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BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.010264
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BIOLOGY OF REPRODUCTION 68, 1584–1589 (2003)
DOI: 10.1095/biolreprod.102.010264
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Growth Hormone-Related Effects on Apoptosis, Mitosis, and Expression of Connexin 43 in Bovine In Vitro Maturation Cumulus-Oocyte Complexes1

Sabine Kölle2,3, Miodrag Stojkovic4, Gudrun Boie3, Eckhard Wolf4, and Fred Sinowatz3

Department of Veterinary Anatomy II,3 University of Munich, 80539 Munich, Germany Department of Molecular Animal Breeding and Biotechnology,4 University of Munich, 81377 Munich, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary LH and FSH are known to be the major regulators of ovarian function. In the last few years, however, there has been evidence that growth hormone (GH) is also involved in ovarian regulation. Therefore, the aim of our study was to elucidate the mechanisms of GH action during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). As shown by detection of the nuclear cell proliferation-associated antigen Ki-67, COCs matured in vitro in the presence of GH revealed a significantly (P < 0.05) higher proportion of proliferating cumulus cells (12.6%) compared with the COCs matured in the control medium TCM 199 (9.9%). In contrast, the percentage of proliferating cells was not increased by supplementation of the medium with a combination of GH and insulin-like-growth factor I (IGF-I). Apoptosis as determined by TUNEL (terminal doxynucleotidyl transferase mediated dUTP nick-end labeling) was significantly (P < 0.05) reduced in the cumulus cells by GH treatment. COCs matured with a combination of GH and IGF-I revealed the lowest percentage of apoptotic cells (11%). The localization and quantification of the gap junction protein connexin 43 (Cx 43) demonstrated that GH induced a significant decrease in the synthesis of the Cx 43 protein in the cumulus cells. Our results imply that GH increases cumulus expansion by promotion of cell proliferation and inhibition of apoptosis. Whereas the increase in cell proliferation is a direct effect of GH, the antiapoptotic effects of GH during in vitro maturation are modulated by IGF-I. Stimulatory effects of GH on oocyte maturation are correlated with changes in the synthesis of gap junction proteins.

apoptosis, assisted reproductive technology, gamete biology, growth hormone, mechanisms of hormone action


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary LH and FSH are known to be the major regulators of ovarian function. In the last few years, however, there has been evidence that growth hormone (GH) is also involved in ovarian regulation. GH-deficient humans and rats reveal a delay in the onset of puberty, which can be induced by GH treatment [1, 2]. In hypophysectomized ewes, growth and ovulation of follicles are not induced by exogenous gonadotropins unless GH is additionally given [3]. Application of exogenous GH results in an increase of folliculogenesis in cows [4, 5]. Similarly, transgenic mice overexpressing GH show higher ovulation rates compared with control animals [6]. Additionally, numerous in vitro experiments have pointed to the importance of growth hormone in ovarian physiology. Thus, GH enhances follicle-stimulating hormone-induced differentiation of cultured granulosa cells [7, 8]. Perfusion of ovaries with GH results in increased follicular growth, in stimulation of oocyte maturation, and in enhancement of estradiol synthesis [9]. During in vitro culture of granulosa cells, application of GH also increases synthesis of steroid hormones [10, 11]. A distinct stimulatory effect of GH on oocyte maturation has been reported in rats [12], rabbits [9], and pigs [13]. In isolated cumulus-oocyte complexes (COCs), GH accelerates nuclear maturation, induces cumulus expansion, and enhances fertilizability and developmental competence of oocytes matured in vitro [14–16].

The actions of GH are mediated by the growth hormone receptor (GHR). The mRNA encoding GHR has been found both in the cumulus cells and the oocytes of COCs obtained from 2–8-mm-sized follicles [17]. The GHR protein itself is mainly localized to the cumulus cells [18]. The COC, however, is not only a site of GH reception and GH action but also a site of GH production. Thus, both the oocyte and the cumulus cells show GH immunoreactivity, whereas expression of GH mRNA is mainly localized to the oocyte [19].

The mechanisms of GH action in the ovary are largely unknown. GH may either exert direct effects or act indirectly via insulin-like-growth factors (IGFs). In rats, the effect of GH on COCs has been reported to be mediated by IGF-I [12]. In bovine, however, stimulation of COCs with GH is not affected by an anti-IGF-I antibody [17].

