0013.72’27/92/1316-2690903.00/O Endocrinology Copyright 0 1992 by The Endocrine
Vol. 131. No. 6 Printed in U.S.A.
Society
Effects of Luteinizing in Cumulus-Enclosed KURT
A. ZUELKE
AND
BENJAMIN
Hormone on Glucose Metabolism Bovine Oocytes Matured in Vitro* G. BRACKETTT
Department of Physiology and Pharmacology, Athens, Georgia 30602-7389
University
of Georgia, College of Veterinary
Medicine,
ABSTRACT The effects of LH on glucose metabolism within cumulus cellenclosed bovine oocytes were determined. Cumulus cell-enclosed bovine oocytes were matured in vitro (IVM) in control medium alone or supplemented with LH, FSH, or TSH, then individually assayed for the metabolism of D-[5-3H]glucose, D-[l-‘4C]glucose, D-[6-“C]glucose, and D-[U-“C]glucose. Glycolytic activity was unchanged after IVM in 1 rg LH/ml, but was greater (P C 0.05) after culture in 10 and 50 rg LH/ml than the control value (1.34 + 0.13, 1.87 f 0.20, and 1.63 ~fr 0.14 us. 1.19 + 0.13 nmol ‘H20/pg protein.3 h, respectively). Increased glycolytic activity was observed within cumulus cell-enclosed oocytes, but not in cumulus cell complexes from which the oocyte was removed. Also, no glycolysis was detected when denuded oocytes from any IVM treatment were assayed. Glycolytic activity was greater (P < 0.01) after
IVM in LH (10 pg/ml) us. TSH (0.5 pg/ml), FSH (1.0 pg/ml), and control treatments (3.04 * 0.10, 2.44 + 0.10, 2.33 + 0.10, and 2.13 + 0.10 nmol 3H,0/pg protein. 3 h, respectively). Treatment with TSH also increased (P c 0.05) glycolytic activity compared to control values. Relative to control values afte; IVM, pentose cycle activity was 73.6% less, 2.9% higher, and 33.9% higher with LH, FSH, and TSH treatments, respectively. Total WO2 generated from D-[U-“C]glucose did not differ between treatments. Glucose oxidation by the pentose cycle accounted for 30.5%, 29.7%, 40.7%, and 11.1% of the total W02 production after IVM in control medium, FSH, TSH, or LH, respectively. Data indicate that IVM with LH results in increased glycolytic activity and mitochondrial glucose oxidation within cumulus cell-enclosed bovine oocytes, and that this may represent a mechanism by which LH enhances oocyte maturation. (Endocrinology 131: 26902696, 1992)
I
oocytes is through modifying the nutritional environment of the oocyte. That LH alters glucose metabolism within the bovine cumulus-oocyte complex was reported in preliminary form (9). The objective of the present study was to characterize the effects of LH on glucosemetabolism within bovine cumulus cell-enclosed oocytes matured in vitro.
MMATURE mammalian oocytes remain arrested in the meiotic prophase until puberty, at which time select oocytes resume meiosis and cytoplasmic maturation before ovulation. Theseimmature oocytes are surrounded by several layers of cumulus granulosa cells, which mediate hormonally induced changeswithin the cumulus-oocyte complex during oocyte maturation (1, 2). In vitro maturation (IVM) of bovine oocytes in the presence of bovine LH (bLH) resulted in increased embryonic development after in vitro fertilization (1, 3, 4). Oocytes denuded of surrounding cumulus cells did not respond to LH, thereby implicating the cumulus cells as the mediator of the LH effect (1, 3). An important role of the cumulus cells is to provide nutritional support to the developing oocyte (2,5, 6). In early work, oocyte maturation did not occur in glucose-containing medium unless cumulus cells or pyruvate were also present (5). Subsequent demonstration that cumulus cells synthesized pyruvate from glucose provided additional evidence that these cells were able to influence the nutritional environment of the maturing oocyte (7, 8). More recently, pyruvate was reported to directly affect nuclear maturation (i.e. meiotic resumption) in mouse oocytes (6). One mechanismby which LH may enhance IVM of bovine Received June 4, 1992. * This work was supported by the Program in Biological Resources and Biotechnology and the Veterinary Medical Experiment Station (92008) of the University of Georgia; the Atlantic Breeders Cooperative (Lancaster, PA); the USDA Animal Hormone Program and National Pituitary Program, NIDDK, University of Maryland School of Medicine (Baltimore, MD); and the Shapiro Packing Co. (Augusta, GA). t To whom all correspondence and requests for reprints should be addressed.
