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Molecular Endocrinology, doi:10.1210/me.2007-0495
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Molecular Endocrinology 22 (12): 2643-2654
Copyright © 2008 by The Endocrine Society

Decreased Oocyte-Granulosa Cell Gap Junction Communication and Connexin Expression in a Type 1 Diabetic Mouse Model

Ann M. Ratchford, Cybill R. Esguerra and Kelle H. Moley

Department of Obstetrics and Gynecology, Washington University in St. Louis, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Dr. Kelle H. Moley, Department of Obstetrics and Gynecology, Washington University in St. Louis, 660 South Euclid Avenue, St. Louis, Missouri 63110. E-mail: moleyk{at}wudosis.wustl.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In women, type 1 diabetes is associated with an increased risk of poor prenatal outcomes such as congenital anomalies and early miscarriage. In murine models of type 1 diabetes, impaired oocyte meiotic maturation, abnormal oocyte metabolism, and increased granulosa cell apoptosis have been noted. because gap junction communication is critical for the regulation of oocyte growth and meiotic maturation, we investigated the level of communication between the oocyte and surrounding cumulus cells in a streptozotocin-induced type 1 diabetic B6SJL/F1 mouse model and the expression of gap junction proteins known as connexins. Fluorescence recovery after photobleaching analyses of cumulus cell-enclosed oocytes (CEOs) from diabetic mice showed a 60% decrease in communication as compared with CEOs from nondiabetic mice. Real-time RT-PCR analyses confirmed the presence of Cx26, Cx37, and Cx57 mRNA and revealed a significant decrease in Cx37 mRNA expression in oocytes from diabetic mice compared with nondiabetic mice. Western analyses detected Cx26 expression in CEO but not denuded oocyte (DO) samples, and Cx37 in DO samples. Cx26 protein levels were decreased by 78% in CEOs from diabetic mice, and Cx37 protein levels were decreased 36% in DOs from diabetic mice. This decrease in connexin expression and gap junction communication in CEOs from diabetic mice may be responsible for the impaired oocyte meiotic maturation and poor pregnancy outcomes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MATERNAL TYPE 1, or insulin-dependent, diabetes has been linked to complications in pregnancies, often resulting in miscarriages, poor embryo development, and congenital malformations that persist at 3 to 4 times the control rate (1, 2, 3). Previous work in our laboratory and others has shown that multiple female reproductive problems occur in mouse models for type 1 diabetes, including reduced ovulation rates, poor embryo development, and delayed oocyte maturation (4, 5, 6).

During growth of the mammalian oocyte, paracrine, autocrine, and gap junction communication between the oocyte (germ cell) and the surrounding granulosa cells (GCs; somatic cells) is required for proper development (7, 8, 9, 10, 11). This communication is complex and bidirectional, resulting in the coordinated development and function of both the oocyte and the surrounding GCs (12, 13, 14). Whereas GCs provide nutrients and molecular signals that regulate oocyte development, the oocytes promote the organization of the follicle, proliferation of GCs, and the differentiation and function of cumulus cells, the subset of GCs surrounding the oocyte that differentiate from mural GCs during the preantral to antral follicle transition (7, 12). This metabolic cooperativity between oocyte and surrounding GCs appears to be under the control of the oocytes and is probably mediated by paracrine factors from the oocyte, such as growth differentiation factor 9 and bone morphogenetic protein (BMP)15, and by signals transmitted through gap junctions (13, 15, 16).

Gap junction channels connect adjacent cells and, when open, allow the exchange of nutrients, ions, and regulatory molecules of less than 1 kDa (17, 18). Molecules that are known to be transferred through gap junctions between cumulus cells and the growing oocyte are amino acids, glucose metabolites and nucleotides (7), and signals that regulate meiotic maturation of the fully grown oocyte (19, 20, 21, 22). Gap junction channels are composed of two symmetrical structures, termed "connexons," which are contributed by each adjacent cell. A single connexon is composed of six connexin proteins and can be homotypic (composed of one type of connexin) or heterotypic (comprised of a mixture of two or more types of connexins).

To date, there are at least 20 known connexin proteins. In ovarian tissues of different mammalian species, there are at least eight types of connexin proteins that have been identified by Northern blotting, in situ hybridization, RT-PCR, and immunohistochemical analyses: Cx26, Cx30.3, Cx32, Cx37, Cx40, Cx43, Cx45, and Cx57 (for excellent reviews see Refs. 10, 11 , and 23). Cx32, Cx43, and Cx45 have been localized between GCs in mouse ovarian follicles, whereas Cx37 is present in gap junctions between the oocyte and surrounding cumulus cells and has been localized to the oocyte surface (24, 25, 26, 27). Cx43, the predominant connexin expressed in GCs of adult mice, is known to form homotypic gap junction channels as well as heterotypic gap junction channels with Cx45 in vivo in GCs of adult mice and rats (11, 28). Cx32 is not thought to play an important role in folliculogenesis and oogenesis because disruption of Cx32 in mice resulted in viable and fertile females (29). Rather, Cx43 and Cx37 are thought to play more critical roles in ovarian function because the absence of either connexin causes a loss of cell coupling and disruption of folliculogenesis (24, 25, 26, 27).