Therefore, the purpose of our study was to analyze the mechanisms of GH action during in vitro maturation of bovine COCs. Because GH has been shown to significantly increase cumulus expansion [14], the effects of GH on cell proliferation and apoptosis of cumulus cells were investigated. Because recent experiments have demonstrated that GH affects metabolism in preimplantation embryos [20] and modulates expression of connexin 43 (Cx 43) in neural tissue [21], the analysis of the gap junction protein Cx 43 was included in our studies. Thus, COCs matured with or without supplementation of GH were examined by Ki-67 labeling, by terminal deoxynucleotidyl transferase-mediated dUTP labeling (TUNEL), and by immunohistochemical detection of the gap junction protein Cx 43.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Cumulus-Oocyte Complexes

Ovaries of cows (Deutsches Fleckvieh breed) collected from a local slaughterhouse were transported to the laboratory in PBS at 30°C. COCs were aspirated from 2- to 8-mm follicles using a micro/macrosuction apparatus (Labotect, Göttingen, Germany) and a 20-gauge needle. Under microscopic control, only oocytes with a multilayered compact cumulus oophorus and a dark, evenly granulated cytoplasm were selected for further maturation.

In Vitro Maturation

Maturation was performed in tissue culture medium 199 (TCM 199; Seromed, Berlin, Germany) supplemented with 2 mM sodium pyruvate, 2.92 mM calcium lactate, 0.01 units of bovine FSH (Sioux Biochemicals, Sioux Center, IA), 0.01 units of bovine LH (Sioux Biochemicals), and 60 µg/ml gentamicin (Sigma, St. Louis, MO). For analyzing the effects of GH and IGF-I on in vitro maturation, five different in vitro maturation treatments were used: a) modified TCM199, b) modified TCM 199 + 100 ng/ml medium recombinant bovine GH (Elanco, Greenfield, IN), c) modified TCM 199 + 100 ng/ml medium IGF-I (Boehringer, Mannheim, Germany), d) modified TCM199 + 100 ng/ml medium GH and 100 ng/ml medium IGF-I, and e) modified TCM 199 + 10% (v/v) estrous cow serum (ECS), which is routinely used in standard in vitro maturation of oocytes. Maturation was performed in a humidified atmosphere of 5% CO2 in air at 39°C for 24 h.

Preparation of COCs

After 24 h of culture, the COCs were removed from the maturation medium, washed in PBS, and fixed in Bouin solution for 20 min. For detection of cell proliferation and expression of connexin 43, specimens were dehydrated in an ascending series of ethanols (50%–100%), treated with xylene, and embedded in paraffin. Serial sections (5 µm) were cut and transferred to 3-aminopropylene-ethoxysilane-coated slides. For the TUNEL studies, the COCs were fixed in Bouin solution and stored in 70% ethanol at 4°C until use.

In each experiment, five groups of COCs were investigated: a) COCs matured in the control medium TCM 199, b) COCs matured with GH (100 ng/ml), c) COCs matured with IGF-I (100 ng/ml), d) COCs matured with GH and IGF-I (each 100 ng/ml), e) COCs matured in the routinely used medium TCM 199/10% ECS. All experiments were carried out three times. In each experiment, sets of 20 COCs were investigated.

Cell Proliferation

After deparaffinization, the slides were hydrated in a descending series of ethanols (100%–70%), washed in distilled water, and put in heated Target Retrieval Solution (DAKO, Hamburg, Germany) for 20 min. After washing in Tris buffer, specimens were incubated with the monoclonal antibody MIB1 (Dianova, Hamburg, Germany; dilution 1:50 in Tris buffer, 1 h), which detects the nuclear cell proliferation-associated antigen Ki-67. As secondary antibody, rabbit-anti-mouse IgG (DAKO) was used in a dilution of 1:25 for 30 min. The antigen Ki-67 was detected using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex (DAKO) for 30 min in a dilution of 1:50. Incubations of the specimens with the secondary antibody and the APAAP complex were repeated once. Between all steps, the COCs were washed in Tris buffer. Visualization of the antigen Ki-67 was performed with Sigma Fast Red (Sigma, Deisenhofen, Germany) according to the manufacturer's instructions. Specimens were mounted with glycerol gelatin (Merck, Ismaning, Germany). Cell counting was performed by using a Leitz Orthoplan microscope.

Negative controls were performed by omitting MIB1 and omitting the secondary antibody. Sections of gut tissues revealing a high percentage of proliferating cells were used as positive controls.

