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Biology of Reproduction 60, 763-768 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Activities of Glucose Metabolic Enzymes in Human Preantral Follicles: In Vitro Modulation by Follicle-Stimulating Hormone, Luteinizing Hormone, Epidermal Growth Factor, Insulin-Like Growth Factor I, and Transforming Growth Factor ß11

Shyamal K. Roy2,a, and Dalores M. Terada3,a

a Leland J. and Dorothy H. Olson Center for Women's Health, Department of OB/Gyn, and Department of Physiology and Biophysics, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, Nebraska 68198-4515


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulation of glucose metabolic capacity of human preantral follicles in vitro by gonadotropins and intraovarian growth factors was evaluated by monitoring the activities of phosphofructokinase (PFK) and pyruvate kinase (PK), two regulatory enzymes of the glycolytic pathway, and malate dehydrogenase (MDH), a key mitochondrial enzyme of the Krebs cycle. Preantral follicles in classes 1 and 2 from premenopausal women were cultured separately in vitro in the absence or presence of FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF-I), or transforming growth factor ß1 (TGFß1) for 24 h. Mitochondrial fraction was separated from the cytosolic fraction, and both fractions were used for enzyme assays. FSH and LH significantly stimulated PFK and PK activities in class 1 and 2 follicles; however, a 170-fold increase in MDH activity was noted for class 2 follicles that were exposed to FSH. Although both EGF and TGFß1 stimulated glycolytic and Krebs cycle enzymes for class 1 preantral follicles, TGFß1 consistently stimulated the activities of both glycolytic enzymes more than that of EGF. IGF-I induced PK and MDH activities in class 1 follicles but negatively influenced PFK activity for class 1 follicles. In general, only gonadotropins consistently stimulated both glycolytic and Krebs cycle enzyme activities several-fold in class 2 follicles. These results suggest that gonadotropins and ovarian growth factors differentially influence follicular energy-producing capacity from glucose. Moreover, gonadotropins may either directly influence glucose metabolism in class 2 preantral follicles or do so indirectly through factors other than the well-known intraovarian growth factors. Because growth factors modulate granulosa cell mitosis and functionality, their role on energy production may be related to specific cellular activities.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Follicular development through preantral stages requires several cycles of granulosa cell division [1], which is an energy-dependent process. Under in vitro condition, glycolysis is the predominant source of ATP for mouse preantral follicles [2]. Because an oxygen concentration gradient exists in ovarian follicles [3], glucose-handling capacity may differ in developing follicles because the layers of granulosa cells increase as follicles grow. Increased follicular glycolytic activity has been reported in the rat treated with LH [4], and increased lactic acid production in vitro by rat eCG-treated follicles has been observed in response to FSH [5, 6]. Boland et al. [7] have shown that mouse preantral follicles grown in vitro produce more lactate when exposed to FSH and LH; however, pyruvate production does not change. Because follicular cell multiplication and differentiation are significantly influenced by intraovarian growth factors [8] and both of these cellular events require energy, modulation of follicular glucose metabolic enzymes by mitogenic and differentiation-inducing factors during preantral follicle development is likely. However, the effect of gonadotropins or intraovarian growth factors on human preantral follicular glucose metabolism is not known.

