A differential autophagic response to hyperglycemia in the developing murine embryo

in Reproduction
Authors:
Katie L Adastra Departments of, Obstetrics and Gynecology, Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8064, Saint Louis, Missouri 63110, USA

Search for other papers by Katie L Adastra in
Current site
Google Scholar
PubMed
Close
,
Maggie M Chi Departments of, Obstetrics and Gynecology, Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8064, Saint Louis, Missouri 63110, USA

Search for other papers by Maggie M Chi in
Current site
Google Scholar
PubMed
Close
,
Joan K Riley Departments of, Obstetrics and Gynecology, Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8064, Saint Louis, Missouri 63110, USA

Search for other papers by Joan K Riley in
Current site
Google Scholar
PubMed
Close
, and
Kelle H Moley Departments of, Obstetrics and Gynecology, Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8064, Saint Louis, Missouri 63110, USA
Departments of, Obstetrics and Gynecology, Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8064, Saint Louis, Missouri 63110, USA

Search for other papers by Kelle H Moley in
Current site
Google Scholar
PubMed
Close

Free access

Sign up for journal news

Autophagy is critical to the process of development because mouse models have shown that lack of autophagy leads to developmental arrest during the pre-implantation stage of embryogenesis. The process of autophagy is regulated through signaling pathways, which respond to the cellular environment. Therefore, any alteration in the environment may lead to the dysregulation of the autophagic process potentially resulting in cell death. Using both in vitro and in vivo models to study autophagy in the pre-implantation murine embryo, we observed that the cells respond to environmental stressors (i.e. hyperglycemic environment) by increasing activation of autophagy in a differential pattern within the embryo. This upregulation is accompanied by an increase in apoptosis, which appears to plateau at high concentrations of glucose. The activation of the autophagic pathway was further confirmed by an increase in GAPDH activity in both in vivo and in vitro hyperglycemic models, which has been linked to autophagy through the activation of the Atg12 gene. Furthermore, this increase in autophagy in response to a hyperglycemic environment was observed as early as the oocyte stage. In conclusion, in this study, we provided evidence for a differential response of elevated activation of autophagy in embryos and oocytes exposed to a hyperglycemic environment.

Abstract

Autophagy is critical to the process of development because mouse models have shown that lack of autophagy leads to developmental arrest during the pre-implantation stage of embryogenesis. The process of autophagy is regulated through signaling pathways, which respond to the cellular environment. Therefore, any alteration in the environment may lead to the dysregulation of the autophagic process potentially resulting in cell death. Using both in vitro and in vivo models to study autophagy in the pre-implantation murine embryo, we observed that the cells respond to environmental stressors (i.e. hyperglycemic environment) by increasing activation of autophagy in a differential pattern within the embryo. This upregulation is accompanied by an increase in apoptosis, which appears to plateau at high concentrations of glucose. The activation of the autophagic pathway was further confirmed by an increase in GAPDH activity in both in vivo and in vitro hyperglycemic models, which has been linked to autophagy through the activation of the Atg12 gene. Furthermore, this increase in autophagy in response to a hyperglycemic environment was observed as early as the oocyte stage. In conclusion, in this study, we provided evidence for a differential response of elevated activation of autophagy in embryos and oocytes exposed to a hyperglycemic environment.

Introduction

Autophagy is a programmed method of protein degradation and recycling of the necessary cellular building blocks of glucose, amino acids, and fatty acids. This process has been shown to be critical to cell survival during the periods of nutrient and specifically glucose deprivation, as well as during the development and differentiation (Aki et al. 2003, Mizushima et al. 2004, Scott et al. 2004, Singh et al. 2009). During the developmental progression from oocyte to cleavage stage embryo, maternal proteins and RNAs are degraded and quickly replaced by newly synthesized embryonic counterparts. Mizushima et al. have recently elucidated a physiological role of autophagy to aid in this process (Tsukamoto et al. 2008a, 2008b). By creating an oocyte-specific Atg5 (autophagy-related 5, a critical component in the formation of autophagosomes) knockout mouse, the authors determined that fertilization of Atg5 null oocytes with Atg5 null sperm resulted in developmental arrest between the four- and eight-cell stage. Fertilization of the Atg5 null oocyte with wild-type sperm; however, resulted in normal embryo development. They also demonstrated that protein-recycling rates were abnormal in the embryos entirely devoid of ATG5 and concluded that proper autophagic degradation within early embryos is essential for pre-implantation development. Results suggesting a role for autophagy during embryoid cavitation were also recently published (Qu et al. 2007). This group reported that loss of either Ambra1 (Beclin-1, required for the initiation of autophagosome formation) or Atg5, both essential autophagic genes, leads to failure of cavitation due to the persistence of cellular corpses in murine ESCs. These cells fail to display signals to indicate removal. This dysregulation is also associated with low levels of ATP. Overall, the lethality associated with the removal of this pathway indicates the critical roles that this pathway plays in embryo development, many of which may not be elucidated yet.