Apoptosis (TUNEL)

To determine late-stage apoptosis in the COCs, the Apop Tag Fluorescein Kit (Intergen, Oxford, UK) was used. The manufacturer's instructions for tissues had to be modified for the COCs as follows: The COCs were removed from ethanol (70%), washed in PBS, and subsequently treated with equilibration buffer for 5 min. Then the COCs were incubated with the TdT enzyme (dilution 3:7 in reaction buffer) for 1 h at 37°C. The reaction was terminated by treatment with stop solution. Following washing three times in PBS, the COCs were incubated with a FITC-labeled antidigoxigenin antibody at room temperature for 30 min. In order to determine the total cell number, cell nuclei were stained with DAPI (diamidinophenylindole dihydrochloride hydrate; Sigma-Aldrich, Deisenhofen, Germany), which was added to the antibody solution in a concentration of 1 µg/ml. Specimens were mounted with 40% polyvinylalcohol in ethylene glycol (dilution 1.4 in 0.1 M Tris, pH 8.5). The COCs were examined by using an LSM-410 confocal laser scanning microscope (Zeiss, Oberkochen, Germany) equipped with a Capo-Zeiss 40 x 1.2 water-immersion objective (Zeiss, Jena, Germany). The fluorescence was observed by using filters of 454 nm (DAPI) and 520 nm (TUNEL). Photomicrographs were used for counting apoptotic and nonapoptotic cumulus cells.

Positive controls were treated with RQ1 DNase (50 U/ml; Promega, Southampton, UK) for 20 min at 37°C. Negative controls were incubated with the FITC-labeled anti-digoxigenin antibody in the absence of TdT.

Expression of Connexin 43

Sections were deparaffinized in xylene and rehydrated in a descending series of ethanols. Endogenous peroxidase was eliminated by incubation in 0.5% H2O2 in PBS for 30 min, following which specimens were put in water for 10 min and treated with 0.1% Triton X in PBS for 15 min. After washing with PBS, the slides were incubated in Protein Block Serum (DAKO) for 10 min. Then the COCs were incubated with the monoclonal Cx 43 antibody (Dianova) in a dilution of 1:100 overnight at 4°C. The secondary antibody was biotinylated rabbit-anti-mouse IgG (DAKO; dilution 1:300). After incubation of the slides in avidin-streptavidin-biotin horseradish peroxidase complex (DAKO) for 1 h at 20°C (dilution 1:150 in PBS-1% BSA), Cx 43 was visualized by 0.05% diaminobenzidine in PBS containing 1% H2O2. The percentage of gap junctions was defined as the number of stained Cx 43 dots in relation to the total cell number. Total cell counting and counting of Cx 43 dots were carried out using a Leitz Aristoplan microscope.

Controls were performed by a) omission of the primary antibody and b) omission of the secondary antibody.

Statistical Analysis

The percentages of proliferating cells in COCs matured in TCM 199 with growth hormone and growth factors were analyzed by the Student t-test with the Levene test for equality of variances. Percentages of apoptotic cumulus cells and of Cx 43 dots were compared by using the ANOVA followed by least significant differences (LSD). Data are presented as the mean ± SEM. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Proliferation

The percentage of proliferating cells was significantly (P < 0.05) higher in COCs matured in the presence of GH (12.6%) compared with the COCs matured in the control media TCM 199 and TCM 199/ECS (9.9% and 9.0%, respectively) (Figs. 1 and 2). As shown in Figure 2, the percentage of Ki-67-positive cells as well as the cumulus expansion were increased by supplementation of GH compared with the control medium TCM 199. The supplementation of the maturation medium with a combination of GH and IGF-I or IGF-I alone did not significantly alter the proliferation rate compared with the control groups (Fig. 1). When the maturation medium was supplemented with GH and IGF-I, the average proportion of proliferating cells was similar (8.9%) to supplementation with IGF-I alone (8.7%) (Fig. 1). In the negative controls (testing the specificity of the used antibodies), no signal was visible.