Cx43 null mice die after birth from cardiac defects, but analyses of the fetal gonads showed a severe reduction in the number of germ line cells (30). Additionally, failure to properly develop GC cell layers in Cx43 null mice caused severe retardation of oocyte development and failure of meiotic maturation (24). Cx37-deficient mice lack mature (Graafian) follicles, fail to ovulate, develop numerous inappropriate corpora lutea, and oocyte development is arrested before meiotic competence is achieved (26, 31). Thus Cx43 and Cx37 appear to have an indispensable role in oocyte to GC communication, oocyte development, and folliculogenesis. Cx26 knockout embryos die 11 d post coitum, possibly due to placental dysfunction (32, 33). Although Cx26 function has not been determined in adult murine ovaries, Cx26 protein was detected in adult mouse oocytes and theca cells by immunolabeling studies, although attempts to detect Cx26 mRNA by others using RT-PCR were unsuccessful (23). However, Cx26 mRNA expression was noted by real-time PCR analyses in granulosa and theca cells of preovulatory follicles and corpora lutea in sheep, suggesting a possible role for Cx26 in folliculogenesis (34). Little is known about the function of Cx57 in ovarian tissue; however, expression of Cx57 mRNA has been detected at low levels in murine ovary tissue by RT-PCR (35), and in the porcine ortholog, Cx60, mRNA was found to be expressed in cumulus and theca cells by in situ hybridization (36).

Previous work in our laboratory has shown that streptozotocin-induced maternal murine diabetes delays germinal vesicle breakdown, a marker of oocyte maturation, and resumption of meiosis I in oocytes (37), which was later confirmed by others (4, 38). A delay in oocyte maturation has been associated with abnormal metabolism of the preovulatory oocyte (39, 40, 41, 42), as well as abnormal metabolism and increased apoptosis in the GCs surrounding the oocyte (4, 43). Furthermore, these changes in the oocyte have been correlated with poor preimplantation embryo quality and pregnancy outcome in other conditions (44). Because diabetes adversely affects oocyte maturation, and gap junction communication plays such an important role in oocyte development, the goal of these studies was to ascertain whether levels of oocyte and GC gap junction communication are altered in diabetic mice, and whether the levels of expression of connexin proteins possibly involved in oogenesis are decreased in diabetic mice. Our laboratory has previously examined the expression of Cx43 in cumulus cell-enclosed oocytes (CEOs) from diabetic and nondiabetic mice and observed a decrease in Cx43 protein in diabetic samples by both Western and immunofluorescence experiments (4).

The studies presented here examine the level of gap junction communication between the oocyte and surrounding cumulus cells in CEO complexes from diabetic and nondiabetic mice. Because of its indispensable role in oocyte development, the levels of Cx37 mRNA and protein expression were analyzed. Additionally, because Cx37 may be complemented by other connexins, and because Cx26 and Cx57 are expressed in ovarian tissue of other species, their expression levels were analyzed. Thus the levels of expression for Cx26, Cx37, and Cx57 were examined in these studies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Decreased Communication in Cumulus CEOs from Diabetic Mice as Compared with Nondiabetic Mice
To determine levels of gap junction communication between oocytes and surrounding cumulus cells, CEOs were collected from nondiabetic and diabetic female B6/SJL mice and analyzed by fluorescence recovery after photobleaching (FRAP) experiments. CEOs were preloaded with an acetoxymethyl (AM) ester derivative of the fluorescent indicator Calcein, which once inside the cell, produces a fluorescent Calcein molecule that is able to pass between cells connected via gap junctions and does not leak out of cells across cell membranes. Examples of individual experiments are shown in Fig. 1Go, A and B, for CEOs from nondiabetic and diabetic mice, respectively. In these experiments, the oocytes were photobleached and the fluorescence recovery from the surrounding cumulus cells to the oocyte was recorded. As shown in Fig. 1CGo, gap junction communication, as measured by the percentage of fluorescence recovery, was lower in oocytes from diabetic mice (13.6 ± 2.6%) as compared with nondiabetic mice (34.0 ± 4.1%). This fluorescence recovery was inhibited by incubating the CEO complexes with a gap junction blocker carbenoxolone (CBX) for 1 h before measuring FRAP, suggesting that the observed fluorescence recovery from the cumulus cells to the oocyte is through functional gap junctions. No difference was noted in GC to GC fluorescence recovery from either mural or cumulus GC clusters of diabetic mice and nondiabetic mice.