The activation of autophagy has been characterized as a survival mechanism in the prevention of cell death (Boya et al. 2005). This role for autophagy has been reviewed elsewhere (Maiuri et al. 2007, Thorburn 2008). Briefly, the interplay between survival and autophagy is demonstrated by studies showing that lack of AMBRA1 expression leads to excessive apoptotic cell death with embryo lethality at a peri-implantation stage (Yue et al. 2003). Although the role for autophagy as a pro-survival mechanism is well accepted, the idea that excess autophagy with elevated levels of autophagosomes leads to cell death is still in debate. However, it is accepted that excessive autophagy leads to cell death during ischemia (Akazawa et al. 2004). Therefore, there may be certain circumstances in which this interrelationship exists. And while the mechanisms may not be entirely clear, recent studies suggest that oxidative stress plays a role in this switch from survival to death (Sakaida et al. 1990, Grune et al. 2003, Rodriguez-Enriquez et al. 2004, Kiffin et al. 2006).

In diabetes models, it has been postulated that autophagy is activated in certain tissues as a mechanism of protection against cellular damage resulting from oxidative stress. This activation is evident in the pancreatic β cells of diabetic animal models and humans (Fujitani et al. 2009, Masini et al. 2009). Other tissues that exhibit an increased level of autophagy in diabetic models include the muscle, neuronal tissue, and liver (Amherdt et al. 1974, Towns et al. 2005). However, a decrease in autophagy-mediated protein turnover is observed in the renal cortex of the diabetic kidney (Sooparb et al. 2004). Therefore, it appears that tissues may regulate autophagy differently in response to a diabetic environment, and thus, investigation of this pathway is important as a potential therapeutic target for problems associated with diabetes.

The uterine milieu plays an important role in the development and implantation of an embryo. Previously, we have determined that a maternal diabetic state in mice results in an increase in number of fetal resorptions and an increase in congenital malformations of the fetus. These deleterious effects resulted with only a 96-h exposure to the diabetic environment, as blastocyst stage embryos transferred from diabetic mothers into non-diabetic control mothers also displayed these phenotypes (Wyman et al. 2008). In addition, we have shown that the hyperglycemic environment of the uterus, by treatment with high glucose, leads to an increase in apoptosis in the embryo (Moley et al. 1998a). Therefore, we wanted to investigate the autophagic pathway in a diabetic milieu.

In this study, we present evidence that the pre-implantation embryo alters autophagy in response to the external environment. The embryos respond to stressors including a known activator of autophagy and a hyperglycemic environment by increasing the activation of autophagy, identifying autophagy as a pathway for survival in the blastocyst. Furthermore, the embryonic response is differentially activated in what appears to be specific cell niches within the embryos.

Results

Embryonic autophagy is influenced by environment

Our laboratory and others have observed a basal level of autophagic activation during all stages of pre-implantation embryonic development in mice (Cooper et al. 2008, Tsukamoto et al. 2008a, 2008b). However, an autophagic response due to environmental stress or a known inhibitor has yet to be studied during this crucial developmental time period. It has been well established that MTOR is an upstream repressor of autophagy. Therefore, the MTOR inhibitor rapamycin is widely used to activate autophagy within in vitro systems. In order to investigate the embryonic response in the presence of an autophagic activator, we exposed morula stage embryos (76 h post-human chorionic gonadotropin (hCG)) to culture media containing either 1 or 5 μM rapamycin for 30 h and observed the levels of AMBRA1 and LC3 expression by immunofluorescence compared with the embryos treated with DMSO (vehicle control) (Fig. 1A and C). We observed autophagic activation as an increase in the levels of AMBRA1 protein expression in the treated groups. This activation was confirmed by LC3 protein expression, a major component of the autophagosomal membrane (Fig. 1D and E). While the increase in autophagic response was expected, there was an unusual variable patterning of response between the single cells within the blastocyst. AMBRA1 protein expression was limited to the TE of the blastocyst, with the inner cell mass (ICM) seemingly unaffected (Fig. 1C). In addition, we examined the expression of LC3, a well-known marker for autophagy, as it is localized to the autophagosomal membrane throughout the bulk of the autophagic process. The expression of LC3 was also detected in a sporadic pattern throughout the embryo; however, this pattern was different than that of AMBRA1 (Fig. 1E). This pattern may represent a differential autophagic response in certain cell niches.

Figure 1
Figure 1

AMBRA1 and LC3 expression in rapamycin-treated embryos. In each group, ten mouse control blastocysts were incubated for 30 h: DMSO control (A), 1 μM (B), or 5 μM (C) rapamycin. This experiment was repeated three times. AMBRA1 expression is increased in a variable pattern, with the most expression observed in the trophectoderm (TE) cells. LC3 expression in mouse control blastocysts incubated in DMSO (D, n=10) or 5 μM rapamycin (E, n=10). This experiment was conducted three times.

Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0265

In addition, a cell death assay was used to determine whether the treatment affected cell quality. We found no significant difference in cell death, the percentage of apoptotic cells, between the rapamycin treated and the control groups; however, there was a difference in total cell number (Supplementary Figure 1A and B, see section on supplementary data given at the end of this article). This difference may be due to either a decrease in proliferation or a non-apoptotic cell death.

The amount of glucose in the environment is critical to baseline autophagy levels

Due to the importance of autophagy in energy homeostasis in other tissues and the alterations of this pathway in diabetic models, we investigated autophagy in the developing embryo exposed to different glucose levels in vitro.