Previously we have shown that class 1 and 2 human preantral follicles grow in vitro in response to FSH and produce estradiol [9]. Moreover, epidermal growth factor (EGF) modulation of transforming growth factor ß (TGFß) receptor induction in human preantral follicles in vitro has been demonstrated [10]. The presence of EGF and TGFß in the human ovary has been documented [11]. Although the effect of EGF on ovarian glucose metabolism is unknown, EGF has been shown to transiently increase hepatic Krebs cycle and gluconeogenesis via a calcium-sensitive metabolic flux through mitochondrial 2-oxoglutarate dehydrogenase systems and inhibition of pyruvate kinase (PK) [12, 13]. On the other hand, EGF administration in vitro induces glycolysis and the pentose phosphate pathway, and it decreases gluconeogenesis in rat proximal tubular cells before they proliferate [14]. Moreover, EGF stimulates phosphofructokinase (PFK) activity in A431 cells [15] and in rat oocytes during their maturation in vitro [16]. These lines of evidence suggest that EGF action may differ depending on cell type and developmental condition. On the other hand, virtually nothing is known about the insulin-like growth factor-I (IGF-I) effect on follicular glucose metabolism during folliculogenesis. Nevertheless, the presence of IGF-I in mammalian ovaries, including those of the human, and its role in ovarian steroidogenesis is well documented [17–20]. Similarly, the presence of TGFß in the ovary and its influence on a variety of ovarian functions [8, 11, 21–26] are well known, but the effect of TGFß on follicular glucose metabolic enzymes, especially in the human, remains unclear.

The objective of the present studies was to determine whether gonadotropins, EGF, IGF-I, or TGFß1 influence the activities of some important glycolytic and Krebs cycle enzymes in human preantral follicles as a mechanism to regulate folliculogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian pieces were collected from women <= 35 yr of age who were undergoing surgery because of clinical conditions other than ovarian pathology. Human tissue collection was approved by the Institutional Review Board. Ovary samples were rinsed with Dulbecco's Modified Eagle's medium (DMEM) supplemented with 100 U/ml penicillin, 10 µg/ml streptomycin, 50 µg/ml amphotericin B, and 0.5% BSA and dissected free of any broken antral follicle and luteal remnant. Ovarian pieces from each woman were processed separately for follicle isolation, and class 1 and 2 follicles were dissociated and collected as described previously [9]. Class 1 follicles were <= 90 µm in diameter with 1–2 layers of granulosa cells, and class 2 follicles were <= 220 but > 90 µm in diameter with >= 3–4 layers of granulosa cells. Moreover, class 2 follicles were without an antral cavity and any definitive thecal layer. Previously, we have shown that these classes of human follicles can produce steroid hormones in response to FSH [9]. Preantral follicles from each ovary were rinsed twice with DMEM supplemented with ITS+ ([9]; insulin, 6.25 µg/ml; transferrin, 6.25 µg/ml; selenium, 6.25 µg/ml; BSA, 1.25 mg/ml; and linoleic acid, 5.35 µg/ml; Collaborative Research, Bedford, MA). Follicles from each ovary were cultured separately (to get n = 3) for 24 h in DMEM with ITS+ under 5% CO2 in air at 37°C in a Heraeus (Life Scientific, Inc., St. Louis, MO) incubator. Follicles were exposed to either 100 ng/ml ovine FSH (NIH-18; Bethesda, MD), 50 ng/ml ovine LH (NIH-25), 50 ng/ml recombinant murine IGF-I (Mallinckrodt, Phillipsburg, NJ), 50 ng/ml murine EGF (Mallinckrodt), or 10 ng/ml human TGFß1 (R&D Systems, Minneapolis, MN). Control culture received no treatment. Follicles were retrieved from the culture and rinsed with ice-cold homogenization buffer (10 mM Tris-HCl, pH 7.0, 0.25 M sucrose, 10% glycerol) supplemented with 1 mM PMSF and 10 µg/ml each of pepstatin, antipain, soybean trypsin inhibitor, and benzidine-HCl to minimize proteolysis; they were then homogenized in 35 µl homogenization buffer using a handheld micro glass-glass homogenizer. Protein content was determined in 1 µl of homogenate using a protein assay kit (ISS, Natick, MA). The rest of the homogenate was centrifuged at 26 000 x g for 30 min in an Eppendorf microfuge at 4°C to separate the mitochondrial fraction. The supernatant was used to determine the activities of PFK and PK, two key regulatory enzymes of glycolysis, and the pellet was resuspended in 25 µl of homogenization buffer to determine the activity of malate dehydrogenase (MDH), an important enzyme of the Krebs cycle. All chemicals for enzyme assay were from Sigma Chemical Company (St. Louis, MO) except those that were from Boehringer-Mannheim GmbH (Mannheim, Germany).

