Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
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16 t h International Congress on Animal <strong>Reproduction</strong><br />
Poster Abstracts 145<br />
Materials and methods Four group <strong>in</strong>clud<strong>in</strong>g eight adult male<br />
Balb/C mice weigh<strong>in</strong>g 30 5g were used <strong>in</strong> this study. Normal sal<strong>in</strong>e<br />
adm<strong>in</strong>istered as placebo to control group and saffron extract <strong>in</strong> doses<br />
of 25 , 50 and 100 were <strong>in</strong>jected <strong>in</strong>traperitoneally for 20 days to<br />
experimental groups. FSH, LH and testosterone <strong>in</strong> serum samples,<br />
were measured by ELISA.<br />
Results In comparison with plasebo the level of FSH, LH and<br />
testosterone significantly <strong>in</strong>creased <strong>in</strong> 100 saffron treated group, but<br />
no significant differences were observed between other treatments and<br />
placebo.<br />
The results of this study <strong>in</strong>dicate the efficacy of saffron extract <strong>in</strong> the<br />
male reproductive endocr<strong>in</strong>e function, and showed that it can modify<br />
the reproduction activities.<br />
Discussion The results of this research show that the extract of saffron<br />
<strong>in</strong> the mass of 100mg/kg has an important effect on level of FSH and<br />
LH and testosterone hormone. Saffron can have a key role <strong>in</strong><br />
<strong>in</strong>creas<strong>in</strong>g the generative power <strong>in</strong> male mice.<br />
Poster 13 - Molecular Biology of <strong>Reproduction</strong><br />
P357<br />
Goat as mo<strong>de</strong>l for study<strong>in</strong>g ovarian differentiation <strong>in</strong><br />
mammals<br />
Auguste, A*; Montazer-Torbati, F; Kocer, A; Pannetier, M; Renault, L;<br />
Mandon-Pep<strong>in</strong>, B; Cot<strong>in</strong>ot, C; Pailhoux, E<br />
INRA – UMR Biologie du Développement et <strong>Reproduction</strong> – 78350 Jouy en<br />
Josas - France<br />
In mammals, once the chromosomal sex XY or XX is set up at<br />
fertilization, the presence of SRY gene (Sex-<strong>de</strong>term<strong>in</strong><strong>in</strong>g Region of Y)<br />
will <strong>de</strong>term<strong>in</strong>e the fate of the gonad by <strong>in</strong>itiat<strong>in</strong>g testis differentiation.<br />
Follow<strong>in</strong>g SRY expression, SOX9 the key testis differentiat<strong>in</strong>g factor<br />
is up-regulated <strong>in</strong> the embryonic XY gonad thus trigger<strong>in</strong>g testis<br />
differentiation. In the female counterpart, key genes for ovarian<br />
differentiation have been isolated from genetics studies of female-tomale<br />
XX sex-reversal. Three loci fit <strong>in</strong> the class of early ovarian<br />
differentiat<strong>in</strong>g factors, the PIS locus (Polled Intersex Syndrome)<br />
discovered <strong>in</strong> goat, RSPO1 <strong>in</strong> human and Wnt4 <strong>in</strong> mouse.<br />
In goat, the pleiotropic PIS mutation leads to a phenotype without<br />
horn <strong>in</strong> both sexes (dom<strong>in</strong>ant) and to a female-to-male sex-reversal <strong>in</strong><br />
all XX <strong>in</strong>dividuals homozygous for the mutation (recessive). This<br />
mutation is a 11.7 kb <strong>de</strong>letion encompass<strong>in</strong>g any genes, but act<strong>in</strong>g on<br />
the transcriptional regulation of at least 3 genes, PISRT1, PFOXic and<br />
FOXL2, ly<strong>in</strong>g at 25 kb (PISRT1) and 300 kb (PFOXic/FOXL2) apart<br />
the <strong>de</strong>letion. When the PIS region is present (PIS +/+ and PIS +/-<br />
<strong>in</strong>dividuals), the 3 genes are highly expressed <strong>in</strong> the <strong>de</strong>velop<strong>in</strong>g ovary,<br />