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  		FIG. 1. Percentage of proliferating cumulus cells of COCS after in vitro maturation with different supplements. COCs matured in modified TCM 199 were used as control groups. Additionally, COCs matured in the routinely used standard medium TCM 199 supplemented with ECS (estrous cow serum) were included in the study. Data were analyzed by using the Student t-test with Levene test for equality of variances. Significant differences between different groups are indicated by different superscripts (P < 0.05)



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  		FIG. 2. Detection of cell proliferation by Ki-67 labeling of COCs after in vitro maturation with different supplements. a) COC matured in the control medium TCM 199 (original magnification, x150). b) COC matured in TCM 199 supplemented with GH (original magnification, x150). After treatment with GH, the cell proliferation of the cumulus cells is significantly increased

Apoptosis

Apoptosis was significantly (P < 0.05) reduced in all COCs that had been matured with GH, IGF-I, or a combination of GH and IGF-I compared with the control COCs matured in TCM 199 (Fig. 3). Treatment with GH and IGF-I resulted in the lowest percentage of apoptotic cumulus cells (11%). The highest incidence of apoptosis (36.3%) was found in COCs matured with the routinely used standard medium TCM 199/ECS (Figs. 3 and 4). Compared with this medium, the supplementation of GH regularly resulted in a significant and very distinct decrease of apoptosis in the cumulus cells of the COCs (Fig. 4). The negative controls did not reveal any signal, whereas the positive controls treated with RQ1 DNase regularly showed strong staining.



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  		FIG. 3. Percentage of apoptotic cumulus cells of COCs after in vitro maturation with different supplements. Data were analyzed using ANOVA followed by LSD. Significant differences between different groups are indicated by different superscripts (P < 0.05)



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  		FIG. 4. Detection of apoptosis in COCs by TUNEL after in vitro maturation with different supplements. a) COC matured in the routinely used medium TCM 199/10% ECS (original magnification, x160). b) COC matured in TCM 199 supplemented with GH (original magnification, x160). Treatment with GH results in a significant decrease of apoptosis in the cumulus cells

Expression of Connexin 43

Percentage of gap junctions (defined as the number of immunoreactive dots per total cell number) was significantly (P < 0.05) reduced in COCs cultured with GH (49.3%) compared with the controls (94.2%) (Figs. 5 and 6). After treatment with GH, a different localization pattern of Cx 43 was identified. Thus, in the COCs matured with GH, Cx 43 was mainly localized to the cells of the corona radiata, whereas in the COCs of the other groups, Cx 43 dots were regularly distributed all over the cumulus cells (Fig. 6). The highest percentage of gap junctions (95.0%) was seen in the COCs matured with IGF-I. They showed a significantly higher expression of Cx 43 compared with the COCs treated with GH (Fig. 6). When the COCs had been matured with GH and IGF-I, the percentage of gap junctions was higher (74.7%) than with supplementation of GH alone (49.3%) but was lower than with supplementation of IGF-I (95.0%). COCs matured in the control medium TCM 199 revealed a very similar expression of Cx 43 compared with the COCs treated with IGF-I (Fig. 5). In the negative controls, there was regularly no staining.



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  		FIG. 5. Percentage of gap junctions in COCs after in vitro maturation with different supplements. Data were analyzed using ANOVA followed by LSD. Significant differences between different groups are indicated by different superscripts (P < 0.05)



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  		FIG. 6. Immunohistochemical detection of the gap junction protein connexin 43. a) COC matured in TCM 199 supplemented with IGF-I (original magnification, x185). b) COC matured in TCM 199 supplemented with GH (original magnification, x185). Synthesis of connexin 43 is significantly decreased by treatment with GH


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the last years, it has been shown that GH accelerates nuclear maturation in oocytes and induces cumulus expansion in COCs matured in vitro [14, 15]. As shown by our studies, cumulus expansion induced by GH is a result of both increased proliferation and decreased apoptosis. Ki-67 labeling demonstrated that treatment with GH resulted in a significantly higher percentage of proliferating cumulus cells compared with the controls. The proliferation rate was not increased by supplementing the maturation medium with a combination of GH and IGF-I. These results imply that the effects of GH on bovine cumulus cell proliferation are not mediated by IGF-I. Thus, GH is able to directly modulate cell proliferation. This is supported by the fact that cumulus expansion induced by GH treatment is not inhibited by an IGF-I antibody [17]. Similarly, direct effects of GH on proliferation and differentiation have also been documented in numerous other cell types of the organism, such as osteoblasts and chondroblasts [22, 23]. Cumulus cells play an essential role in transportation processes and nutrition of the oocyte. Therefore, the higher number of cumulus cells in the presence of GH is likely to stimulate the feeding of the oocyte, resulting in a higher viability of the oocyte. This could also contribute to the fact that fertilizability and developmental capacity of oocytes are increased by GH treatment [14, 15].