As an embryo transitions from the morula to blastocyst stage, the amount of environmental glucose is critical as the embryo switches using pyruvate and lactate as energy to glucose (Brown & Whittingham 1991, Leese 1991). In previous papers, we have determined that the hyperglycemic conditions present in the milieu of diabetic mothers leads to a decrease in glucose transporter expression, deceased intracellular glucose, and an increase in apoptosis in murine blastocysts (Moley et al. 1998a, 1998b). In addition, when embryos were exposed to a hyperglycemic environment for 72 h and then transplanted to a control pseudo-pregnant foster mouse, an increased number of resorptions and malformations in the fetuses were observed (Wyman et al. 2008). Therefore, the maternal environment and, in particular, the amount of glucose present are critical to fetal outcome.

To investigate whether the amount of glucose present in the environment alters the basal level of autophagy present in the embryos, we exposed the developing embryos to varying concentrations of glucose in vitro during morula to blastocyst transition for 30 h. We have previously determined that a level of 52 mM d-glucose closely mimics the phenotypic effects we observe in the embryos exposed to an in vivo diabetic environment, with similar apoptosis, glucose transporter downregulation, and decreased intracellular glucose (Moley et al. 1998a). Following this exposure to high glucose, we then attempted to detect LC3 by immunofluorescence, as a marker for autophagosomes. At more physiological levels (5 mM glucose), a minimal level autophagy is observed. However, the levels of LC3 appear to increase as the embryos are cultured in higher concentrations of glucose (Fig. 2A–E). We anticipate that the drop in intracellular glucose triggers an autophagic response in a rescue attempt to recycle cellular substrates and generate alternative energy substrates.

Figure 2
Figure 2

Autophagic and apoptoic responses to varying levels of excess glucose. LC3 expression in embryos. Negative control (A), physiologic 5 mM (B), 20 mM (C), 35 mM (D), and 52 mM (E) glucose. Cell death detection assay for apoptosis. Positive control of DNase-treated embryos (F), physiologic 5 mM (G), 20 mM (H), 35 mM (I), 52 mM (J) glucose. Each group consisted of at least 30 embryos and the experiment was repeated three times.

Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0265

To investigate the interchange between autophagy and apoptosis in the developing blastocyst, we repeated the experiment and used a cell death detection assay to determine the level of cell death occurring in each of the conditions. Previously, we have published results indicating an increase in apoptosis in embryos exposed to a hyperglycemic environment in vivo or in vitro (Moley et al. 1998a). While we observed a similar result, the levels of apoptosis appear to plateau above 20 mM glucose concentrations (Fig. 2F–J).

Therefore, we concluded that higher levels of cell stress induced by a diabetic milieu result in a dose-dependent increase of autophagy, as measured by the autophagosomal marker LC3. Interestingly, the embryos appear to display an internal rheostat that controls the level of apoptosis that occurs. We speculate that this persistence of the cells within the blastocyst that do not undergo apoptosis but display a compensatory increase in autophagy may result in significant changes in these specific cells, thereby permanently altering the lineages derived from this cell of the blastocyst. These cell-specific lineage changes could be responsible for the occurrence of malformations and/or miscarriages of these fetuses.

Changes in GAPDH activity reflect autophagic activation and inhibition

Although it is well established that increased GAPDH activity triggers flux through the glycolytic pathway in general and triose metabolism specifically, a recent study has demonstrated a previously unknown role for GAPDH in signaling the activation of autophagy (Colell et al. 2007). By overexpressing GAPDH, the investigators were able to protect the cells from caspase-independent cell death and even promote cell survival. In addition, they showed that increased GAPDH activity leads to an elevation in intracellular ATP as well as upregulation of ATG12, an autophagic protein involved in the formation of the autophagosome. We chose GAPDH activity as a metabolic marker of the autophagic response in these embryos.

To test whether GAPDH activity could be used as a marker of autophagy in blastocysts, we measured the activity using microanalytic enzymatic assays (Passonneau & Lowry 1993) in individual blastocysts exposed to either DMSO as vehicle control or 5 μM rapamycin for 30 h to activate autophagy (Supplementary Figure 2A, see section on supplementary data given at the end of this article). The activity increased significantly over vehicle. Conversely, blastocysts were exposed to control media versus media with added 4 vs 8 mM 3-methyladenine, a known inhibitor of autophagy for 30 h. Activity of GAPDH was significantly decreased in a dose-dependent manner by 3-MA (Supplementary Figure 2B). After the confirmation that GAPDH activity could serve as an accurate measure of autophagic activation in the embryos, blastocysts were collected from both in vivo and in vitro diabetic conditions and GAPDH activity was measured in each group (Fig. 3A and B). Our results indicate that these diabetic conditions result in an increase in the GAPDH activity, and we believe that this increase signifies an upregulation of autophagy in an attempt to protect the cell from damage as our other results suggest.

Figure 3
Figure 3

GAPDH activity and autophagy are increased in embryos exposed to diabetic environments both in vivo and in vitro. GAPDH activity (in vivo): embryos from control versus streptozocin-induced diabetic mothers (A) *P value <0.0001. GAPDH activity (in vitro): embryos exposed to 2.78 vs 52 mM d-glucose (B), **P value <0.0005 (n=at least 50 for each group).

Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0265

Autophagosomes are more abundant in oocytes of diabetic mice

Previously, our laboratory has published results indicating that in vivo maternal diabetes leads to an increase of apoptosis in the ovarian follicles of mice (Chang et al. 2005). Recently, our laboratory has also concluded that the mitochondria of diabetic oocytes display an abnormal morphology and distribution leading to a decrease in the metabolites. Furthermore, these oocytes display meiotic spindles defects and chromatin misalignment (Wang et al. 2009). Using GFP-LC3 mice, Mizushima et al. observed a basal level of autophagy occurring at the unfertilized oocyte stage; however, much lower than that of the fertilized oocyte (Tsukamoto et al. 2008a). Therefore, we hypothesize that the metabolic milieu of diabetes may lead to a dysregulation of autophagy in the oocytes of these mice.

By using transmission electron microscopy, we were able to obtain a more accurate estimation of the prevalence of autophagy during this stage (Fig. 4). Our results indicate the presence of basal level autophagosomes in control oocytes. The numbers of autophagosomes per area increase in oocytes obtained from diabetic mice. This increase suggests an increased activation of the autophagic pathway.

Figure 4
Figure 4

A representative autophagosome in a control (A) and diabetic (B) oocyte. There is a 43% increase in number of autophagosomes in the diabetic oocyte per area (sample size=100, ten sections from each group quantitated).

Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0265

Discussion

Although it has been determined that genetic removal of key players in the autophagic pathway leads to developmental arrest and embryonic malformations (Zheng et al. 2006, Fimia et al. 2007, Cecconi et al. 2008), the dysregulation of this pathway in response to embryonic environmental stressors has yet to be elucidated. This study suggests that an autophagic response to the environmental stress of a hyperglycemic milieu occurs during the oocyte and blastocyst stages of development and that this event is heterogeneously triggered at the blastocyst stage with some cells undergoing cell death and others surviving, but significantly and perhaps permanently altered by the autophagic process. We speculate that these remaining cells may alter their fate, thereby changing the originally assigned cell lineages and possibly having developmental consequences.

By exposing the embryos to an autophagic activator, rapamycin, we were able to observe an increase in autophagic proteins, AMBRA1 and LC3, as indicators of an increased activation of autophagy. Interestingly, the embryos display a heterogeneous autophagic response to culture conditions. This differential display of activation of a molecular pathway in embryos has been detailed before; however, this was in regard to apoptosis (Pampfer 2000). In those studies, it was suggested that the ICM, which will develop into the embryo, and the TE, which develops into the extraembryonic tissues including the placenta, consists of separate ‘micro-environments.’ This internal separation establishes differential gene expression, different levels of ion and glucose transport, and a varying response to cytotoxic agents (Pampfer 2000). Furthermore, cells within each of these compartments display a disparity. It has been reported, using cell lines derived from either the ICM or the TE, that the TE is more viable in a hyperglycemic environment, whereas the ICM is more susceptible to hyperglycemic environments, leading to an increased incidence in apoptosis (Pampfer 2000). This is recapitulated in vivo as the number of cells in the ICM of embryos exposed to maternal hyperglycemia is reduced compared with controls (Lea et al. 1996, Pampfer et al. 1997).

In this study, we show a different pattern of expression than apoptosis. Although, the expression of AMBRA1 appears exclusively in the TE, the autophagic activation by LC3 is seen in random individual cells of the blastocyst. Murine embryos inherit a pool of maternal transcripts that are progressively degraded and replaced by the products of embryonic transcription. This embryonic genomic activation is triggered when the blastomeres still exhibit developmental plasticity and can change their cell fate (Zernicka-Goetz et al. 2009). It is possible that this differential autophagic response in single cells in the embryo during pre-implantation development affects the subsequent lineage allocation in that cell and its resulting daughter cells. We postulate that this early change in lineage patterning may result not only in the growth abnormalities of infants and placentas but also in the malformations commonly seen in fetuses from diabetic mothers.

While the effect of a maternal hyperglycemic environment on embryo development has been studied, much is still left unanswered. Our laboratory has previously published data establishing a result of increased apoptosis in murine blastocysts exposed to a hyperglycemic diabetic environment in vivo or in vitro (Moley et al. 1998a). Furthermore, when transferred into pseudo-pregnant females, these exposed blastocysts display an increased rate of resorptions and malformations (Wyman et al. 2008). Surprisingly, one-cell zygotes when transferred from diabetic to non-diabetic mice also demonstrated a significantly higher rate of malformations and growth retardation, suggesting an even earlier period of vulnerability of the zygote to hyperglycemia. More recent studies by our laboratory have confirmed this hypothesis. Recently, we determined that GV and MII oocytes from diabetic mice have biochemical and meiotic abnormalities that could predispose them to developmental problems post-fertilization and perhaps implantation (Ratchford et al. 2007, Wang et al. 2009). We established that oocytes from diabetic mice have significantly lower ATP levels at both the GV and the MII stages (Ratchford et al. 2007). In addition, we demonstrate a significantly higher number of autophagosomes in GV stage oocytes by electron microscopy (Wang et al. 2009). Other groups have shown that the autophagic protein LC3 is not detected until after fertilization (Tsukamoto et al. 2008a); however, we show a significant difference in activation in the unfertilized oocyte from diabetic mice. This raises the possibility that maternal proteins at this very early stage may be prematurely degraded by autophagy, resulting in oocytes deficient in protein and perhaps predisposed to increased autophagy and other abnormalities, as we see in the blastocysts from diabetic mice. Future studies will be designed to test this hypothesis.

