2
are located on the
mouse Y short arm (Yp) and for the present study we created three mouse models with a limited Y gene content, and
diminishing
Zfy
gene complements: (1) an almost complete Yp gene complement -
Zfy2
,
Zfy1
,
Rbmy1a1, H2al2y, Sry, Usp9y,
Ddx3y, Uty, Eif2s3y, Kdm5d, Ube1y1
; (2)
a highly deleted Yp retaining
Rbmy1a1, H2al2y, Sry, Eif2sy
and creating a fusion
gene
Zfy2/1
by an ectopic recombination event between
Zfy1
and
Zfy2
;
and (3) a limited Y gene content restricted
to
Sry
and
Eif2s3y
.
All three models lack the Y long arm that normally mediates pairing and exchange with the X via their pseudo
autosomal regions (PAR), so we added a minute PAR-bearing X chromosome derivative to each thus enabling the formation of a
sex bivalent to avoid any apoptosis triggered by a univalent X chromosome. These three models were first used to substantiate
previous findings suggesting a role for a Yp gene(s) in promoting the second meiotic division (
Vernet et al. 2011
),
and also to
confirm that a Yp gene(s) is required for the initiation of sperm morphogenesis (specifically the round spermatid to elongating
spermatid transition;
Vernet et al. 2012
).
We have then used a Y transgene addition strategy to show that it is
Zfy1
and
Zfy2
that
promote the second meiotic division and that sperm morphogenesis requires
Zfy2
but not
Zfy1
.
Thus these Y-encoded zinc finger
transcription factors, in addition to meiotic quality control functions, have important roles in normal
spermatogenesis.
References
:
Vernet, N
,
et al. (2011) The Y-encoded gene Zfy2 acts to remove cells with unpaired
chromosomes at the first meiotic metaphase in male mice. Current Biology 21 787-793.
Vernet, N
,
et al. (2012) Spermatid
development in XO male mice with varying Y chromosome short arm gene content: evidence for a Y gene controlling the
initiation of sperm morphogenesis. Reproduction (Submitted).
25
FGFR2IIIc Is Required for Testis Determination but Is Dispensable for Subsequent Testis Cord Differentiation and
Spermatogenesis.
Stefan Bagheri-Fam
1
,
Meiyun Yong
1
,
Anja Dietrich
1
,
Terje Svingen
2
,
Peter Koopman
2
,
Veraragavan
Eswarakumar
3
,
Vincent R. Harley
1
. 1)
Molecular Genetics & Development, Prince Henry's Inst, Clayton, VIC, Australia; 2)
Institute for Molecular Bioscience, University of Queensland, QLD, Australia; 3) School of Medicine, Yale.
Loss of Fibroblast Growth Factor Receptor 2 (FGFR2) in mice leads to XY gonadal sex reversal, indicating that FGFR2 acts as
the receptor for FGF9 during testis development. FGFR2 exists in two isoforms, FGFR2IIIb and FGFR2IIIc which differ in their
FGF ligand-binding specificity. FGFR2IIIb is a low affinity receptor for FGF9 and is not normally expressed in Sertoli cells, and
XY Fgfr2IIIb knockout mice do not show abnormalities in testis development. FGFR2IIIc is a high affinity receptor for FGF9
and is expressed in Sertoli cells. However, Fgfr2IIIc knockout male mice were reportedly fertile suggestive of normal testis
development. It was speculated that over-expression of the FGFR2IIIb isoform could compensate for the loss of FGFR2IIIc.
Alternatively, Fgfr2IIIc knockout male mice might show testicular defects at fetal stages that resolve postnatally. To investigate