Data di Pubblicazione:
2009
Citazione:
In vitro meiosis resumption of feline oocytes isolated from vitrified ovarian tissue / M. Apparício, E. Ruggeri, G.C. Luvoni. ((Intervento presentato al 6. convegno Annual Symposiun European Veterinary Society for Small Animal Reproduction tenutosi a Wroclaw nel 2009.
Abstract:
Introduction and aim. In the preovulatory follicle, canine oocytes are exposed to high
concentrations of progesterone (P4) and estradiol (E2), and different concentrations of
gonadotrophins are present in the pre- and post-ovulatory environment, i.e. oviduct, where full
nuclear maturation occurs (1,2,3). Thus, two microenvironments characterized by the presence of
different hormonal concentrations contribute to the achievement of Metaphase II (MII) stage. In
vitro, maturation conditions should be based on these different pre- and post-ovulatory
environments and on the fact that cultural requirements for oocyte maturation may vary along the
time. Hence, the aim of this work was to study the influence of different bi-phasic systems with
gonadotrophins and steroids on in vitro maturation rates of canine oocytes.
Materials and Methods. Ovaries were collected from 14 anestrous bitches (various breeds, age 1-
7 y) by routine ovariohysterectomy and sliced in PBS-PVA to release the cumulus-oocyte
complexes (COCs). A total of 363 COCs grade I (two or more dense layers of cumulus cells and
darkly pigmented cytoplasm) were selected for culture. Oocytes were randomly allocated in three
different cultural systems with the base medium (BM) consisting of TCM-199 (Sigma Chemical
Co., USA), with antibiotics, 10% FBS, 2.2 mg/ml sodium bicarbonate and 20 μl/ml pyruvic acid.
The systems were as follows: in system A (control) oocytes (n. 91) were matured for 72h in BM
with 10 i.u./ml hCG (Sigma), 1 μg/ml P4 (Sigma), 1 μg/ml E2 (Sigma); in bi-phasic system B
oocytes (n. 120) were matured for 48h in BM with hCG and for 24h in BM with P4; in bi-phasic
system C oocytes (n. 124) were matured for 48h in BM with hCG, P4 and E2, and for 24h in BM
with P4. In systems B and C hormones were supplemented at the same doses as in system A.
Cumulus-oocyte complexes were incubated at 38°C, 5% CO2 in air and, at the end of maturation
(72h), were transferred to PBS containing 0.2% hyaluronidase (Sigma) for removal of cumulus cells
by repeated pipetting. Oocytes were stained with Hoechst 33342 (Sigma) for evaluation of meiotic
configuration. Data were analyzed by Chi-square test.
Results. Anaphase/Metaphase I rates were higher (P<0.02) in oocytes cultured in bi-phasic system
B (27.5%; 33/120) and C (29%; 36/124) when compared to control (13.2%; 12/91). Only three
oocytes from control group reached MII stage (3.3%), while higher rates (P<0.02) of oocytes
cultured in system B (11.7%; 14/120) and C (13.7%; 17/124) were able to complete meiosis.
Degeneration rate was remarkably lower (P<0.05) in C system (4%; 5/124) compared to B (12.5%;
15/120) and control (19.8%; 18/91).
Conclusions. These results suggest that the use of bi-phasic systems is beneficial for oocyte meiosis
in vitro, when compared to the continuous exposition (72h) to the same hormonal association
(control). This is the first time that a bi-phasic system with gonadotrophins and steroids had been
studied. As no differences were observed between the two associations of hormones (system B and
C), further investigation on the effect of other hormonal combinations and concentrations are
needed.
References.
1) De los Reyes et al., Theriogenology 2005;64:1-11. 2) Willingham-Rocky et al., Reproduction
2003;126:501-8. 3) Luvoni et al., Theriogenology 2005;63:41-59.
Tipologia IRIS:
14 - Intervento a convegno non pubblicato
Keywords:
cat; ovarian tissue; vitrification
Elenco autori:
M. Apparício, E. Ruggeri, G.C. Luvoni
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