Recent studies have implicated GAPDH as a possible mediator and biochemical indicator of autophagic activation. Although the mechanism is not entirely elucidated, those studies demonstrated that overexpressing GAPDH in cells induced to undergo autophagy prevented this process. The conclusions were that GAPDH activity not only triggered metabolism of trioses within the glycolytic pathway but also, directly or indirectly, induced an increase in the transcription of ATG12, a key autophagic protein. Our study demonstrated a significant increase in the GAPDH activity within the whole blastocyst in response to maternal diabetes or in vitro high glucose. In addition, we confirmed that changes in this enzyme activity are predictive of either activation of autophagy by rapamycin or inhibition of autophagy by 3-methyladenine.

In conclusion, we show in this paper that the levels of autophagy are altered during oocyte and pre-implantation development in response to environmental stressors (Fig. 5). With the addition of the autophagy activator rapamycin, the levels of autophagy are increased in a cell-specific manner. In addition, the embryos cultured in a hyperglycemic environment mimicking maternal diabetes result in the elevation of the autophagic pathway. This elevation is also seen as an increase in the GAPDH activity, which has recently been suggested as an activator of the autophagic pathway. Furthermore, it appears that the autophagic pathway may be altered as early as the oocytes as a result of maternal diabetes. This early alteration in autophagy may result in changes in quality of the embryo after fertilization. However, further studies need to be conducted to validate this hypothesis. Overall, our data suggest that the hyperglycemic environment leads to an increased activation of autophagy during the oocyte growth phase and pre-implantation development, resulting in a differential response by the individual cells within the embryo.

Figure 5
Figure 5

Cartoon of the level of autophagy thought to exist in control versus diabetic oocytes and embryos. Although no autophagy was detected in MII oocytes from control mice (Tsukamoto et al. 2008a, 2008b), we determined increased autophagosomes (in this study) and decreased ATP in MII oocytes from diabetic mice (Ratchford et al. 2007). In addition, although some autophagic proteins have been detected in control blastocysts, diabetic blastocysts demonstrated increased LC3 and AMBRA1 protein (this study) as well as decreased ATP (Chi et al. 2002). The sinusoidal pattern of autophagy is an estimation based on prior work as well as this study. Furthermore, we speculate that increased autophagy at the oocyte stage may prematurely degrade maternal proteins leading to developmental arrest, and increased autophagy at the blastocyst stage may lead to abnormal degradation of embryonic proteins essential for proper development.

Citation: REPRODUCTION 141, 5; 10.1530/REP-10-0265

Materials and Methods

Oocyte and embryo retrieval

To collect ovulated oocytes, control and diabetic B6SJL mice (Jackson Laboratory, Bar Harbor, ME, USA) received an injection of 10 IU hCG 2 days after PMSG priming (PSMG 2000 IU, National Hormone & Peptide Program, Torrance, CA, USA; hCG, Sigma). Oocytes were recovered from oviductal ampullae 13.5 h post-hCG, and cumulus cells were removed by incubating briefly in 1 mg/ml hyaluronidase. For embryo retrieval, the mice received the hormonal stimulation mentioned earlier and were then mated overnight with males of proven fertility. Mating was confirmed by the presence of a vaginal plug. Embryos for the in vivo blastocyst study (Fig. 3A) were obtained by flushing the uterine horns 96 h post-hCG. For the in vitro hyperglycemia and autophagy activation studies (Figs 1A–E, 2A–J and 3B), the embryos were recovered from control mice 76 h post-hCG and mating and were cultured for 30 h in KSOM/0.25% BSA (v/v) (KSOM, Millipore Specialty Media, Danvers, MA, USA) and the indicated culture conditions at 37 °C in 5% CO2.

Immunofluorescence staining

Embryos were collected and fixed in 3% paraformaldehyde in PBS/2% BSA (v/v) for 20 min at room temperature. The embryos were permeabilized in 0.5% Triton X-100 in PBS/2% BSA for 30 min. To block, the embryos were placed in 5% normal goat serum in PBS/2% BSA for 1 h. Then, the embryos were incubated in the primary antibody (1:250) in PBS/2% BSA overnight at 4 °C (LC3: Novus Biologicals, Littleton, CO, USA; AMBRA1: Cell Signaling Technology, Danvers, MA, USA). The embryos were washed three times in PBS/2% BSA and then incubated in secondary antibody (Alexa Fluor goat-anti-rabbit IgG 488; Molecular Probes, Eugene, OR, USA) for 45 min at room temperature. The embryos were rinsed three times again and placed in To-pro-3-iodide (Molecular Probes; 1:500) in PBS for 15 min. After three more rinses in PBS, the embryos were mounted on slides using Vectashield (Vecta, Burlingame, CA, USA) and visualized by confocal microscopy. All experimental groups (Figs 1A–C or D, E and 2) were conducted on the same day in order to use the same prepared secondary antibody preparation. Relative fluorescence was quantified by a blinded observer as described previously (Jungheim et al. 2010).

Cell death assay

Apoptosis was detected using the In Situ Cell Death Detection kit, TMR red (Roche). The embryos were fixed and permeabilized as indicated in the immunofluorescence staining method. A positive control was obtained by treating embryos with DNase (0.5 μl/ml in PBS) for 20 min at 37 °C. Then, the embryos were incubated in the reaction mix, as per the manufacturer's specifications for 1 h at 37 °C in the dark. All subsequent reactions were also carried out in the dark. After three rinses in PBS/2% BSA, the embryos were counterstained with To-pro-3-iodide as mentioned earlier, rinsed, and then mounted on slides. The slides were viewed on a confocal microscope.