Figure 1
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  		Fig. 1. Decreased Gap Junction Communication in CEOs from Diabetic Mice as Compared with CEOs from Nondiabetic Mice

Oocytes or GC clusters were photobleached, and the fluorescence recovery in the photobleached region was monitored. The FRAP of four CEOs from control mice (A) and diabetic mice (B) are shown as examples. C, Data were graphed showing the average percentages ± SEM of fluorescence recovery in CEOs, GC (mural and cumulus) complexes (GC), and in CEOs treated with a gap junction inhibitor CBX from nondiabetic mice (black bars) and diabetic mice (white bars). Bars with identical letters are significantly different from each other, P < 0.0001. D, Diabetic; ND, nondiabetic.

 
To demonstrate the relationship between this gap junction communication and nuclear maturation, experiments were performed in which in vitro maturation of control oocytes was measured in the presence or absence of CBX at the same concentration used for the FRAP assay (200 µM). A significant decrease in percent germinal vesicle breakdown was seen after culturing in CBX for 1 h (23 ± 7% vs. 58 ± 9%; P < 0.004).

Connexin mRNA Expression in Oocytes from Nondiabetic and Diabetic Mice
Because gap junction communication is decreased in CEOs from diabetic mice, and because Cx26, Cx37, and Cx57 have been shown to be expressed in ovarian tissues of different species, we wished to analyze their expression levels in oocytes from diabetic mice, compared with nondiabetic mice. Therefore, we analyzed connexin mRNA expression by real-time RT-PCR with connexin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers, using RNA extracted from CEO, denuded oocyte (DO), or mural GC samples from nondiabetic and diabetic mice. mRNA for all three connexins (Cx26, Cx37, and Cx57) was expressed in all RNA samples analyzed (Fig. 2Go). However, mRNA for the connexins was found to be more highly expressed in DO-enriched samples from both nondiabetic and diabetic mice when compared with respective CEO samples; mural GC samples had the least amount of expression (Fig. 2AGo for Cx26; Fig. 2CGo for Cx57; and Fig. 2EGo for Cx37). To analyze any changes in mRNA expression in oocytes undergoing maturation, CEO samples were collected before or 6 h after human chorionic gonadotropin (hCG) administration. No significant differences in mRNA expression levels for Cx26 (Fig. 2AGo) and Cx57 (Fig. 2CGo) were detected after hCG administration. Cx37 mRNA expression appeared to increase in CEOs from nondiabetic mice 6 h after hCG (compared with 0 h), although this increase was not significant (Fig. 2EGo). When comparing DO samples from diabetic mice to nondiabetic, there were no significant differences in Cx26 (Fig. 2BGo) mRNA levels. Although not statistically significant, the mRNA expression levels for Cx37 (Fig. 2FGo) and Cx57 (Fig. 2DGo) appeared to be lower in DO samples from diabetic mice compared with nondiabetic mice (Fig. 2FGo) in a manner that trended toward significance (P = 0.08 and P = 0.09, respectively). To confirm purity of sample preparations in each experiment, real-time PCR was performed with specific primers for an oocyte-specific marker, BMP 15 (Fig. 3AGo) (45). Has2 primers were used as a cumulus cell-specific marker, which also showed maturation of the CEO samples from nondiabetic and diabetic mice because Has2 mRNAs increased in the CEO samples 6 h after hCG (Fig. 3BGo). No significant difference was found between the nondiabetic and diabetic samples for Has2.


Figure 2
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  		Fig. 2. Real-Time RT-PCR Analyses of Connexin mRNA Transcripts

Real-time RT-PCR was performed as described in Materials and Methods. To compare the levels of connexin transcript expression between RNA samples from CEOs, DOs, and GCs at 0 h hCG, or CEOs at 6 h hCG, the data are normalized to respective nondiabetic (ND) or diabetic (D) CEO 0 h hCG values for Cx26 (A), Cx57 (C), and Cx37 (E). To compare the levels of connexin transcript expression between nondiabetic and diabetic DO RNA samples, the data are normalized to the nondiabetic values for Cx26 (B), Cx57 (D), and Cx37 (F). A minimum of three experiments were performed for real-time RT-PCR analysis, and data are presented as the mean ± SEM of the fold changes. *, P ≤ 0.005 compared with ND CEO 0 h hCG; **, P ≤ 0.005 compared with D CEO 0 h hCG; #, P < 0.0001 compared with ND CEO 0 h hCG; ##, P = 0.02 compared with D CEO 0 h hCG.

 

Figure 3
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  		Fig. 3. Real-Time RT-PCR Analyses of BMP15 and Has2 mRNA Transcripts

Real-time PCR was performed with BMP15 (A)- and Has2 (B)-specific primers. Data are normalized to the respective nondiabetic (ND) or diabetic (D) CEO 0 h hCG samples.

 
Connexin Protein Expression in Oocytes from Diabetic and Nondiabetic Mice
Connexin 43 expression, the predominant connexin expressed in GCs, has been previously analyzed in our laboratory, and our findings showed a decrease in Cx43 protein in CEOs from diabetic compared with nondiabetic mice via Western blot analyses. The Cx43 protein expression was localized to the cumulus GCs when analyzed by immunofluorescence experiments (4). In the studies presented here, we analyze expression of three other connexin proteins: Cx26, Cx37, and Cx57.