Determination of PFK Activity

PFK is one of the major regulatory enzymes of glycolysis and regulates the second important control point in glycolysis [27]. Therefore, measurement of its activity in cell-free follicular preparation will indicate the status of this control point in glycolysis at that time. Enzyme activity was assessed essentially as described by Ling et al. [28] with minor modification to fit 1-ml reaction volume. The reaction mixture contained (final concentrations) 33 mM Tris-HCl, pH 8.0, 2 mM ATP, 5 mM MgSO4, 2 mM fructose-6 phosphate (potassium salt), 0.16 mM NADH, 1 mM dithithreitol, 0.05 mM KCl, and 66.6 µl of an auxiliary enzyme solution (aldolase, triose phosphate isomerase, and glycerophosphate dehydrogenase). The enzyme reaction was conducted at 37°C in a temperature-controlled quartz cuvette, and the absorbance of NADH was recorded at 340 nM in a Milton Roy (Rochester, NY) spectrophotometer. After recording of the background rate of NADH oxidation for 5 min without samples, 10 µl of supernatant was added to the reaction mixture and mixed, and the rate of NADH oxidation was recorded at 1-min intervals for 5 min. The linearity of NADH oxidation throughout the recording duration indicated that the fall in absorbance was not due to substrate limitation. The enzyme activity was expressed as millimoles NADH oxidized per minute per milligram protein.

Determination of PK Activity

PK activity was determined essentially as described by Valentine and Tanaka [29] with modification to fit 1-ml reaction volume. The pyruvate kinase reaction is a secondary control point in glycolysis, controlling the transfer of the high-energy phosphate group from phosphoenolpyruvate to ADP. It is an allosteric enzyme, and under the intracellular condition, PK reaction is essentially irreversible [27]. Because intracellular ATP concentration negatively influences PK activity, increased ATP utilization due to heightened cellular activity will result in enhanced PK activity. Therefore, hormones and growth factors that stimulate cellular growth may stimulate PK activity for ATP supply. PK activity was determined in 1 ml of 50 mM triethanolamine buffer, pH 7.5, containing 2.25 M KCl, 0.24 M MgSO4, 6 µM ADP, 18 U/ml lactic dehydrogenase, 1.4 µmol NADH, and 5 µl of follicular supernatant at 340 nm at 37°C. After recording of the background rate of NADH oxidation for 5 min without substrate, 33.3 µl of a 45 mM phosphoenol pyruvate was added to the mixture and mixed immediately. The rate of NADH oxidation was recorded at 1-min intervals for 5 min. The rate of NADH oxidation was linear during the reaction period, and the enzyme activity was expressed as millimoles NADH oxidized per minute per milligram protein.

Determination of MDH Activity

MDH in mitochondrial matrix controls the availability of oxaloacetate, a critical factor for the progress of the Krebs cycle [30]. Therefore, factor(s) influencing the MDH activity can alter the rate of ATP generation in cells. Moreover, because the production of ATP by the Krebs cycle depends on the oxidative phosphorylation, increased MDH activity will indicate increased mitochondrial activity as well as the maturation of glucose metabolic pathways in cells. Enzyme activity was determined as described by Paria et al. [31] in the mitochondrial fraction from the rate of NADH oxidation at 340 nm. The reaction mixture consisted of 1 ml of 100 mM potassium phosphate buffer, pH 7.5, containing 50 mM oxaloacetate and 20 mM NADH. The rate of NADH oxidation following the addition of 10 µl of follicular pellet fraction was recorded as described in the previous section. MDH activity was expressed as millimoles NADH oxidized per minute per milligrams protein.