Materials
and Methods
Materials Phenol red-free TCM-199, BSA, Ca- and Mg-free Hanks’ Balanced Salt Solution, 2-[N-cyclohexylaminolethane sulfonic acid (CHES), 2,4dinitrophenol (DNP), HEPES, phenazine ethosulfate (PES), and polyvinylalcohol (mol wt, 30,000-70,000) were purchased from Sigma Chemical Co. (St. Louis, MO). Coomassie blue was purchased from BioRad Co. (Richmond, CA). n-[5-3H]Glucose (10.5 Ci/mmol), 3H20 (1 mCi/ ml), and Triton X-100 were acquired from Amersham (Arlington Heights, IL). D-[I-‘4C]Glucose (55.9 mCi/mmol), n-[6-‘4C]glucose (55.8 mCi/mmol), o-[U-‘4C]glucose (286.9 mCi/mmol), and NaH14C03 (8.4 mCi/mmol) were purchased from New England Nuclear-DuPont (Boston, MA). Cell culture plasticware and microtiter plates were purchased from Beckton Dickinson Co. (Lincoln Park, NJ). Scintillation fluid (Scintiverse BD) and paraffin oil were purchased from Fisher Chemical Co. (Norcross, GA). Bovine LH (USDA bLH B-5; Cl % FSH and between 24% TSH activity), porcine FSH (USDA pFSH B-l; Cl% LH and TSH activity), and bovine TSH (USDA bTSH l-l;
Vol. 131. No. 6 Printed in U.S.A.
Society
Effects of Luteinizing in Cumulus-Enclosed KURT
A. ZUELKE
AND
BENJAMIN
Hormone on Glucose Metabolism Bovine Oocytes Matured in Vitro* G. BRACKETTT
Department of Physiology and Pharmacology, Athens, Georgia 30602-7389
University
of Georgia, College of Veterinary
Medicine,
ABSTRACT The effects of LH on glucose metabolism within cumulus cellenclosed bovine oocytes were determined. Cumulus cell-enclosed bovine oocytes were matured in vitro (IVM) in control medium alone or supplemented with LH, FSH, or TSH, then individually assayed for the metabolism of D-[5-3H]glucose, D-[l-‘4C]glucose, D-[6-“C]glucose, and D-[U-“C]glucose. Glycolytic activity was unchanged after IVM in 1 rg LH/ml, but was greater (P C 0.05) after culture in 10 and 50 rg LH/ml than the control value (1.34 + 0.13, 1.87 f 0.20, and 1.63 ~fr 0.14 us. 1.19 + 0.13 nmol ‘H20/pg protein.3 h, respectively). Increased glycolytic activity was observed within cumulus cell-enclosed oocytes, but not in cumulus cell complexes from which the oocyte was removed. Also, no glycolysis was detected when denuded oocytes from any IVM treatment were assayed. Glycolytic activity was greater (P < 0.01) after
IVM in LH (10 pg/ml) us. TSH (0.5 pg/ml), FSH (1.0 pg/ml), and control treatments (3.04 * 0.10, 2.44 + 0.10, 2.33 + 0.10, and 2.13 + 0.10 nmol 3H,0/pg protein. 3 h, respectively). Treatment with TSH also increased (P c 0.05) glycolytic activity compared to control values. Relative to control values afte; IVM, pentose cycle activity was 73.6% less, 2.9% higher, and 33.9% higher with LH, FSH, and TSH treatments, respectively. Total WO2 generated from D-[U-“C]glucose did not differ between treatments. Glucose oxidation by the pentose cycle accounted for 30.5%, 29.7%, 40.7%, and 11.1% of the total W02 production after IVM in control medium, FSH, TSH, or LH, respectively. Data indicate that IVM with LH results in increased glycolytic activity and mitochondrial glucose oxidation within cumulus cell-enclosed bovine oocytes, and that this may represent a mechanism by which LH enhances oocyte maturation. (Endocrinology 131: 26902696, 1992)
I
oocytes is through modifying the nutritional environment of the oocyte. That LH alters glucose metabolism within the bovine cumulus-oocyte complex was reported in preliminary form (9). The objective of the present study was to characterize the effects of LH on glucosemetabolism within bovine cumulus cell-enclosed oocytes matured in vitro.
