Maternal diabetes causes abnormal dynamic changes of endoplasmic reticulum during mouse oocyte maturation and early embryo development

Women with poorly controlled type I diabetes encounter a higher prevalence of reproductive problems, such as infertility, miscarriage and offspring with congenital malformations [1]. Earlier studies showed that the diabetic condition adversely affects the development of pre- and post-implantation embryos in rodents [2‐5]. Furthermore, numerous reports have suggested that in vitro-cultured two-cell stage embryos that were recovered from diabetic mice still experience significant delay in their progression to the blastocyst stage and about 50% of two-cell stage embryos isolated from sub-diabetic rats were unable to develop to the eight-cell stage, even in a non diabetic tract [4, 6]. Other studies revealed that preovulatory oocytes from chemically induced diabetic mice experience delayed germinal vesicle (GV) breakdown and abnormal cellular metabolism [4, 7‐9]. Oocytes from diabetic mice displayed a higher frequency of spindle defects and chromosome misalignment in meiosis, resulting in increased aneuploidy rates in ovulated oocytes [10]. To date, however, the effects of maternal diabetes on cytoplasmic structures of oocytes and early embryos remain poorly understood.

During maturation, the oocyte undergoes numerous cytoplasmic changes in preparation for successful fertilization and early embryonic development. One of these changes involves acquiring the ability of the mature oocyte to release Ca²⁺ which is critical for preventing polyspermy and stimulating the oocyte to complete meiosis and begin early development [11]. An important component of the Ca²⁺ release system is the endoplasmic reticulum (ER), which is a multifunctional organelle that consists of a network of membraneous tubules extending throughout the cell [12]. In maturing oocytes, the ER undergoes distribution changes that are associated with the ability of the oocyte to be fertilized successfully [13‐20]. ER reorganization is characterized by the development of cortical clusters of ER; this formation correlates with the ability of the maturing oocyte to generate Ca²⁺ transients in response to sperm and inositol 1,4,5-trisphosphate (InsP3) [19]. The presence of cortical ER clusters in mammalian oocytes has been proposed to account for the susceptibility of the cortex to sperm factors and InsP3 [21] and the spatial organization of the sperm-induced Ca²⁺ wave [22]. The similar distribution of the ER clusters and InsP3 receptors (Insp3Rs) further suggests that the ER clusters are specialized sites for the initiation and propagation of Ca²⁺ waves in oocytes [15, 17, 20]. Changes in the ER distribution patterns also take place after fertilization [12, 19]. The spindle-associated ER is seen in most mitotic cells, including those in early stage embryos and in somatic cells during development [12, 20].

The objective of this study was to examine how diabetes affects oocyte and embryo quality in relation to the ER distribution pattern as a cytoplasmic criterion. By employing time-lapse live cell imaging confocal microscopy, we revealed dynamic changes of ER structure and found that the diabetic condition adversely affects the distribution pattern of ER during mouse oocyte maturation, fertilization and early embryo development.