Statistical Analysis

There were enough samples from each group to run each enzyme assay in duplicate. An average of the two replicates represented the mean enzyme activity for each pooled sample (n = 1) for each ovary. All assays were done separately using homogenates of classes 1 and 2 preantral follicles obtained from 3 premenopausal women (n = 3), and values representing 3 ovaries were used to calculate the mean ± SEM. The results were analyzed by one-way ANOVA with Scheffe's test, and Student's t-test whenever necessary. The level of significance was at 5%.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The activities of glycolytic and Krebs cycle enzymes were evaluated to provide an understanding of the extent of glucose metabolic capacity of human preantral follicles and their regulation by gonadotropins and intraovarian growth factors. Under the basal condition, follicular PFK activity was low for both class 1 and class 2 follicles. However, the enzyme activity increased significantly in response to both FSH and LH compared to control (Fig. 1). Whereas LH was more potent than FSH in stimulating PFK activity for class 1 follicles, the opposite was true for class 2 follicles (Fig. 1). Nevertheless, the degree of stimulation by both gonadotropins was greater for class 2 follicles (FSH, 8-fold; LH, 6-fold) than for class 1 follicles (FSH, 2.1-fold; LH, 3-fold). EGF apparently did not influence the activity of PFK for either class (Fig. 1). Whereas IGF-I exerted a negative influence on PFK activity in class 1, a 2-fold stimulation (p < 0.05) was observed for class 2 follicles (Fig. 1). Likewise, TGFß1 significantly stimulated PFK activity in class 1 but had no effect on class 2 follicles. TGFß1 stimulation of PFK activity in class 1 follicles was comparable to the FSH and LH effect (Fig. 1).



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  		FIG. 1. PFK activity in human class 1 (A) and class 2 (B) preantral follicles. Follicles were incubated for 24 h in the presence or absence of gonadotropins or growth factors, and the enzyme activity was determined in the cytosol. Gonadotropins and TGFß1 significantly stimulated PFK activity for class 1, whereas the effect of gonadotropins on class 2 follicles was considerably greater than that of any growth factor. Note a reversal of the FSH and LH effects between class 1 and 2 follicles. Values with the same letter are significantly (p < 0.05) different from each other.

The basal PK activity was almost undetectable in follicles cultured for 24 h without any hormonal stimulus; however, significant (p < 0.05) stimulation was noted in response to FSH and LH (Fig. 2). As with the PFK activity, FSH stimulation of PK activity for class 2 follicles was more pronounced (55-fold) than for class 1 follicles (6-fold). Similarly, LH was more effective than FSH for class 1 in comparison to class 2 follicles (Fig. 2, A and B); however, the degree of LH stimulation for class 2 follicles was significantly (p < 0.5) greater than for class 1. Both EGF and IGF-I stimulated PK activity almost similarly for class 1 and class 2 follicles (Fig. 2, A and B). TGFß1, on the other hand, augmented the enzyme activity 19-fold for class 1 as compared to class 2 follicles (8-fold; Fig. 2).



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  		FIG. 2. PK activity in human preantral follicles in class 1 (A) and class 2 (B) follicles cultured in vitro for 24 h in the presence or absence of gonadotropins and growth factors. Both gonadotropins and growth factors stimulated enzyme activity for class 1 and 2 follicles. The degree of TGFß1 stimulation was greater for class 1 than for class 2 follicles; however, the TGFß1 effect on class 2 follicles was significant (p < 0.05). The reversal of FSH and LH effects between class 1 and 2 follicles was very prominent. Values with the same letter are significantly (p < 0.05) different from each other.

Gonadotropins also significantly (p < 0.05) stimulated MDH activity for both class 1 and 2 follicles (Fig. 3). Whereas FSH stimulated MDH activity 170-fold for class 2 follicles (16 fold for class 1), the degree of LH action was almost similar for the two classes of follicles. The degree of MDH stimulation by EGF and IGF-I for class 1 and class 2 follicles, respectively, was almost similar; however, a 13-fold stimulation of MDH activity by TGFß1 for class 2 follicles was observed (Fig. 3B). TGFß1 also augmented MDH activity by 3-fold for class 1 follicles (Fig. 3A). LH and TGFß1 showed almost identical degrees of stimulation of MDH activity for class 2 follicles. Interestingly, IGF-I was more effective than EGF in inducing MDH activity for class 1 follicles.