GAPDH activity assay

This assay has been previously described for individual cell activity measurements (Passonneau & Lowry 1993). Individual mouse blastocysts were extracted in 20 mM phosphate buffer (pH 7.4), 0.02% BSA, 0.5 mM EDTA (pH 7.0), 5 mM B-Me, 0.25% glycerol, and 0.5% Triton X-100 (v/v) at room temperature for 120 min under oil and stored at −75 °C. A 0.1 μl aliquot was added to 1 μl GAPDH reagent containing 50 mM imidazole HCl (pH 7.0), 0.05% BSA, 1 mM EDTA (pH 7.0), 1 mM B-Me, 1 mM Na2HASO4, 100 μM NAD, and 100 μM glyceraldehyde 3-phosphate at room temperature for 1 h. NADH standards in GAPDH reagent were added in this step. The reaction was stopped by the addition of 1 μl of 0.12 M NaOH and heated to 80 °C for 25 min. To amplify, a 0.5 μl aliquot was taken out and added to 10 μl NAD cycling reagent at room temperature overnight under oil for 15 000-fold amplification, as performed previously by our group (Chi et al. 2002). The reaction was stopped by the addition of 1 μl 1 M NaOH and heated at 80 °C for 25 min. To read the samples, a 10 μl aliquot was taken out and added to 1 ml malate indicator reagent, as performed previously to obtain the NADH final reading in an A-1 Filter Fluorometer (Farrand Optical Components and Instruments, Valhalla, NY, USA; Chi et al. 2002). All calculations were based on the internal NADH standards.

Generation of diabetic mice

To generate an in vivo diabetic model, 3-week-old female B6SJLF1 mice received a single injection of streptozotocin at a dose of 190 mg/kg. A tail-blood sample was measured for glucose concentrations via a Contour TS One Touch Glucometer (Bayer) 4 days after injection. Glucose levels >300 mg/dl were considered diabetic. Age-matched controls injected with PBS were used.

Transmission electron microscopy

For ultrastructural analysis of autophagy, 100 oocytes of each group were processed for transmission electron microscopy as described previously in the Molecular Microbiology Imaging Facility at Washington University in St Louis (Gualtieri et al. 2009). The presence of autophagosomes was determined from electron micrographs at 10 000× magnification. To quantify the autophagosomes, the number of autophagosomes and the area in ten random sections of each group were recorded using ImageJ (National Institutes of Health, Bethesda, MD, USA). The following criteria for identifying autophagosomes were used: vesicles with a double membrane, between 0.3 and 2 μm, with clearly recognizable cytoplasmic contents and are not multilamellar bodies.

Statistical analysis

All experiments were completed in triplicate with at least ten embryos or oocytes per group in each experiment. The images in the figures are representative of the group of embryos in each group. For the GAPDH assay, a Student's t-test was performed. Significance was defined as P<0.05.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-10-0265.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

K L Adastra: National Institute of Health (T32 GM07067), K H Moley: National Institute of Health (R01 HD40390).

Acknowledgements

The authors would like to acknowledge Wandy Beatty in the Molecular Microbiology Imaging Facility at the Washington University in St Louis for the transmission electron microscopy imaging and The Bakewell Neuroimaging Laboratory for the use of the confocal microscopy for imaging.

References

  • Akazawa H, Komazaki S, Shimomura H, Terasaki F, Zou Y, Takano H, Nagai T & Komuro I 2004 Diphtheria toxin-induced autophagic cardiomyocyte death plays a pathogenic role in mouse model of heart failure. Journal of Biological Chemistry 279 4109541103 doi:10.1074/jbc.M313084200.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Aki T, Yamaguchi K, Fujimiya T & Mizukami Y 2003 Phosphoinositide 3-kinase accelerates autophagic cell death during glucose deprivation in the rat cardiomyocyte-derived cell line H9c2. Oncogene 22 85298535 doi:10.1038/sj.onc.1207197.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Amherdt M, Harris V, Renold AE, Orci L & Unger RH 1974 Hepatic autography in uncontrolled experimental diabetes and its relationships to insulin and glucagon. Journal of Clinical Investigation 54 188193 doi:10.1172/JCI107742.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Boya P, Gonzlez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Mtivier D, Meley D, Souquere S & Yoshimori T et al. 2005 Inhibition of macroautophagy triggers apoptosis. Molecular and Cellular Biology 25 10251040 doi:10.1128/MCB.25.3.1025-1040.2005.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Brown JJ & Whittingham DG 1991 The roles of pyruvate, lactate and glucose during preimplantation development of embryos from F1 hybrid mice in vitro. Development 112 99105.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cecconi F, Piacentini M & Fimia GM 2008 The involvement of cell death and survival in neural tube defects: a distinct role for apoptosis and autophagy? Cell Death and Differentiation 15 11701177 doi:10.1038/cdd.2008.64.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chang AS, Dale AN & Moley KH 2005 Maternal diabetes adversely affects preovulatory oocyte maturation, development, and granulosa cell apoptosis. Endocrinology 146 24452453 doi:10.1210/en.2004-1472.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chi MM, Hoehn A & Moley KH 2002 Metabolic changes in the glucose-induced apoptotic blastocyst suggest alterations in mitochondrial physiology. American Journal of Physiology. Endocrinology and Metabolism 283 E226E232 doi:10.1152/ajpendo.00046.2002.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Colell A, Ricci JE, Tait S, Milasta S, Maurer U, Bouchier-Hayes L, Fitzgerald P, Guio-Carrion A, Waterhouse NJ & Li CW et al. 2007 GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation. Cell 129 983997 doi:10.1016/j.cell.2007.03.045.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cooper ASR, Boehle K, Riley J & Moley K 2008 Autophagy is a physiologic process regulated by glucose availability in the murine preimplantation blastocyst. Reproductive Sciences 15 73A.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fimia GM, Stoykova A, Romagnoli A, Giunta L, Di Bartolomeo S, Nardacci R, Corazzari M, Fuoco C, Ucar A & Schwartz P et al. 2007 Ambra1 regulates autophagy and development of the nervous system. Nature 447 11211125 doi:10.1038/nature05925.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fujitani Y, Kawamori R & Watada H 2009 The role of autophagy in pancreatic beta-cell and diabetes. Autophagy 5 280282 doi:10.4161/auto.5.2.7656.