To analyze connexin 26 protein expression, CEOs were collected from nondiabetic and diabetic mice at 0 or 6 h after hCG administration and subjected to Western analyses. The purchased antibody for Cx26 recognized multiple bands, including a 26-kDa band in CEO samples and liver samples (data not shown). Shown in Fig. 4AGo, when 100 CEOs are loaded per lane, a band at 26 kDa appeared in both nondiabetic and diabetic samples both before and after hCG administration, with an increase in expression being seen 6 h after hCG. No significant differences in Cx26 protein expression was noted between nondiabetic and diabetic samples at 0 h hCG; however, there was a significant decrease (78%) in Cx26 protein expression in CEOs 6 h after hCG in diabetic mice compared with nondiabetic mice (Fig. 4CGo).


Figure 4
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  		Fig. 4. Cx26 Protein Expression in Oocytes from Nondiabetic and Diabetic Mice

Western blot analyses were used to detect Cx26 protein expression in CEOs (A) and DOs (B) from nondiabetic (ND) and diabetic (D) mice before (0 h) and 6 h after hCG administration as described in Materials and Methods. CEOs (100/lane) and DOs (180/lane) were used, and levels of GAPDH are shown as an internal control. Western blot shown is representative of the results from three independent experiments. Western blots for Cx26 expression in CEO samples were quantitated, and Cx26 expression relative to GAPDH levels were graphed (C). Data are shown as the mean ± SEM. *, P < 0.002 compared with all other samples.

 
Because Cx26 mRNA was more highly expressed in DO purified samples, Westerns were performed to analyze Cx26 protein expression with DOs from nondiabetic and diabetic DOs before or after (6 h) hCG administration. As shown in Fig. 4BGo, Cx26 was not detectable in DOs (with 180 DOs loaded per lane), suggesting that Cx26 protein may be more highly expressed in the cumulus cells surrounding the oocyte as opposed to the oocyte itself. This is in contrast to the mRNA expression that is higher in oocyte rather than the GCs. However, this is no surprise because protein expression is highly regulated by selective translation and/or degradation during oocyte maturation and transition to zygote stages (46). Immunofluorescent analyses of ovarian sections for Cx26 showed a general staining including in the oocyte and the cumulus cells; however, much of the staining appeared to be background (data not shown).

Cx37 was likewise analyzed in DO samples from nondiabetic and diabetic mice before or after hCG administration. Our results show that Cx37 protein expression was decreased (36%) in DOs from diabetic mice compared with nondiabetic mice (Fig. 5Go, A and B). Additionally, after hCG administration in both nondiabetic and diabetic mice, levels of Cx37 decreased (Fig. 5Go, A and B). Cx37 was barely detectable in CEO samples when 100 CEOs per lane were loaded, and in GC samples (data not shown), suggesting that it is more highly expressed in the oocyte. Immunofluorescent labeling of frozen ovarian sections confirmed localization of Cx37 to the oocytes (Fig. 5CGo), with some possible staining in the zona pellucida, as has been reported previously (26, 27, 47, 48). The zona pellucida staining in our experiments was not as prominent as recently reported (47, 48), possibly due to the variations in staining technique. Localization of Cx37 in the oocytes from nondiabetic mice appeared to be on the oolemma in 24% of the oocytes analyzed, which was only seen in 8% of the oocytes from diabetic mice, suggesting that Cx37 protein levels and localization differ in oocytes of diabetic mice compared with nondiabetic mice. This difference in localization combined with the decrease in protein expression may together explain the decrease in GC-oocyte communication. At lower magnification, Cx37 staining was seen in the theca and stromal cells (data not shown).


Figure 5
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  		Fig. 5. Cx37 Protein Expression in Oocytes from Nondiabetic and Diabetic Mice

Western blot analyses were used to detect Cx37 protein expression in DOs (A) from nondiabetic (ND) and diabetic (D) mice before (0 h) and 6 h after hCG administration as described in Materials and Methods. DOs (180/lane) were used, and GAPDH levels were analyzed as an internal control. Western blot shown is representative of the results from three experiments. The Western blots were quantified, and Cx37 expression was graphed relative to GAPDH levels (B). Data shown are the mean ± SEM. *, P < 0.05 compared with ND DO 0 h sample. Immunofluorescent labeling of Cx37 (C) was performed with frozen ovary sections on glass slides from nondiabetic and diabetic mice. The zoomed images are from the boxes outlined in white from the images to the immediate left. Experiment shown is representative of three experiments. Exp., Experiment.