The cumulus expansion induced by GH treatment is not only due to an increase in cell proliferation but is also caused by a distinct reduction in apoptosis. As shown in our studies, the application of GH resulted in a significantly lower percentage of apoptotic cumulus cells compared with the controls. A combination of GH and IGF-I resulted in the lowest percentage of apoptotic cells, implying that the antiapoptotic action of GH is increased by IGF-I. This is supported by the fact that, in the rat, the antiapoptotic effects of GH on granulosa cells are eliminated by application of IGF-binding proteins [24]. The reduction of apoptosis in the presence of GH has also been described in ovarian cell lines of hamsters. In these cells, GH inhibited apoptosis induced by elimination of essential medium supplements [25]. In these experiments, the antiapoptotic effects of GH were mediated by phosphorylation of the serin-threonin-kinase AKT [25]. In the bovine, supplementation of GH significantly reduced apoptosis in granulosa cells cultured in vitro. The signal transduction pathway included the activation of the cAMP/protein kinase A system [26]. In the ovary, apoptosis plays an essential role in follicular atresia and luteolysis [27–30]. Generally, apoptosis was seen in the granulosa cells of all follicular stages. The highest number of apoptotic cells occurred in tertiary follicles, especially in the granulosa cells surrounding the antrum [31]. In the ovary, cells of the cumulus oophorus revealed no apoptosis [31]. In our studies, the COCs matured in vitro regularly showed apoptotic cumulus cells. Whether apoptosis is a typical event of in vitro maturation or occurs physiologically after ovulation and migration of the oocyte through the oviduct needs to be further investigated.

The antiapoptotic effects of GH have not only been demonstrated in vitro but also in vivo. Thus, in the ovaries of transgenic mice overexpressing GH, apoptosis in tertiary follicles was significantly reduced compared with controls. As additionally the number of preovulatory follicles was increased in the transgenic mice, ovulation rates were higher compared with controls [32].

An essential component for establishing a precise balance between cell proliferation, differentiation, and apoptosis is intercellular communication. Cumulus cells communicate through an intricate network of transmembrane channels, the so-called gap junctions [33]. The cumulus cells do not only communicate with each other but also extend cytoplasmic processes through the zona pellucida and form gap junctions with the oocyte [34]. Gap junctions play an essential role for the exchange of ions and metabolites up to 1 kDa between neighboring cells and mediate the signal transduction of hormones (for a review, see [35]). Synthesis of connexins is generally regulated by hormones [36]. To investigate the effect of GH on formation of gap junctions and intercellular communication, the expression of Cx 43 was examined in COCs matured in the presence or absence of GH. Our studies showed that the percentage of gap junctions was significantly reduced by GH treatment, with Cx 43 being mainly localized to the cells of the corona radiata. This localization pattern is an indication that the communication between the oocyte and the cumulus cells is maintained in the presence of cumulus cell expansion. Because it has already been demonstrated that GH accelerates nuclear maturation in oocytes [14, 15], the reduction of gap junctions could result in alterations in the transport of signal transducers and metabolites in the cumulus cells. Thus, resumption of meiosis and nuclear maturation of the oocyte might be stimulated. This is supported by the fact that Cx 43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes [37] and that expression of Cx 43 in oocytes is developmentally regulated [38]. Additionally, the reduced expression of Cx 43 might also result in changes of the proportion of the different connexin proteins synthesized in the COCs such as Cx 43, Cx 32, Cx 37, and Cx 26 [39, 40]. This could be related to modifications in intercellular communication resulting in an acceleration of maturation in the oocyte.

Our studies demonstrated that GH induces cumulus expansion during in vitro maturation of oocytes by stimulating cell proliferation and inhibiting apoptosis. Thus, GH is capable of exerting direct and indirect effects on oocytes. GH-induced acceleration of nuclear maturation of oocytes may be partly due to changes in intercellular communication as seen in alterations of Cx 43 expression. Thus, the actions of GH during in vitro maturation of oocytes add new aspects regarding the role of this hormone in differentiation and maturation of the COC.


   ACKNOWLEDGMENTS
 
We wish to thank Dr. Peter Hutzler, Department of Pathology, GSF, Oberschleißheim, for the excellent help with the confocal laser scanning microscope.


   FOOTNOTES
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (FOR 478/1). Back

2 Correspondence: Sabine Kölle, Department of Veterinary Anatomy II, University of Munich, Veterinärstrasse 13, 80539 Munich, Germany. FAX: 49 89 2180 2569; s.koelle{at}anat.vetmed.uni-muenchen.de Back

Received: 12 August 2002.

First decision: 8 September 2002.

Accepted: 20 November 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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