from the onset of their differentiation. This expression is abolished <strong>in</strong><br />
the ovaries of XX fetuses homozygous for the <strong>de</strong>letion (PIS -/- ).<br />
Concomitantly, the expression of SOX9 <strong>in</strong>creased <strong>in</strong> these mutant<br />
gonads and the first histological sign of sex-reversal appeared. So, it<br />
seems that one of the PIS-regulated genes have an “anti-testis” effect.<br />
Among the three genes, only FOXL2 enco<strong>de</strong>s a conserved prote<strong>in</strong><br />
belong<strong>in</strong>g to the fork-head box family of transcription factors.<br />
In or<strong>de</strong>r to better un<strong>de</strong>rstand the role of these 3 genes, we have<br />
restored PISRT1 expression <strong>in</strong> XX PIS -/- gonad of transgenic goats. As<br />
no effect on the sex-reversal phenotype was observed, our current<br />
work<strong>in</strong>g hypothesis is that FOXL2 is the only gene of the PIS locus<br />
responsible for both pathological phenotypes. However, Foxl2<br />
<strong>in</strong>validation <strong>in</strong> mouse leads to an early block of follicle formation but<br />
not to sex-reversal. Consequently, we hypothesize that FOXL2/Foxl2<br />
could have additional roles <strong>in</strong> goat dur<strong>in</strong>g the early stages of ovarian<br />
differentiation, a period that is clearly different between goat and<br />
mouse. By contrast to mouse, goat fetal ovaries show an early spatial<br />
organization and produce estrogens un<strong>de</strong>r the control of FOXL2<br />
before germ cell meiosis. In or<strong>de</strong>r to <strong>de</strong>f<strong>in</strong>itely precise the role of<br />
FOXL2 <strong>in</strong> ovarian differentiation of non-ro<strong>de</strong>nt mammals, FOXL2<br />
<strong>in</strong>validation is currently un<strong>de</strong>r process <strong>in</strong> goat.<br />
P358<br />
I<strong>de</strong>ntification of genes expressed dur<strong>in</strong>g sheep ovarian<br />
<strong>de</strong>velopment<br />
Baillet, A*; Mandon-Pep<strong>in</strong>, B; Cabau, C; Poumerol, E; Pailhoux, E; Cot<strong>in</strong>ot, C<br />
INRA, UMR 1198; ENVA; CNRS, FRE 2857 Biologie du Développement et<br />
<strong>Reproduction</strong>, Jouy-en-Josas, F 78350, France<br />
In mammals, several genes <strong>in</strong>volved <strong>in</strong> ovarian <strong>de</strong>velopment have<br />
been i<strong>de</strong>ntified but the molecular casca<strong>de</strong> lead<strong>in</strong>g to a functional<br />
female gonad rema<strong>in</strong>s poorly documented. Although, <strong>in</strong> the last<br />
<strong>de</strong>ca<strong>de</strong>, progress has been ma<strong>de</strong> towards the i<strong>de</strong>ntification of genes<br />
controll<strong>in</strong>g ovarian differentiation us<strong>in</strong>g transcriptomic approaches<br />
and gene <strong>in</strong>activation, most of theses studies have been achieved <strong>in</strong><br />
mouse.<br />
The aim of this work is to i<strong>de</strong>ntify genes <strong>in</strong>volved <strong>in</strong> the two ma<strong>in</strong><br />
steps of ovarian <strong>de</strong>velopment <strong>in</strong> sheep, germ cells meiosis and follicle<br />
formation. In contrast to ro<strong>de</strong>nts both steps occur dur<strong>in</strong>g fetal period<br />
<strong>in</strong> several mammals (human, sheep, pig) promot<strong>in</strong>g sheep as a good<br />
mo<strong>de</strong>l to study human ovogenesis and early folliculogenesis.<br />
For this study, we used suppressive subtractive hybridization allow<strong>in</strong>g<br />
isolation of genes differentially expressed between two <strong>de</strong>velopmental<br />
stages. This technique was used <strong>in</strong> or<strong>de</strong>r to compare two ovarian<br />