 
Western analyses using a commercial antibody to detect Cx57 were inconclusive (data not shown); therefore, Cx57 protein expression in murine CEOs or DOs remains to be determined.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Gap junction communication between somatic and germ cells of the ovary is crucial for the growth and development of competent germ cells. Evidence is presented here that oocyte-cumulus cell gap junction communication is significantly decreased in diabetic mice, compared with nondiabetic mice. Results from FRAP analyses show a 60% decrease in gap junction communication in CEOs from diabetic mice compared with nondiabetic mice. This decreased gap junction communication could, in turn, be responsible for the delay in oocyte growth and maturation observed in diabetic mice (4). The necessity of pyruvate metabolism for oocyte maturation has been demonstrated in a pyruvate dehydrogenase knockout mouse (49). Furthermore, culture of the Pdha1–/– oocytes with cumulus cells can partially compensate for the enzyme deficiency, presumably through the transfer of energy metabolite transfer from the cumulus cells to the oocyte (49). Therefore, it is plausible that the decrease in gap junction communication in diabetic CEOs could decrease the transfer of energy substrates from the cumulus cells to the oocyte, leading to the adverse oocyte energy metabolism we have recently described, in which alterations in energy substrates negatively correlate with poor oocyte quality and poor maturational competence (42). No difference was observed in GC-GC communication between GC clusters of nondiabetic and diabetic mice, suggesting that the oocyte-cumulus cell gap junction communication defect may have a more important role in the delayed oocyte maturation noted in diabetic mice.

Using a murine model for human oocyte maturation is a well-accepted model (50, 51, 52). Several recent reports validate the similarities in cumulus-oocyte complex composition both in vivo and in vitro (43, 53). Thus our conclusions, specifically that abnormal connexin expression and resulting aberrant communication may result in delayed maturation, could be directly applicable to human oocytes and complexes.

Furthermore, we demonstrate that this decreased communication may be due to decreased connexin protein expression, in particular, decreases in Cx26 and Cx37 as shown in these studies, and Cx43 as shown previously (4). According to one report (54), Cx26 protein is expressed in mouse oocytes and theca cells, although attempts by others to detect Cx26 mRNA in mouse oocytes and GCs by RT-PCR were unsuccessful (23). Cx26 expression has also been found in oocytes of primordial and primary/secondary follicles of cows (55), and in granulosa and/or thecal cell layers of healthy antral follicles of sheep and cows (55, 56). Here we report that Cx26 mRNA is expressed in murine denuded oocytes, CEOs, and GCs as analyzed by real-time PCR. The relative amount of Cx26 mRNA expression did not appear to differ in diabetic and nondiabetic denuded oocyte samples; however, Cx26 protein expression was decreased in CEOs from diabetic mice as shown by Western analyses. Interestingly, Cx26 protein expression increased after hCG administration, which is in contrast to gonadotropin-induced decreases in Cx43 expression that have been reported (57), but in accordance with hCG-induced increases in Cx26 expression that have been shown in rat mammary and uterine tissues (58). Even though decreases in Cx43 and gap junction communication are thought to be responsible for the hCG-induced oocyte maturation, it is possible that some gap junction communication is necessary, perhaps through Cx26-containing gap junction channels, to facilitate the transfer of growth/maturation mediators or energy substrates. Alternatively, there is recent evidence emerging for gap junctional-independent effects of connexins, such as cell growth (for a recent review, see Ref. 59). Perhaps this is why Cx26 expression is increased in response to hCG in nondiabetic CEOs. Our data further demonstrated that Cx26 protein expression was undetectable in DO samples, suggesting that the Cx26 protein expression seen in the CEO samples is contributed mostly, if not completely, by the cumulus cells.

Expression of Cx37 mRNA, the connexin known to be predominantly expressed in the oocyte, was higher in denuded oocyte samples than in CEO and GC samples when analyzed by real-time RT-PCR. When denuded oocyte samples from diabetic and nondiabetic mice were compared, both real-time RT-PCR and Western blot analyses show that Cx37 mRNA and protein expression is decreased in oocyte samples from diabetic mice. Additionally, Cx37 protein expression, as analyzed by immunofluorescence studies, appears to localize to the oolema of ovary sections from nondiabetic mice, which is not seen in ovary sections from diabetic mice. Furthermore, Cx37 protein expression is decreased in denuded oocytes from both nondiabetic and diabetic mice after an hCG stimulus in vivo, suggesting that its expression decreases with gonadotropin stimulation, similar to the gonadotropin-induced decrease in ovarian Cx43 expression that has been reported (57).

Cx43 has been characterized as an important gap junction protein required for GC to GC (both mural and cumulus GCs) communication, which, in turn, is required for follicle and oocyte maturation. Our laboratory has previously shown decreased expression of Cx43 in cumulus cells of diabetic mice (4). Although Cx43 is the predominant Cx in GCs, elegant studies using chimeric mouse ovaries show that it is not required for communication between cumulus GCs and the oocyte (27, 60). Cx37 and Cx43 may not form functional gap junctions at the oocyte-cumulus cell interface; however, Cx37 of the oocyte may form homotypic gap junctions, as has been suggested (23, 27), or may dock with other connexin proteins of the cumulus cell, perhaps Cx26.