The ER is a dynamic structure, capable of changing its cellular organization and distribution patterns remarkably as shown at fertilization of starfish and sea urchin eggs. Here using time-lapse live imaging confocal microscopy, we showed that mouse oocytes undergo a dramatic reorganization of ER during meiotic maturation in vitro and in vivo. GV-stage oocytes contained a fine ER network throughout the interior cytoplasm and cortex. Following GVBD, ER surrounded the spindle during its migration to the oocyte cortex. MII oocytes contained striking ER accumulations at the cortex, with no apparent polarity in relation to the meiotic spindle, similar to those described previously [19, 24, 25]. On the other hand, we first revealed that maternal diabetes is associated with inadequate translocation of ER during oocyte maturation and a high proportion of oocytes from diabetic mice showed morphological abnormalities. Morphological parameters have been widely recognized as an indicator of oocyte quality. In our study, we showed that oocytes with morphological abnormalities degenerated at a high frequency, and only those oocytes with a normal appearance were selected for further analysis. We clearly observed a homogeneous distribution of ER throughout the entire ooplasm during the meiotic maturation process in oocytes from diabetic mice. ER distribution is an indicator for cytoplasmic maturation [26]. Studies have shown that spatial remodeling of endoplasmic reticulum render the oocyte capable of supporting development [24]. The ER is a vast membranous network responsible for protein synthesis and assembly, maturation, and along with the Golgi apparatus, transportation and release of correctly folded proteins. It is also a critical site for Ca²+ homeostasis [10, 27]. Ca²⁺-ATPases, Ca²⁺ storage proteins, and specific Ca²⁺ release channels have been localized to the ER, which permits this organelle to perform a crucial role in the regulation of intracellular Ca²⁺[28, 29]. Moreover, the ER contains InsP3 receptors and, in some cases, ryanodine receptors, both of which mediate Ca²⁺ release from the ER [30]. A specialized ER organization in MII mouse oocytes are the cortical ER clusters which act as pacemaker sites for the generation of Ca²⁺ oscillations at fertilization [31, 32]. The vegetal cortex is more sensitive to InsP3 and Ca²⁺-releasing sperm extracts [21] and acts as the Ca²⁺ wave pacemaker at fertilization [22, 31] and even after fertilization near the spindle [33]. The localization of InsP3Rs to the ER clusters suggests an important role in regulating the initiation of Ca²⁺ release [20, 33]. Clustering of InsP3Rs in ER increases the sensitivity of Ca²⁺ release such that coherent signals can be generated in response to very low levels of stimuli that otherwise would not elicit a response [34]. This may be particularly pertinent to fertilization of mammalian eggs where low InsP3 concentrations have been proposed [35] and where the signaling pathway involves the introduction of a phospholipase C from a very small cell (the sperm) into a very large cell (the egg) [35]. These observations demonstrate that the cortical ER clusters play an important role in the initiation and spatial organization of Ca²⁺ signaling at fertilization. Thus, we can conclude that inadequate redistribution of ER may be one of the important factors contributing to the maturation delay and spindle/chromosome disorganization observed in diabetic oocytes. However, previous studies revealed that ovulated oocytes from diabetic mice displayed an alteration in mitochondrial ultrastructure, and quantitative analysis of mitochondrial DNA copy number demonstrated an increase [10]. Therefore, the defects in diabetic oocytes might be the interaction between the ER and mitochondria. Taken together, the above results suggest that maternal diabetes leads to inadequate redistribution of ER during oocyte maturation in vitro and in vivo.

As a central regulator of protein quality control, folding, trafficking, and targeting, the ability of the ER to adapt its capacity to manage synthetic, metabolic, and other adverse conditions is of paramount importance for the cell [29]. In the present study we found spindle-associated ER as well as larger areas of ER-fluorescence deeper within the cytoplasm in mouse early embryos. ER displayed a homogeneous distribution pattern throughout the entire ooplasm during development of embryos from diabetic mice. We conclude that the impaired organization of the ER could account for the reduced developmental potential observed in early embryos from diabetic mice.

In addition, very large ER aggregations were seen in GV oocytes or in two-cell embryos from diabetic mice. First, we found that GV oocytes from diabetic mice displayed a significantly higher percentage of aggregated ER distribution areas near the nucleus when compared with controls. These oocytes were not able to resume meiotic maturation and completely deteriorated within a short time. Second, we found that two-cell embryos from diabetic mice showed a higher percentage of very large aggregated ER throughout the cytoplasm when compared to controls. Importantly, most of them were unable to develop further. The ER elicits an elaborate adaptive response known as the unfolded protein response (UPR). Much of the systemic physiology related to its dysfunctions has been viewed in the context of its luminal adaptation to protein processing and folding. In eukaryotic cells, monitoring of the ER lumen and the canonical branches of the UPR are mediated by three ER membrane-associated proteins. In a well-functioning and “stress-free” ER, these transmembrane proteins are bound by a chaperone in their intraluminal domains and rendered inactive [36, 37]. There are three mechanisms to mitigate ER stress which includes reducing protein synthesis, facilitating protein degradation, and increasing production of chaperones that help proteins in the ER lumen to fold. These mechanisms are implicated in resolving stress; if they fail the cell becomes functionally compromised and may undergo apoptosis. Together with our findings, we propose that the very large aggregated ER in oocytes or early embryos may contribute to ER stress and UPR, and hence commit to cell death which needs further detailed investigation.

In summary, our results show that maternal diabetes disrupts the ability of the ER to reorganize during oocyte maturation and early embryo development, and this could contribute to the reproductive problems experienced by type I diabetic mice as well as women. Our findings may have clinical implications for the assessment of oocyte quality from diabetic women. For example, polarization microscopy [38] can be used to directly recognize those diabetic oocytes with abnormal ER distribution; therefore it may be possible to screen diabetic oocytes to choose oocytes or embryos with optimal quality, finally, targeting drugs to improve ER function in oocytes may have therapeutic potential in treating reproductive failure of diabetic women.