  • Grune T, Merker K, Sandig G & Davies KJ 2003 Selective degradation of oxidatively modified protein substrates by the proteasome. Biochemical and Biophysical Research Communications 305 709718 doi:10.1016/S0006-291X(03)00809-X.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gualtieri R, Iaccarino M, Mollo V, Prisco M, Iaccarino S & Talevi R 2009 Slow cooling of human oocytes: ultrastructural injuries and apoptotic status. Fertility and Sterility 91 10231034 doi:10.1016/j.fertnstert.2008.01.076.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jungheim ES, Schoeller EL, Marquard KL, Louden ED, Schaffer JE & Moley KH 2010 Diet-induced obesity model: abnormal oocytes and persistent growth abnormalities in the offspring. Endocrinology 151 40394046 doi:10.1210/en.2010-0098.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kiffin R, Bandyopadhyay U & Cuervo AM 2006 Oxidative stress and autophagy. Antioxidants & Redox Signaling 8 152162 doi:10.1089/ars.2006.8.152.

  • Lea RG, McCracken JE, McIntyre SS, Smith W & Baird JD 1996 Disturbed development of the preimplantation embryo in the insulin-dependent diabetic BB/E rat. Diabetes 45 14631470 doi:10.2337/diabetes.45.11.1463.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Leese HJ 1991 Metabolism of the preimplantation mammalian embryo. Oxford Reviews of Reproductive Biology 13 3572.

  • Maiuri MC, Zalckvar E, Kimchi A & Kroemer G 2007 Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nature Reviews. Molecular Cell Biology 8 741752 doi:10.1038/nrm2239.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Masini M, Bugliani M, Lupi R, del Guerra S, Boggi U, Filipponi F, Marselli L, Masiello P & Marchetti P 2009 Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia 52 10831086 doi:10.1007/s00125-009-1347-2.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mizushima N, Yamamoto A, Matsui M, Yoshimori T & Ohsumi Y 2004 In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Molecular Biology of the Cell 15 11011111 doi:10.1091/mbc.E03-09-0704.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Moley KH, Chi MM, Knudson CM, Korsmeyer SJ & Mueckler MM 1998a Hyperglycemia induces apoptosis in pre-implantation embryos through cell death effector pathways. Nature Medicine 4 14211424 doi:10.1038/4013.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Moley KH, Chi MM & Mueckler MM 1998b Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos. American Journal of Physiology 275 E38E47.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pampfer S 2000 Apoptosis in rodent peri-implantation embryos: differential susceptibility of inner cell mass and trophectoderm cell lineages – a review. Placenta 21 (Suppl A) S3S10 doi:10.1053/plac.1999.0519.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pampfer S, Vanderheyden I, McCracken JE, Vesela J & De Hertogh R 1997 Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro. Development 124 48274836.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Passonneau JV & Lowry OH 1993 Enzymatic Analysis, Totowa, NJ: Humana Press.

  • Qu X, Zou Z, Sun Q, Luby-Phelps K, Cheng P, Hogan RN, Gilpin C & Levine B 2007 Autophagy gene-dependent clearance of apoptotic cells during embryonic development. Cell 128 931946 doi:10.1016/j.cell.2006.12.044.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ratchford AM, Chang AS, Chi MM, Sheridan R & Moley KH 2007 Maternal diabetes adversely affects AMP-activated protein kinase activity and cellular metabolism in murine oocytes. American Journal of Physiology. Endocrinology and Metabolism 293 E1198E1206 doi:10.1152/ajpendo.00097.2007.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rodriguez-Enriquez S, He L & Lemasters JJ 2004 Role of mitochondrial permeability transition pores in mitochondrial autophagy. International Journal of Biochemistry & Cell Biology 36 24632472 doi:10.1016/j.biocel.2004.04.009.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sakaida I, Kyle ME & Farber JL 1990 Autophagic degradation of protein generates a pool of ferric iron required for the killing of cultured hepatocytes by an oxidative stress. Molecular Pharmacology 37 435442.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Scott RC, Schuldiner O & Neufeld TP 2004 Role and regulation of starvation-induced autophagy in the Drosophila fat body. Developmental Cell 7 167178 doi:10.1016/j.devcel.2004.07.009.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Singh R, Xiang Y, Wang Y, Baikati K, Cuervo AM, Luu YK, Tang Y, Pessin JE, Schwartz GJ & Czaja MJ 2009 Autophagy regulates adipose mass and differentiation in mice. Journal of Clinical Investigation 119 33293339 doi:10.1172/JCI35541.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sooparb S, Price SR, Shaoguang J & Franch HA 2004 Suppression of chaperone-mediated autophagy in the renal cortex during acute diabetes mellitus. Kidney International 65 21352144 doi:10.1111/j.1523-1755.2004.00639.x.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Thorburn A 2008 Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis 13 19 doi:10.1007/s10495-007-0154-9.