Despite a report of Cx57 mRNA expression in mouse ovaries (35), this report has been disputed. However, the porcine ortholog, Cx60, is expressed in cumulus cells and theca cells (36). Our real-time RT-PCR analyses show that Cx57 mRNA is expressed in murine DO, CEO, and GC samples from both nondiabetic and diabetic mice with no significant differences between nondiabetic and diabetic samples. Experiments to analyze Cx57 protein expression by Western blot analyses were unsuccessful; therefore, protein expression of Cx57 remains to be determined. However, because Cx57 knockout mice do not display any ovarian defects (23, 61), it may not have any role in oocyte development and maturation.

The expression and function of specific connexin proteins in ovaries of porcine, ovine, bovine, mouse, and rat species are discussed in a recent review paper (59). Although little is known about connexins in the human ovary, aberrant gap junction communication may be involved in human female infertility (23). In fact, only recently has there been a report of Cx37 expression in human cumulus cells of patients undergoing fertility treatments (62). A recent publication described the use of a gap junction inhibitor in rat follicles to induce meiotic maturation (63). This study was performed in rat follicle enclosed oocytes, which were incubated overnight in the inhibitor CBX. After the overnight incubation, the CEOs were released from the follicle-enclosed oocyte, and the CEO was allowed to mature in vitro in the absence of CBX. In our study, murine CEOs are directly recovered from large preovulatory follicles, and the complexes themselves are incubated in CBX for 1 h. Unlike the rat study, which requires absorption and penetration of the follicle overnight and maintenance of the inhibitory effect during this time interval, our study examines direct inhibition of the GC-oocyte complex for 1 h. Also in contrast to the other study, this study provides proof that this methodology is effective in inhibiting the gap junction communication by FRAP imaging after Calcein labeling, which clearly shows inhibition of communication in the presence of CBX at this concentration. For these reasons, we conclude that direct gap junction complex inhibition of the CEO adversely affects oocyte maturation and resumption of meiosis.

In a variety of tissues, connexin expression and the formation of gap junctions have been shown to be regulated by a variety of factors, including gonadotropins, growth factors, and intracellular regulators (10). In ovaries, gonadotropins have been shown to regulate connexin 43 expression at the transcriptional, translational, and posttranslational levels (57). FSH and the FSH-like hormone pregnant mare serum gonadotropin (PMSG), up-regulated Cx43 mRNA and protein expression whereas LH, or hCG, decreased Cx43 expression (57). In rat endometrium, Cx43 and 26 gene expression is regulated by progesterone and estrogen levels (64). In our experiments, hCG caused no changes in connexin mRNA, although there was an increase in Cx26 protein expression in CEOs, and a decrease in Cx37 protein expression in Dos, as shown by Western analyses. Although not analyzed in these studies, gap junction communication may also be regulated by phosphorylation of connexins by multiple kinases. The phosphorylation may either increase gap junction activity by opening the gap junction channels, or decrease gap junction activity by closing the gap junction channels or tagging the channels for degradation by the proteosomal or lysosomal pathways (57).

Our observations of diabetes-induced decreases in connexin expression and blunted gonadotropin responses may be a result of increases in apoptosis in the cumulus cell population, which we have previously described (4). Alternatively, the decreases in connexin expression may be a result of decreased transcription factor activity and gene expression (especially for Cx37; Fig. 2FGo), or decreased regulation at the protein level, either by alterations in translation, degradation, or translocation. Diabetes-induced decreases in connexin protein expression and communication have also been seen in rat epidermal tissue (65); however, the mechanisms by which diabetes alters connexin protein expression remain unknown.

This study provides evidence of decreased cumulus cell-oocyte gap junction communication in CEOs of diabetic compared with nondiabetic mice. The decrease in gap junction communication may be responsible for the delay in oocyte growth and maturation (4) and the altered energy resources (42) seen in diabetic mice. Furthermore, our data described here suggest that Cx26 may be contributed by cumulus cells to the oocyte-cumulus cell gap junctions, and a decrease in Cx26 protein expression in CEOs from diabetic mice, along with a decrease in cumulus cell Cx43 and oocyte Cx37 expression, may contribute to the decrease in gap junction communication in diabetic mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Oocyte Retrieval
All mouse studies were approved by the Animal Studies Committee at Washington University School of Medicine and conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Female immature B6SJLF1 mice (age 20–24 d; Jackson Laboratories, Bar Harbor, ME) were given free access to food and water and were maintained on a 12-h light, 12-h dark cycle. Diabetic and age-matched controls were superovulated with 10 IU/animal of PMSG (Sigma Chemical Co., St. Louis, MO) by ip injection, and 48 h later, they either were killed or given an injection of 5 IU of hCG. At the appropriate time points (t = 0 h or 6 h), the ovaries were removed and placed in a dish containing 1.5 ml of culture human tubal fluid (HTF) medium (Irvine Scientific, Santa Anna, CA) with 0.25% BSA (Sigma) added. Preovulatory oocytes were isolated by puncturing the antral follicles with sterile needles, and then washed through several changes of HTF medium. Before puncturing, the ovaries were stored briefly in an incubator with the settings of 5% CO2, 5% O2, 90% N2 atmosphere at 37 C. CEOs were collected, or denuded using a small bore pipette and repetitive pipetting.

