MOLECULAR REPRODUCTION AND DEVELOPMENT 31:144-151 (1992)
Thyroid Stimulating Hormone Enhancement of Bovine Oocyte Maturation In Vitro ABDELMONEIM I. YOUNIS AND BENJAMIN G. BRACKETT Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia
Efforts to improve bovine embryABSTRACT onic development in vitro involved study of effects of thyroid stimulating hormone (TSH)alone or in combination with LH on bovine oocyte maturation (IVM). Putative effects were assessed by observing cumulus expansion (CE), fertilization (IVF),and development to morulae/blastocysts (M/B). Effects of prolactin (PRL) were also investigated. Variables for the 24-hr IVM interval were no hormone (control),TSH (0.1,0.5,or 1.0 pg/ml) or PRL (10, 100, or 1000 pg/ml), luteinizing hormone (LH) (0, 10, or 100 pg/ ml) + TSH (0.1 or 0.5 p.g/ml), and serum (20%,v/v) + 0.5 pg TSH/ml; data were from 4-5 trials for each IVM treatment. Higher proportions of oocytes exhibited complete CE with hormones or serum than without (P < 0.05).All oocytes (with and without CE) were inseminated with heparin-capacitated sperm. A higher proportion of inseminated oocytes cleaved after IVM with 0.5 pg TSH/ml (53.4%)than for other TSH treatments ( P < 0.05). The combination of TSH (0.1and 0.5 pg/ml) with 10 pg LH/ml for IVM enabled higher proportions (P < 0.05) of ova to fertilize (67.4 and 69.2%)than did medium alone (28.3%), LH (10 pg/ml) alone (54.1%), or serum + 0.5 pg TSH/ml (55.6%). No improvement in proportions undergoing fertilization was seen after addition of TSH to 100 pg LH/ml for IVM. Frequency of CE and cleavage did not differ among PRL treatments. More M/B developed from cleaved ova after IVM with LH or TSH than with PRL or no hormone (P < 0.05). Data document the beneficial influences of TSH and LH during IVM on the quality of the resulting ova. Key Words: TSH, Oocyte, Cumulus expansion, IVF, Embryos, Bovine
INTRODUCTION Cumulus cell-enclosed bovine oocytes isolated from antral follicles regardless of the donor stage of estrous cycle can be matured, fertilized, and cultured in vitro to stages competent for continued development following transfer into uteri of appropriately synchronized recipients (Lu et al., 1989; Xu et al., 1987; Sirard et al., 1988; Goto e t al., 1988; Brackett et al., 1989; Younis and Brackett, 1990). The addition of gonadotropins, luteinizing hormone (LH)and/or follicle stimulating hormone (FSH), during in vitro maturation has been demonstrated to improve the developmental potential for oocytes of the mouse (Jinno et al., 19891, rat (Shalgi et al., 1979; Vanderhyden and Armstrong, 19901, goat 0 1992 WILEY-LISS, INC.
(Younis et al., 1991), sheep (Moor and Trounson, 1977; Staigmiller and Moor, 19841, and cow (Brackett et al., 1989; Younis et al., 1989; Zuelke and Brackett, 1990). Enhanced bovine embryonic viability after in vitro fertilization (IVF) was demonstrated after in vitro maturation (IVM) of oocytes in M 199 supplemented with 20% (viv) proestrous phase (day 20) bovine serum and a high concentration (100 pgiml) of bovine LH (Brackett et al., 1989). Increased oocyte fertilizability and embryonic viability after IVF following IVM with added LH (50-100 pg/ml) (Zuelke and Brackett, 1990)or with LH, FSH, and estradiol-17P (5.0, 0.5, and 1.0 pgiml, respectively) (Saeki et al., 1991) have been demonstrated in serum-free conditions. Since the purified LH preparations employed were contaminated with TSH the working hypothesis that TSH might be (wholly or in part) responsible for the enhanced oocyte quality, reflected in IVF and developmental viability in vitro, prompted the present investigation. A highly purified bovine TSH has been demonstrated to have intrinsic gonadotropic activity a s evidenced by its ability to bind directly to LH and hCG receptors in rat testis and to both inhibit the binding of [lz5I1-hCG and to increase the concentration of CAMP (Amir et al., 1978), a s well as to cause expansion of cumulus cells surrounding mouse oocytes in vitro (De Silva and Pearl, 1990). Also, LH has been demonstrated to compete with TSH for receptor binding in thyroid plasma membrane (Talidouros et al., 1978; Carayon et al., 1980). Day 20 (proestrous) cow serum was found to be optimal among sera tested for IVM. It was characterized by relatively high concentrations of LH and PRL (Younis et al., 1989). Hence, the possibility of a positive influence of PRL on the quality of bovine IVM, as reflected by ability of oocytes to develop into embryos in vitro, was of interest. Cumulus cells tightly adherent to mammalian oocytes become dispersed within a hyaluronic acid containing matrix in response to the preovulatory surge of LH. That cumulus cells participate in the acquisition of competence by the oocyte for fertilization and embryo-
Received July 1, 1991; accepted September 25,1991. Address reprint requests to Dr. B.G. Brackett, Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602. A. I. Younis is now a t the New England Regional Primate Research Center, Harvard Medical School, Southboro, MA 01772.