<strong>de</strong>velopmental stages <strong>in</strong> sheep: 55dpc (onset of prophase I) and 82dpc<br />
(first follicle formation).<br />
Two subtractive libraries (55/82; 82/55) were constructed and 7296<br />
clones were recovered. Among them, 6080 clones were sequenced.<br />
Us<strong>in</strong>g SIGENAE bio<strong>in</strong>formatics facilities, clones shar<strong>in</strong>g sequence<br />
homology were grouped <strong>in</strong>to 2090 unique contigs. Then all contigs<br />
were blasted aga<strong>in</strong>st databases of different mammalian species<br />
(human, bov<strong>in</strong>e and ov<strong>in</strong>e) and annotad. In both libraries, 99% of<br />
contigs have an Unigene annotation and 1% were unknown.<br />
The comparison of the meiosis library contigs with another study of<br />
SSH concern<strong>in</strong>g mouse male germ cells revealed 30 genes <strong>in</strong> common<br />
with our library. We have <strong>in</strong>vestigated the possible <strong>in</strong>volvement of<br />
these 30 transcripts <strong>in</strong> female meiosis by RT-PCR. Two of them were<br />
never <strong>de</strong>scribed <strong>in</strong> female and presented a differentially expression<br />
pattern.<br />
Their expression profile was performed by RT q-PCR <strong>in</strong> female and<br />
male gonads dur<strong>in</strong>g fetal life <strong>in</strong> sheep. Both genes showed a high<br />
expression level dur<strong>in</strong>g female meiosis I while their expression<br />
rema<strong>in</strong>ed low <strong>in</strong> male and dur<strong>in</strong>g the other stages of ovarian<br />
<strong>de</strong>velopment.<br />
Further <strong>in</strong>vestigations such as chromosomal localisation <strong>in</strong> sheep,<br />
expression pattern <strong>in</strong> mouse gonads, cell type orig<strong>in</strong> are currently <strong>in</strong><br />
progress for these two genes. Moreover, <strong>in</strong>vestigations of the<br />
folliculogenesis library are <strong>in</strong> un<strong>de</strong>r progress. In parallel, these 2090<br />
genes will be spotted on a nylon membrane to constitute a macroarray<br />
<strong>de</strong>dicated to the ov<strong>in</strong>e ovarian differentiation, <strong>in</strong> or<strong>de</strong>r to evaluate<br />
sheep ovarian expression profiles <strong>in</strong> different physiological or<br />
physiopathological conditions.<br />
P359<br />
Changes <strong>in</strong> gene expression of mouse blastocysts<br />
treated with high hydrostatic pressure pulse<br />
Bock, I 1 *; Mamo, S 2 ; Polgar, Zs 3 ; Pribenszky, Cs 4<br />
1BioTalentum Ltd., Hungary; 2 Genetic Reprogramm<strong>in</strong>g Group, Agricultural<br />
Biotechnology Center, Hungary; 3 Faculty of Natural Sciences, Constant<strong>in</strong>e<br />
the Philosopher University, Slovakia; 4 Faculty of Veter<strong>in</strong>ary Science, St.<br />
István University, Hungary<br />
High hydrostatic pressure (HHP) treatment of mouse blastocysts<br />
before vitrification was reported to <strong>in</strong>crease their post-warm<strong>in</strong>g <strong>in</strong><br />
vitro <strong>de</strong>velopmental competence. Similarly, HHP treatment of<br />
gametes or embryos has proved to <strong>in</strong>crease substantially the efficacy<br />
of the subsequent processes such as vitrification of porc<strong>in</strong>e oocytes, <strong>in</strong><br />
vitro produced bov<strong>in</strong>e or cloned pig embryos, somatic cell nuclear<br />
transfer <strong>in</strong> pig, bull and boar semen cryopreservation. Proteomic<br />
studies have revealed changes <strong>in</strong> the prote<strong>in</strong> profiles of boar<br />
spermatozoa that may relate to <strong>in</strong>creased post-thaw survival and<br />
fertility. Here we report the first results of HHP <strong>in</strong>duced alterations <strong>in</strong><br />
the gene expression of mouse blastocysts.