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  		FIG. 3. MDH activity in human preantral follicles in class 1 (A) and class 2 (B) follicles exposed in vitro to gonadotropins and growth factors for 24 h. While both gonadotropins and all tested intraovarian growth factors significantly (p < 0.05) stimulated MDH activity for class 1 follicles, only FSH stimulation was 170-fold for class 2 follicles as compared to other treatment groups. Nevertheless, significant (p < 0.05) stimulation of MDH activity for class 2 follicles by LH and TGFß1 is evident. Values with same letter are significantly (p < 0.05) different from each other.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study provide strong evidence that preantral human follicles utilize both the glycolytic and the Krebs cycle for energy production when exposed to gonadotropins; the findings also show for the first time that intraovarian growth factors, apart from their well-known mitogenic and steroidogenic (see [8] for references) effect, differentially influence glucose metabolic capacity of preantral follicles by modulating the activities of essential regulatory enzymes of the glycolytic and Krebs cycle. Overall, gonadotropins seem to positively influence the key regulatory enzymes of glycolysis and the Krebs cycle in both classes of preantral follicles; however, remarkable differences exist in the case of growth factors. Undoubtedly, cellular glycolytic and Krebs cycle activities in vivo depend on a variety of cellular parameters, such as substrate availability, oxygen gradient, and the activities of several other enzymes. However, direct measurement of the activities of regulatory enzymes in a cell-free system suggests that hormonal stimulus to intact follicles in culture significantly influences major regulatory enzymes of glucose metabolism per se.

We have previously demonstrated that FSH stimulates DNA synthesis and steroidogenesis [9] and growth factor receptor induction [10] in human preantral follicles. Boland et al. [7] have documented that the degree of lactate production in vitro by preantral mouse follicles with surrounding theca/stromal tissue increases steadily as follicles develop into the antral stage, and that FSH either alone or in combination with LH stimulates the rate of lactate production. This result corroborates the increased PFK activity observed in response to gonadotropins in the present studies; however, human preantral follicles also exhibit an increase in PK activity when exposed in vitro to FSH, suggesting that human preantral follicles are able to metabolize glucose to pyruvate under the proper hormonal milieu. Although we do not know the relative levels of pyruvate or lactate in human follicle culture medium, the pyruvate produced by the mouse follicle-stroma complex apparently gets converted to lactate, thus keeping the pyruvate level unchanged throughout culture [7]. In our protocol, human preantral follicles were free of surrounding stromal tissue, and follicles were cultured for 24 h during which no significant follicle growth occurs. Human preantral follicles in class 2 develop into antral follicles by 72–96 h in culture [9].

The exact reason(s) for the differences in FSH and LH action on the activities of PFK and PK versus MDH is not known; however, the results strongly indicate that some of the major regulatory steps of glycolysis and the Krebs cycle may be influenced differently by FSH and LH, and that the influence varies with the maturational status of the follicle. Nilsson [5] has demonstrated that LH significantly stimulates lactic acid production in eCG-treated rat preovulatory follicles in vitro, indicating an increase in glycolysis by gonadotropins; and this mode of energy production may be favored in vitro for preantral follicles as observed in the present studies. Moreover, follicular ability to metabolize pyruvate further via the Krebs cycle seems to depend, at least partly, on the type of hormonal stimulus and developmental status of the follicle. Whereas mouse preantral follicles with theca can grow to the antral stage under experimental anaerobic culture conditions [2], increased follicular MDH activity in the present studies clearly indicates that under normal physiological conditions, endocrine and paracrine factors stimulate the overall energy production cycle resulting in adequate ATP formation for follicle development. A mathematical model also indicates that energy production in more centrally located cells of large preantral follicles may depend on anaerobic glycolysis because of the existence of a rarefied oxygen gradient from the peripheral to central layers of granulosa cells [3]; however, it is important to consider that the availability of oxygen to the central core of preantral follicles with 3–4 layers of granulosa cells, such as class 2 human preantral follicles, may be greater than that to preantral follicles with 6–8 layers of granulosa cells. Therefore, energy production by the Krebs cycle due to gonadotropin and growth factor stimuli in culture is possible. Although the measurement of enzyme activity in a cell-free system under optimal and uniform reaction conditions indicates whether exogenous hormonal stimuli influence the enzyme activity per se, it is important to realize that under in vivo conditions, hormones and growth factors may also affect other factors that influence the flow of glycolysis and the Krebs cycle by acting at various levels. Further studies involving other glycolytic and Krebs cycle enzymes and intermediates may provide some information.