TSH ENHANCES BOVINE IVM AND IVF genesis is well known (Staigmiller and Moor, 1984; Brackett et al., 1989; Vanderhyden and Armstrong, 1989; Younis et al., 1989; Buccione et al., 1990a). Mouse oocytes have been shown to participate in regulation of their cumulus cell expansion and hyaluronic acid synthesis (Buccione et al., 1990b; Salustri et al., 1990). Observations on cumulus expansion are of great interest in efforts to improve oocyte quality via IVM treatments. Preliminary data revealed a beneficial effect of TSH on bovine IVM (Younis and Brackett, 1991). Effects of various concentrations of TSH alone and in combination with different concentrations of LH during IVM on cumulus expansion (CE), in vitro fertilization (IVF), and in vitro development to morulae/blastocysts are reported here. Similarly, results of PRL on IVM as reflected by CE, IVF, and in vitro development are presented.
MATERIALS AND METHODS Oocyte Isolation and Maturation Cow ovaries obtained a t a n abattoir within 15 min postslaughter were placed in a thermos (25-27°C) containing normal saline with Penicillin G (2 x lo6 IU/ ml). Small antral follicles (2-5 mm in diameter) were aspirated within 1h r after slaughter using a n 18-gauge needle and 10-ml syringe. Follicular fluid aspirates were pooled into a sterile 50-ml flask (Falcon, Becton Dickinson Labware, Lincoln Park, NJ) and held a n additional hr during transit to the laboratory. In a 39°C environmental room contents of the flask were transferred into 2-3 sterile glass (15-ml) centrifuge tubes. Oocyte-cumulus complexes (OCC) were recovered from the settled follicular fluid within 15 min using a stereomicroscope ( x 16). Oocytes were washed 6 x in M 199 (Sigma Chemical Co., St. Louis, MO) modified by the addition of sodium pyruvate (50 pg/ml), sodium bicarbonate (2.6 mgiml), glucose (5.5 mgiml), and gentamicin sulfate (50 pgiml). Oocytes with normal complements of surrounding cumulus cells and homogeneousappearing ooplasm (Younis et al., 1989) were randomly assigned to 100-pl droplets of modified M 199 (10-201 drop) for IVM under sterile paraffin oil and a humidified atmosphere of 5% C02, 5% 0,, and 90% N, a t 39°C. Controls included IVM in modified M 199 alone, modified M 199 supplemented with 20% (v/v) day 20 serum, and 0.5 pg TSHlml, where appropriate. Endpoints for IVM (described more completely below) included proportions of oocytes exhibiting cumulus expansion (CE) after the IVM incubation, subsequent fertilization (IVF) assessed by cleavage data 48 h r after in vitro insemination, and proportions of cleaved ova that reached morula and blastocyst stages by 9 days after insemination. Experiment 1. In vitro maturation treatments of 0 , 0.1, 0.5, and 1.0 p,g TSH (NIADDK-bTSH-13)lml were compared to investigate dose reponsiveness in modified M 199. The bovine TSH (NIADDK-bTSH-13, AFP9074c) was characterized with biopotency of 30 IU/mg
145
in terms of the International Standard for bovine TSH by the McKenzie mouse TSH bioassay and by radioimmunoassay (RIA) determined contamination with prolactin (PRL), luteinizing hormone (LH), and growth hormone (GH) to the extent of 0.0005%, 0.7400% and 0.0020% by weight, respectively. Experiment 2. Treatments of oocytes for IVM with 0.1 and 0.5 pg TSH in combination with 0 , 10, or 100 pg LH/ml (USDA-bLH-B5)/ml were evaluated to assess possible synergistic effects. The purified bovine LH (USDA-bLH-B-5, AFP-550a) was characterized by biopotency of 2.1 S1 U/mg with residual biological contamination of
Thyroid Stimulating Hormone Enhancement of Bovine Oocyte Maturation In Vitro ABDELMONEIM I. YOUNIS AND BENJAMIN G. BRACKETT Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia
Efforts to improve bovine embryABSTRACT onic development in vitro involved study of effects of thyroid stimulating hormone (TSH)alone or in combination with LH on bovine oocyte maturation (IVM). Putative effects were assessed by observing cumulus expansion (CE), fertilization (IVF),and development to morulae/blastocysts (M/B). Effects of prolactin (PRL) were also investigated. Variables for the 24-hr IVM interval were no hormone (control),TSH (0.1,0.5,or 1.0 pg/ml) or PRL (10, 100, or 1000 pg/ml), luteinizing hormone (LH) (0, 10, or 100 pg/ ml) + TSH (0.1 or 0.5 p.g/ml), and serum (20%,v/v) + 0.5 pg TSH/ml; data were from 4-5 trials for each IVM treatment. Higher proportions of oocytes exhibited complete CE with hormones or serum than without (P < 0.05).All oocytes (with and without CE) were inseminated with heparin-capacitated sperm. A higher proportion of inseminated oocytes cleaved after IVM with 0.5 pg TSH/ml (53.4%)than for other TSH treatments ( P < 0.05). The combination of TSH (0.1and 0.5 pg/ml) with 10 pg LH/ml for IVM enabled higher proportions (P < 0.05) of ova to fertilize (67.4 and 69.2%)than did medium alone (28.3%), LH (10 pg/ml) alone (54.1%), or serum + 0.5 pg TSH/ml (55.6%). No improvement in proportions undergoing fertilization was seen after addition of TSH to 100 pg LH/ml for IVM. Frequency of CE and cleavage did not differ among PRL treatments. More M/B developed from cleaved ova after IVM with LH or TSH than with PRL or no hormone (P < 0.05). Data document the beneficial influences of TSH and LH during IVM on the quality of the resulting ova. Key Words: TSH, Oocyte, Cumulus expansion, IVF, Embryos, Bovine
INTRODUCTION Cumulus cell-enclosed bovine oocytes isolated from antral follicles regardless of the donor stage of estrous cycle can be matured, fertilized, and cultured in vitro to stages competent for continued development following transfer into uteri of appropriately synchronized recipients (Lu et al., 1989; Xu et al., 1987; Sirard et al., 1988; Goto e t al., 1988; Brackett et al., 1989; Younis and Brackett, 1990). The addition of gonadotropins, luteinizing hormone (LH)and/or follicle stimulating hormone (FSH), during in vitro maturation has been demonstrated to improve the developmental potential for oocytes of the mouse (Jinno et al., 19891, rat (Shalgi et al., 1979; Vanderhyden and Armstrong, 19901, goat 0 1992 WILEY-LISS, INC.
(Younis et al., 1991), sheep (Moor and Trounson, 1977; Staigmiller and Moor, 19841, and cow (Brackett et al., 1989; Younis et al., 1989; Zuelke and Brackett, 1990). Enhanced bovine embryonic viability after in vitro fertilization (IVF) was demonstrated after in vitro maturation (IVM) of oocytes in M 199 supplemented with 20% (viv) proestrous phase (day 20) bovine serum and a high concentration (100 pgiml) of bovine LH (Brackett et al., 1989). Increased oocyte fertilizability and embryonic viability after IVF following IVM with added LH (50-100 pg/ml) (Zuelke and Brackett, 1990)or with LH, FSH, and estradiol-17P (5.0, 0.5, and 1.0 pgiml, respectively) (Saeki et al., 1991) have been demonstrated in serum-free conditions. Since the purified LH preparations employed were contaminated with TSH the working hypothesis that TSH might be (wholly or in part) responsible for the enhanced oocyte quality, reflected in IVF and developmental viability in vitro, prompted the present investigation. A highly purified bovine TSH has been demonstrated to have intrinsic gonadotropic activity a s evidenced by its ability to bind directly to LH and hCG receptors in rat testis and to both inhibit the binding of [lz5I1-hCG and to increase the concentration of CAMP (Amir et al., 1978), a s well as to cause expansion of cumulus cells surrounding mouse oocytes in vitro (De Silva and Pearl, 1990). Also, LH has been demonstrated to compete with TSH for receptor binding in thyroid plasma membrane (Talidouros et al., 1978; Carayon et al., 1980). Day 20 (proestrous) cow serum was found to be optimal among sera tested for IVM. It was characterized by relatively high concentrations of LH and PRL (Younis et al., 1989). Hence, the possibility of a positive influence of PRL on the quality of bovine IVM, as reflected by ability of oocytes to develop into embryos in vitro, was of interest. Cumulus cells tightly adherent to mammalian oocytes become dispersed within a hyaluronic acid containing matrix in response to the preovulatory surge of LH. That cumulus cells participate in the acquisition of competence by the oocyte for fertilization and embryo-
Received July 1, 1991; accepted September 25,1991. Address reprint requests to Dr. B.G. Brackett, Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602. A. I. Younis is now a t the New England Regional Primate Research Center, Harvard Medical School, Southboro, MA 01772.