Induction of Hyperglycemia
To generate type 1 diabetic mice, female B6SJLF1 mice (age 20–24 d) received a single injection of streptozotocin (Sigma) at a dose of 190 mg/kg (dissolved in sodium citrate buffer, pH 4.4; Sigma). A tail-blood sample was measured 4 d after injection for glucose concentrations via a Hemocue B glucose analyzer (Quest Diagnostics, Madison, NJ). If glucose levels were more than 300 mg/dl, these mice were selected and received a priming injection of PMSG. A few control mice were also randomly selected, and their blood sugar was checked to ensure that it was less than 240 mg/dl.

FRAP Analyses
To assess the level of intercellular gap junctional connection between the oocyte and its cumulus cells, gap junctional dye transfer from the cumulus cells to the oocyte was measured using the AM ester derivative of the fluorescent indicator Calcein (Calcein-AM; Molecular Probes, Inc., Eugene, OR). Calcein-AM is a nonfluorescent molecule that can rapidly permeate into the cytoplasm through the cell membrane (66). Once inside the cell, endogenous esterases cleave the lipophilic AM groups, producing fluorescent calcein molecule that is unable to leak out of cells across cell membranes, but is able to pass between cells connected via gap junctions.

CEOs were collected and loaded with 2 µM Calcein-AM, in HTF culture medium with 0.2% BSA for 25 min. The concentration of DMSO in the media from the Calcein-AM solution was less than 0.01%. The CEOs were then transferred to HTF + BSA medium without Calcein-AM and incubated for 20 min, to allow dye transfer from cumulus cells to the oocyte. Next the CEOs were transferred to clear tissue culture media (M199/Earles medium with no phenol red) in the presence or absence of a known gap junction blocker, CBX (200 µM; Sigma) (67).

FRAP was performed on a Zeiss LSM 510 confocal microscope (Carl Zeiss, Inc., Thornwood, NY) using the Edit Bleach function in combination with Time Series LSM 510 software. A region of interest (ROI), either the oocyte or a GC cluster, was chosen, and 150 iterations of the 488 nM laser line at 98.1% emission strength were used to photobleach the fluorescence within the ROI. These were the conditions determined for optimal bleaching of the oocytes. The progression of FRAP was followed by continuously acquiring images with a time interval of 30 sec for 10 min of total imaging time. Fluorescence of the mobile fraction was quantified using the Mean ROI function within the Zeiss LSM 510 software. Fluorescence intensities of ROIs were recorded before photobleaching, immediately after photobleaching, and at 30-sec intervals after photobleaching. Postbleach intensities were corrected for any residual, nonbleached fluorescence, as described previously, and the percentage of fluorescence recovery was calculated using the equation for determining the mobile fraction (68).

Western Analyses
CEOs, DOs, or mural GC samples were added directly to Laemmli sample buffer, boiled for 5 min, subjected to SDS-PAGE on 10% acrylamide gels, and transferred to nitrocellulose. Membranes were blocked for 1 h with 5% (wt/vol) nonfat dry milk in TBST [Tris-buffered saline containing 0.1% (wt/vol) Tween 20] and immunoblotted overnight at 4 C in 3% (wt/vol) BSA in TBST with antibody (1:250 dilution for polyclonal anti-Cx26 (Zymed Laboratories, Inc., South San Francisco, CA), 1:250 dilution for polyclonal anti-Cx57 (Zymed), 1:100 dilution for polyclonal anti-Cx37 (Zymed), 1:1000 dilution for polyclonal anti-GAPDH (AbCam, Danvers, MA). Membranes were washed three times with TBST (15 min/wash) and incubated with antirabbit antibody from a SuperSignal West Dura ECL kit (Pierce Chemical Co., Rockford, IL) at a dilution of 1:500 in 5% (wt/vol) nonfat dry milk in TBST at room temperature (RT) for 1 h. After three more washes with TBST (15 min/wash), the blots were subjected to enhanced chemiluminescence (SuperSignal West Dura ECL, Pierce), and the protein bands detected on x-ray films were quantitated using an {alpha}-imager and Image J.