  • Towns R, Kabeya Y, Yoshimori T, Guo C, Shangguan Y, Hong S, Kaplan M, Klionsky DJ & Wiley JW 2005 Sera from patients with type 2 diabetes and neuropathy induce autophagy and colocalization with mitochondria in SY5Y cells. Autophagy 1 163170 doi:10.4161/auto.1.3.2068.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tsukamoto S, Kuma A & Mizushima N 2008a The role of autophagy during the oocyte-to-embryo transition. Autophagy 4 10761078.

  • Tsukamoto S, Kuma A, Murakami M, Kishi C, Yamamoto A & Mizushima N 2008b Autophagy is essential for preimplantation development of mouse embryos. Science 321 117120 doi:10.1126/science.1154822.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Wang Q, Ratchford AM, Chi MM, Schoeller E, Frolova A, Schedl T & Moley KH 2009 Maternal diabetes causes mitochondrial dysfunction and meiotic defects in murine oocytes. Molecular Endocrinology 23 16031612 doi:10.1210/me.2009-0033.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Wyman A, Pinto AB, Sheridan R & Moley KH 2008 One-cell zygote transfer from diabetic to nondiabetic mouse results in congenital malformations and growth retardation in offspring. Endocrinology 149 466469 doi:10.1210/en.2007-1273.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yue Z, Jin S, Yang C, Levine AJ & Heintz N 2003 Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. PNAS 100 1507715082 doi:10.1073/pnas.2436255100.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zernicka-Goetz M, Morris SA & Bruce AW 2009 Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo. Nature Reviews. Genetics 10 467477 doi:10.1038/nrg2564.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zheng B, Tang T, Tang N, Kudlicka K, Ohtsubo K, Ma P, Marth JD, Farquhar MG & Lehtonen E 2006 Essential role of RGS-PX1/sorting nexin 13 in mouse development and regulation of endocytosis dynamics. PNAS 103 1677616781 doi:10.1073/pnas.0607974103.

    • PubMed
    • Search Google Scholar
    • Export Citation

 

  • Collapse
  • Expand
  • AMBRA1 and LC3 expression in rapamycin-treated embryos. In each group, ten mouse control blastocysts were incubated for 30 h: DMSO control (A), 1 μM (B), or 5 μM (C) rapamycin. This experiment was repeated three times. AMBRA1 expression is increased in a variable pattern, with the most expression observed in the trophectoderm (TE) cells. LC3 expression in mouse control blastocysts incubated in DMSO (D, n=10) or 5 μM rapamycin (E, n=10). This experiment was conducted three times.

  • Autophagic and apoptoic responses to varying levels of excess glucose. LC3 expression in embryos. Negative control (A), physiologic 5 mM (B), 20 mM (C), 35 mM (D), and 52 mM (E) glucose. Cell death detection assay for apoptosis. Positive control of DNase-treated embryos (F), physiologic 5 mM (G), 20 mM (H), 35 mM (I), 52 mM (J) glucose. Each group consisted of at least 30 embryos and the experiment was repeated three times.

  • GAPDH activity and autophagy are increased in embryos exposed to diabetic environments both in vivo and in vitro. GAPDH activity (in vivo): embryos from control versus streptozocin-induced diabetic mothers (A) *P value <0.0001. GAPDH activity (in vitro): embryos exposed to 2.78 vs 52 mM d-glucose (B), **P value <0.0005 (n=at least 50 for each group).

  • A representative autophagosome in a control (A) and diabetic (B) oocyte. There is a 43% increase in number of autophagosomes in the diabetic oocyte per area (sample size=100, ten sections from each group quantitated).

  • Cartoon of the level of autophagy thought to exist in control versus diabetic oocytes and embryos. Although no autophagy was detected in MII oocytes from control mice (Tsukamoto et al. 2008a, 2008b), we determined increased autophagosomes (in this study) and decreased ATP in MII oocytes from diabetic mice (Ratchford et al. 2007). In addition, although some autophagic proteins have been detected in control blastocysts, diabetic blastocysts demonstrated increased LC3 and AMBRA1 protein (this study) as well as decreased ATP (Chi et al. 2002). The sinusoidal pattern of autophagy is an estimation based on prior work as well as this study. Furthermore, we speculate that increased autophagy at the oocyte stage may prematurely degrade maternal proteins leading to developmental arrest, and increased autophagy at the blastocyst stage may lead to abnormal degradation of embryonic proteins essential for proper development.