MMATURE mammalian oocytes remain arrested in the meiotic prophase until puberty, at which time select oocytes resume meiosis and cytoplasmic maturation before ovulation. Theseimmature oocytes are surrounded by several layers of cumulus granulosa cells, which mediate hormonally induced changeswithin the cumulus-oocyte complex during oocyte maturation (1, 2). In vitro maturation (IVM) of bovine oocytes in the presence of bovine LH (bLH) resulted in increased embryonic development after in vitro fertilization (1, 3, 4). Oocytes denuded of surrounding cumulus cells did not respond to LH, thereby implicating the cumulus cells as the mediator of the LH effect (1, 3). An important role of the cumulus cells is to provide nutritional support to the developing oocyte (2,5, 6). In early work, oocyte maturation did not occur in glucose-containing medium unless cumulus cells or pyruvate were also present (5). Subsequent demonstration that cumulus cells synthesized pyruvate from glucose provided additional evidence that these cells were able to influence the nutritional environment of the maturing oocyte (7, 8). More recently, pyruvate was reported to directly affect nuclear maturation (i.e. meiotic resumption) in mouse oocytes (6). One mechanismby which LH may enhance IVM of bovine Received June 4, 1992. * This work was supported by the Program in Biological Resources and Biotechnology and the Veterinary Medical Experiment Station (92008) of the University of Georgia; the Atlantic Breeders Cooperative (Lancaster, PA); the USDA Animal Hormone Program and National Pituitary Program, NIDDK, University of Maryland School of Medicine (Baltimore, MD); and the Shapiro Packing Co. (Augusta, GA). t To whom all correspondence and requests for reprints should be addressed.
Materials
and Methods
Materials Phenol red-free TCM-199, BSA, Ca- and Mg-free Hanks’ Balanced Salt Solution, 2-[N-cyclohexylaminolethane sulfonic acid (CHES), 2,4dinitrophenol (DNP), HEPES, phenazine ethosulfate (PES), and polyvinylalcohol (mol wt, 30,000-70,000) were purchased from Sigma Chemical Co. (St. Louis, MO). Coomassie blue was purchased from BioRad Co. (Richmond, CA). n-[5-3H]Glucose (10.5 Ci/mmol), 3H20 (1 mCi/ ml), and Triton X-100 were acquired from Amersham (Arlington Heights, IL). D-[I-‘4C]Glucose (55.9 mCi/mmol), n-[6-‘4C]glucose (55.8 mCi/mmol), o-[U-‘4C]glucose (286.9 mCi/mmol), and NaH14C03 (8.4 mCi/mmol) were purchased from New England Nuclear-DuPont (Boston, MA). Cell culture plasticware and microtiter plates were purchased from Beckton Dickinson Co. (Lincoln Park, NJ). Scintillation fluid (Scintiverse BD) and paraffin oil were purchased from Fisher Chemical Co. (Norcross, GA). Bovine LH (USDA bLH B-5; Cl % FSH and between 24% TSH activity), porcine FSH (USDA pFSH B-l; Cl% LH and TSH activity), and bovine TSH (USDA bTSH l-l;
