- Email: [email protected]
The role of estradiol-17β in the development of zona pellucida-intact pig embryos
THERIOGENOLOGY
THE ROLE Of ESTRADIOL-17B IN THE DEVELOPMENT Of ZONA PELLUCIDAINTACT PIG EMBRYOS
Institut
H. Niemann, f. Elsaesser, D. Smidt fiir Tierzucht und Tierverhalten (FAL), 3057 Neustadt, F.R.G.
Mariensee,
A specific uptake of radiolabelled estradiol-17B in day-5 pig blastocysts has recently been demonstrated and the presence of specific binding sites for estrogens was suggested (Niemann and Elsaesser, Theriogenology 2l, 84-102, 1984). In order to elucidate the physiological significance of this finding, three experiments were conducted to study the effects of estradiol-17B withdrawal on the development of pig morula stages. Morphologically intact morulae (1040) were cultured in vitro in Krebs-Ringer-bicarbonate (KRB) supplemented with 10% heat inactivated lamb serum. Development to blastocysts was recorded after 24 or 48 hrs. Exp. 1: Morulae (218) were cultured in normal culture medium (controls), in KRB-solution supplemented with charcoal stripped lamb serum to remove steroids or in KRB-solution supplemented with charcoal stripped lamb serum and 1 nM estradiol-1713.However, no treatment differences could be determined, since 93.6 2 4.7%; 93.1 2 4.0%; 95.4 + 2.0% (x 2 SEM) respectively, developed to blastocysts. Exp. 2: Morulae (351) were incubated in standard culture medium (controls), in medium supplemented with either Nafoxidine (15 yg/ml) or with Nafoxidine and different concentrations of estradiol-17B, progesterone or cortisol. The antiestrogen Nafoxidine is known to compete with estradiol-17B for the same binding sites. Blastocyst formation was significantly (p < 0.001) reduced (13.3 + 5.8%) at a concentration of 15 pg/ml Nafoxidine when compared to controls (93.3 + 4.2%). The inhibitory effect of Nafoxidine could be overcome by the supplementation with 1 nM estradiol-17B sinceblastocyst formation rate was significantly (p < 0.001) increased to 57.2 f 8.9%. The stimulatory effects of estradiol-17B were specific since Nafoxidine reduced blastocyst formation rate remained low in the presence of progesterone (10.0 t 4.5%) or cortisol (3.3 + 3.3%). Exp. 3: Morulae (471) were incubated in culture medium supplemented with either 5% normal rabbit serum (controls), 5% estradiol-17B antiserum, or 5% estradiol-17D antiserum and 100 FM estradiol-17B. The antiserum had been produced in a rabbit and had been proven to be highly specific for estradiol-170. Blastocyst formation rate was significantly (p < 0.001) reduced in the presence of 5% estradiol-17B antiserum (51.9 + 6.7%) when compared to controls (93.1 + 2.2%). The percentage of blastocysts was significantly (p < 0.0051 increased after supplementation with 100 FM estradiol-170 (79.7 - 4.7%). Results from the present investigation indicate that estradiol-17B in vitro taken up from the medium and in vivo presumably from the uterine fluid is an essential factor for the transformation of the compacted morula to the cavitated blastocyst stage and most likely exerts its effect through specific binding sites for estrogens. The lack of any detectable morphological effect following incubation with charcoal stripped lamb serum indicates that only very small amounts of estrogens are necessary for undisturbed early embryonic development.
JANUARY 1986 VOL. 25 NO. 1
177
THE ROLE Of ESTRADIOL-17B IN THE DEVELOPMENT Of ZONA PELLUCIDAINTACT PIG EMBRYOS
Institut
H. Niemann, f. Elsaesser, D. Smidt fiir Tierzucht und Tierverhalten (FAL), 3057 Neustadt, F.R.G.
Mariensee,
A specific uptake of radiolabelled estradiol-17B in day-5 pig blastocysts has recently been demonstrated and the presence of specific binding sites for estrogens was suggested (Niemann and Elsaesser, Theriogenology 2l, 84-102, 1984). In order to elucidate the physiological significance of this finding, three experiments were conducted to study the effects of estradiol-17B withdrawal on the development of pig morula stages. Morphologically intact morulae (1040) were cultured in vitro in Krebs-Ringer-bicarbonate (KRB) supplemented with 10% heat inactivated lamb serum. Development to blastocysts was recorded after 24 or 48 hrs. Exp. 1: Morulae (218) were cultured in normal culture medium (controls), in KRB-solution supplemented with charcoal stripped lamb serum to remove steroids or in KRB-solution supplemented with charcoal stripped lamb serum and 1 nM estradiol-1713.However, no treatment differences could be determined, since 93.6 2 4.7%; 93.1 2 4.0%; 95.4 + 2.0% (x 2 SEM) respectively, developed to blastocysts. Exp. 2: Morulae (351) were incubated in standard culture medium (controls), in medium supplemented with either Nafoxidine (15 yg/ml) or with Nafoxidine and different concentrations of estradiol-17B, progesterone or cortisol. The antiestrogen Nafoxidine is known to compete with estradiol-17B for the same binding sites. Blastocyst formation was significantly (p < 0.001) reduced (13.3 + 5.8%) at a concentration of 15 pg/ml Nafoxidine when compared to controls (93.3 + 4.2%). The inhibitory effect of Nafoxidine could be overcome by the supplementation with 1 nM estradiol-17B sinceblastocyst formation rate was significantly (p < 0.001) increased to 57.2 f 8.9%. The stimulatory effects of estradiol-17B were specific since Nafoxidine reduced blastocyst formation rate remained low in the presence of progesterone (10.0 t 4.5%) or cortisol (3.3 + 3.3%). Exp. 3: Morulae (471) were incubated in culture medium supplemented with either 5% normal rabbit serum (controls), 5% estradiol-17B antiserum, or 5% estradiol-17D antiserum and 100 FM estradiol-17B. The antiserum had been produced in a rabbit and had been proven to be highly specific for estradiol-170. Blastocyst formation rate was significantly (p < 0.001) reduced in the presence of 5% estradiol-17B antiserum (51.9 + 6.7%) when compared to controls (93.1 + 2.2%). The percentage of blastocysts was significantly (p < 0.0051 increased after supplementation with 100 FM estradiol-170 (79.7 - 4.7%). Results from the present investigation indicate that estradiol-17B in vitro taken up from the medium and in vivo presumably from the uterine fluid is an essential factor for the transformation of the compacted morula to the cavitated blastocyst stage and most likely exerts its effect through specific binding sites for estrogens. The lack of any detectable morphological effect following incubation with charcoal stripped lamb serum indicates that only very small amounts of estrogens are necessary for undisturbed early embryonic development.
JANUARY 1986 VOL. 25 NO. 1
177