TSH ENHANCES BOVINE IVM AND IVF genesis is well known (Staigmiller and Moor, 1984; Brackett et al., 1989; Vanderhyden and Armstrong, 1989; Younis et al., 1989; Buccione et al., 1990a). Mouse oocytes have been shown to participate in regulation of their cumulus cell expansion and hyaluronic acid synthesis (Buccione et al., 1990b; Salustri et al., 1990). Observations on cumulus expansion are of great interest in efforts to improve oocyte quality via IVM treatments. Preliminary data revealed a beneficial effect of TSH on bovine IVM (Younis and Brackett, 1991). Effects of various concentrations of TSH alone and in combination with different concentrations of LH during IVM on cumulus expansion (CE), in vitro fertilization (IVF), and in vitro development to morulae/blastocysts are reported here. Similarly, results of PRL on IVM as reflected by CE, IVF, and in vitro development are presented.
MATERIALS AND METHODS Oocyte Isolation and Maturation Cow ovaries obtained a t a n abattoir within 15 min postslaughter were placed in a thermos (25-27°C) containing normal saline with Penicillin G (2 x lo6 IU/ ml). Small antral follicles (2-5 mm in diameter) were aspirated within 1h r after slaughter using a n 18-gauge needle and 10-ml syringe. Follicular fluid aspirates were pooled into a sterile 50-ml flask (Falcon, Becton Dickinson Labware, Lincoln Park, NJ) and held a n additional hr during transit to the laboratory. In a 39°C environmental room contents of the flask were transferred into 2-3 sterile glass (15-ml) centrifuge tubes. Oocyte-cumulus complexes (OCC) were recovered from the settled follicular fluid within 15 min using a stereomicroscope ( x 16). Oocytes were washed 6 x in M 199 (Sigma Chemical Co., St. Louis, MO) modified by the addition of sodium pyruvate (50 pg/ml), sodium bicarbonate (2.6 mgiml), glucose (5.5 mgiml), and gentamicin sulfate (50 pgiml). Oocytes with normal complements of surrounding cumulus cells and homogeneousappearing ooplasm (Younis et al., 1989) were randomly assigned to 100-pl droplets of modified M 199 (10-201 drop) for IVM under sterile paraffin oil and a humidified atmosphere of 5% C02, 5% 0,, and 90% N, a t 39°C. Controls included IVM in modified M 199 alone, modified M 199 supplemented with 20% (v/v) day 20 serum, and 0.5 pg TSHlml, where appropriate. Endpoints for IVM (described more completely below) included proportions of oocytes exhibiting cumulus expansion (CE) after the IVM incubation, subsequent fertilization (IVF) assessed by cleavage data 48 h r after in vitro insemination, and proportions of cleaved ova that reached morula and blastocyst stages by 9 days after insemination. Experiment 1. In vitro maturation treatments of 0 , 0.1, 0.5, and 1.0 p,g TSH (NIADDK-bTSH-13)lml were compared to investigate dose reponsiveness in modified M 199. The bovine TSH (NIADDK-bTSH-13, AFP9074c) was characterized with biopotency of 30 IU/mg
145
in terms of the International Standard for bovine TSH by the McKenzie mouse TSH bioassay and by radioimmunoassay (RIA) determined contamination with prolactin (PRL), luteinizing hormone (LH), and growth hormone (GH) to the extent of 0.0005%, 0.7400% and 0.0020% by weight, respectively. Experiment 2. Treatments of oocytes for IVM with 0.1 and 0.5 pg TSH in combination with 0 , 10, or 100 pg LH/ml (USDA-bLH-B5)/ml were evaluated to assess possible synergistic effects. The purified bovine LH (USDA-bLH-B-5, AFP-550a) was characterized by biopotency of 2.1 S1 U/mg with residual biological contamination of