Real-Time RT-PCR
Total RNA was extracted from 400 CEOs or 500 DOs per group using an RNeasy kit (QIAGEN, Chatsworth, CA), followed by treatment with ribonuclease-free deoxyribonuclease (Ambion, Inc., Austin, TX). The quantity of RNA was assessed by spectrophotometry after deoxyribonuclease treatment. cDNA synthesis was performed per instructions using a Superscript III kit (Invitrogen, Carlsbad, CA) with the additional items not included in the kit: oligo deoxythymidine primer (Invitrogen), ribonuclease inhibitor (Promega), and ribonuclease H (Promega). A quantity of 200 ng of RNA was used for cDNA synthesis. Real-time quantitative PCR assays were performed using Applied Biosystems 7000 Sequence Detecting system, and Power SYBER Green PCR Master Mix (Applied Biosystems, Foster City, CA). cDNA (20 ng per group) was used to perform duplicate PCR analyses per experiment. For amplification of Cx26 cDNA, the upstream primer, 5'-GACCCGCTTCAGACCTGCTCCTTAC-3', and the downstream primer, 5'-GCCTGGAAATGAAGCAGTCCACTGT-3', were used to amplify a 658-bp product (69). For amplification of Cx57 cDNA, the upstream primer, 5'-AATTTACTGGGTGGCATCCTAGA-3', and the downstream primer, 5'-GGGAAAGCATCATCGTAACAGAT-3', were used to amplify a 200-bp product [primer pair identification no. 6753990a1 (70)]. For amplification of Cx37 cDNA, the upstream primer, 5'-ACGGTCGTCCCCTCTACAT-3', and the downstream primer, 5'-GGTAGATCAGGGTGGGTGTG-3', were used (27). For amplification of BMP15 cDNA, the upstream primer, 5'-GCACGATTGGAGCGAAAATG-3', and the downstream primer, 5'-CGTACGCTACCTGGTTTGATGC-3', were used to amplify a 123-bp product (71). For amplification of GAPDH cDNA, the upstream primer, 5'-AGTGGAGATTGTTGCCATCAACGA-3', and the downstream primer, 5'-GGGAGTCGCTGCTGTTGAAGTCGCAGGA-3', were used. The threshold cycle (Ct) was used for determining the relative expression level of each gene, by normalizing to the Ct of GAPDH. The method of ddCT was used to calculate the relative fold change of each gene. For example, the calculating equation for analyzing Cx26 gene expression in nondiabetic DOs normalized to nondiabetic CEOs is as follows: fold change = 2–ddCt, where ddCt = (CtDO,Cx26 – CtDO,GAPDH) – (CtCEO,Cx26– CtCEO,GAPDH). Because SYBER Green binding is not sequence specific, a dissociation curve analysis was performed at the end of the amplification process, and the PCR products were subjected to agarose gel electrophoresis to verify the specificity of the PCR products. Real-time RT-PCR experiments were repeated at least three times independently. Data are represented as the mean ± SEM.

Immunofluorescent Studies
Ovaries were frozen, stored at –80 C and cryosectioned into 10-µm slices. Multiple ovarian slices from both nondiabetic and diabetic mice were fixed side by side on Superfrost microscope glass slides (Fisher Scientific Inc., Pittsburgh, PA) with 3% paraformaldehyde for 15 min at RT. Slides were washed three times in PBS for 5 min/wash, and tissue sections were permeabilized with 0.1% triton in PBS for 5 min. After three additional 5-min washes in PBS, slides were incubated with a blocking solution of 8% BSA in PBS for 1 h at RT. Tissue sections were then incubated with primary antibody to Cx37 (1:250 dilution in 8% PBS/BSA; Alpha Diagnostic International, San Antonio, TX) or 8% PBS/BSA for a negative control overnight at 4 C in a hybridized chamber. The next day, slides were rinsed three times in PBS and incubated with goat antirabbit Alexa Fluor 488 secondary antibody (1:200 in PBS/BSA) for 30 min at RT and washed twice in PBS, and nuclear staining was performed in 2.5 µM To-Pro-3-iodide. After two rinses in PBS/BSA, slides were mounted in vectashield and sealed with a coverslip and nail polish. Fluorescence of the tissue sections was viewed with confocal immunofluorescent microscopy (Olympus FV500 laser scanning microscope; x40 magnification).

Statistics
Differences between the groups with regard to gap junction communication, mRNA expression, or protein expression were compared by one-way ANOVA (Statview 4.5, Abacus Concepts, Inc., Berkeley, CA). For gap junction communication and protein expression, Fisher’s post hoc test was used. For statistical differences between two groups, such as the real-time RT-PCR data with DO samples of diabetic and nondiabetic mice, a t test was performed. Results are expressed as a mean ± SEM of at least three separate experiments.


   FOOTNOTES
 
This work was supported by the National Institutes of Child Health and Human Development Cooperative Program on Female Health and Egg Quality, U01 HD044691 (to K.H.M.), T32 HD049305 (to A.M.R.), and the National Institutes of Health Neuroscience Blueprint Core Grant NS057105 to Washington University and the Bakewell Family Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 1, 2008

Abbreviations: AM, Acetoxymethyl; BMP, bone morphogenetic protein; CBX, carbenoxolone; CEO, cumulus cell-enclosed oocyte; DO, denuded oocyte; FRAP, fluorescence recovery after photobleaching; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, granulosa cell; HTF, human tubal fluid; PMSG, pregnant mare serum gonadotropin; ROI, region of interest; RT, room temperature; TBST, Tris-buffered saline containing 0.1% (wt/vol) Tween 20.

Received for publication October 29, 2007. Accepted for publication September 25, 2008.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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