Although no report is available on the role of intraovarian growth factors on follicular glucose metabolism for any species, EGF has been studied for its role in glucose metabolism in the liver [12, 13, 16, 32] and renal tubular cells [14]. EGF stimulation of MDH activity for class 1 follicles may indicate a transient rise in mitochondrial glucose oxidation as observed in perfused rat liver preparation [13]. EGF stimulates mitochondrial gluconeogenetic enzymes in rat hepatocytes [32] and PFK mRNA expression in cultured rat fibroblasts via mitogen-activated protein kinase [33]. IGF-I induction of PK and MDH activities in class 1 follicles, as well as PFK and PK activities in class 2 follicles, suggests that intraovarian growth modulators involving the tyrosine kinase signal transduction system may affect follicular glucose metabolism in a similar fashion. Moreover, IGF-I may indeed augment glycolytic activity in multilayered preantral follicles, in which the existence of an oxygen gradient is likely. In cultured rat hepatocytes, IGF-I stimulates the formation of [14C]lactate from [14C]glucose up to 3-fold [34]. IGF-I induction of enzyme activities strongly indicates that insulin present in the culture medium does not significantly affect follicular IGF-I receptor activity.

The results of the present studies provide the first evidence of TGFß modulation of follicular glucose metabolism, especially in human preantral follicles. The results clearly indicate that TGFß modulates both glycolysis and Krebs cycle activities during human preantral follicle development. TGFß has been shown to stimulate glycolytic activity in rat kidney cells [35] and glucose uptake by cell monolayers [36]. The induction of key glycolytic enzymes in human preantral follicles by TGFß1 may reflect an additional level of regulation by this growth modulator to promote follicular differentiation. TGFß1 potentiates FSH action on hamster follicular cells [37, 38] and estrogen-induced DNA synthesis in rat granulosa cells [39]. FSH induces TGFß receptor type II in human preantral follicles in classes 1 and 2 [10] and in hamster preantral follicles [37], and TGFß2 in hamster preantral follicles [40]. Granulosa cells of human preovulatory follicles exposed in vivo to exogenously administered gonadotropins express TGFß receptor types I and II protein [41], and TGFß1 ligand has been detected in human follicular fluid [11]. Development of class 1 follicles to class 2 requires cell division as well as the onset of functionality, such as steroidogenesis [9]. The unique regulation of some of the key enzymes of glucose metabolism by TGFß in class 1 and class 2 follicles indicates that this growth factor differentially influences preantral follicles at different stages of development.

In summary, we provide here direct in vitro evidence to suggest that gonadotropins and growth factors significantly influence several regulatory enzymes of glucose metabolism in human preantral follicles. Considering the importance of these enzymes for the flow of the glycolytic and Krebs cycles, glycolysis appears to be favored at early preantral stages, but energy production by Krebs cycle activity may also be augmented.


   FOOTNOTES
 
1 This research was funded by grants HD 28165 from the National Institute of Child Health and Human Development, NIH, and Olson Foundation to S.K.R. Back

2 Correspondence. FAX: 402 559 6164; skroy{at}mail.unmc.edu Back

3 Current address: Dalores M. Terada, Department of Pharmacy, Creighton University Medical Center, Omaha, NE 68178. Back

Accepted: October 27, 1998.

Received: July 16, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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