Among various biological imaging methods, FIB-SEM exhibits obvious advantages in obtaining meticulous organelle information at a subcellular level with 3D view revealing entire cellular spatial distribution. Amira's precise algorithm not only produce 3D images efficiently, but also facilitates the measurement, calculation and comparation of number, area and volume on target research objectives. In this study, combining computer image processing with FIB‐SEM, a large amount of comprehensive 3D information on cell substructures is obtained. The OCC, a special complex comprised of an oocyte and granulosa cells are described in 3D space. This is an elementary unit for female reproductive outcome because it is where oocyte growth, maturation, meiosis and ovulation take place. FIB-SEM 3D imaging technology helps us to further understand the mechanism of functional realization in OCCs.

Cells and extracellular matrix can be clearly distinguished in FIB-SEM images. 1) Based on the continuity of the structure. The cell structure was continuous in all images of FIB-SEM without interruption from the first slice to the last. Non cellular substances, however, were non-continuous and appeared randomly. 2) Based on the difference in electron density. The electron density of cellular structures was high, while the electron density of non-cellular structures was low. Under electron microscopy, the contrast between cellular structures and non-cellular structures was significant. 3) Based on the different morphology The cellular structure normally exists as an integrated structure, in the form of sheets or blocks, while the non-cellular structures (impurities) were mostly sparsely distributed dots.

As can be seen in our results, OCC is a multicellular complex rich in intercellular connections. These connections are an important basis for interaction and synergy between adjacent cells. In general, there are four main types of cell connections: a) tight junctions with blocking effect, which usually close the intercellular space at tissue surface; b) intermediate junctions with adhesion effect, which maintain cell shapes and transmit contractile force from cell to cell; c) desmosomes with anchoring effect, which fix and support cells. d) gap junctions with communication effect, which facilitates intercellular exchange of certain small molecules and ions. Among the four types, only gap junctions were observed between cGCs and between oocytes and cGCs. On our 3D images, the adjacent plasma membranes at the junctions are very close to each other and extremely parallel, the distance is as short as 3 nm. The density and abundance in the microvilli-like protrusions on the surface of cGCs and oocytes facilitate the formation of extensive gap junctions between cells.

Previously confirmed by X-ray diffraction technology, the gap junction is a hexameric structure of membrane protein molecules. Six monomers constitute a central tubule protruding outwards from the cell membrane, directly opposite to the central tubule of the adjacent cell in a spatial symmetry pattern. These central tubules may help facilitate exchanges of ions and small molecules between cells, such as amino acids, glucose, nucleotides, vitamins, hormones, growth factors, cAMPs, etc. Unfortunately, the hexamer could not be observed at the FIB-SEM resolution level.

Granulosa cells differentiate throughout follicular development from flat to columnar. They first form monolayers then stratified layers, and then synthesize and secrete mucopolysaccharides surrounding the oocyte to assist zona pellucida formation. Meanwhile, the innermost layer consistently remains close contact with the oocyte to allow two-way communication [18]. The abundance in microvilli in both cGCs and oocytes were greater than we expected. The characteristic microvilli on the granulosa cells adjacent to ZP are called transzonal projections (TZPs). TZPs can pass through the ZP as thick as 3–5 μm and reach oolemma, forming gap junctions connecting cGCs and the oocyte.

As the follicle develops, the plentiful cross-belt membrane protrusions provide extensive gap junctions for better nutrients and signal transmission to regulate oocyte maturation [19]. The existence of gap junctions in OCCs is essential for follicles to develop, mature and respond to endocrine signals as a whole. In addition, gap junction is a dynamic structure [20], whose opening and closing can be regulated by many factors. For example, the pores close upon a decrease in membrane potential or pH, or an increase in calcium ions (Ca2+) concentration, to protect cells from damage.

CGCs are the driver of follicular growth as they are responsible for energy supply and signal transmission [21]. Although appeared sphericalunder light microscopy observation, they are in fact in irregular shape. With the high resolution of the electron microscope, a large number of tentacles and pseudopods with different lengths can be seen on the cell membranes of cGCs. Oocytes, on the other hand are generally spherical even under electron microscopy, because they are plump and possess abundant microvilli on the surface of the cell membrane. In this study, we confirmed that cGCs have abundant typical energy and steroid producing organelles, such as mitochondria, ER and lipid droplets. Interaction is observed between these organelles.

In response to the need of follicular growth, oxidative phosphorylation is intensified in cGCs to produce sufficient ATP [22], accompanied with high mitochondrial membrane potential [23] and high ROS levels [24, 25]. In our images, the mitochondria possess tightly-packed tubular vesicle cristae and distribute close to ER, which is evidence of highly active oxidative phosphorylation. However, pathological granulosa cells are characterized with mitochondrial crista dissolution, fracture, and vacuole formation. More seriously, pathological granulosa cells exhibit obvious features of apoptosis [26]. Besides, the 3D images also revealed a lot of M-SER and MV structures, which may be involved in material or membrane reservoirs, and work as one of the predictive factors for subsequent fertilization and early embryogenesis. In cGCs, the mitochondria tightly surround the lipid droplets. The mitochondrial-associated membrane (MAM) not only forms a special part of ER but also constitutes the lipid synthesis center. Membrane contact sites between lipid droplets and mitochondria play a role in mitochondrial fatty acid transport and beta oxidation [27].

ER is the intracellular reservoir of Ca2+ and contains most of the biosynthetic enzymes involved in the synthesis of lipids. By regulating Ca2+ signal transduction, ER controls lipid synthesis and mitochondrial biogenesis. The cGCs with vigorous ER provide more energy and materials to the oocyte. The abundance in functional ER is an indicator of good-quality cGCs.

The association between oocyte ultrastructural abnormalities and infertility was first reported by Afzeliusin in 1955 [28]. Since then, EM technology has made a great contribution to the diagnosis of infertility-related diseases, but the majority only focuses on abnormal structure of oocytes. As our understanding of the whole follicle structure deepens, the abnormal morphology of cGCs may also provide valuable diagnostic information in the future.

In this study, we discovered for the first time a special ciliary structure in granulosa cells. Cilia are microtubule-based hair-like organelles with different functions at different stages of a cell. During cell division, it acts as a centriole; during functional phase, it acts as a sensor. Cilia protrude out of cell surface to help cell move and sense signals. It can thus be divided into two categories: motor cilia and non-motor cilia [29]. Motor cilia have a typical 9 + 2 microtubule structure. Take the tail of sperm as an example, its dysfunction is related to impaired male reproductive tract development and male infertility [30]. Non-motor cilia are also called sensory cilia, made up of microtubules. They may act as antennas, receiving and converting signals in intercellular communication [31], which is essential in the development of tissues and organs.

Although cilia are reported to be ubiquitous in cells, their existence and function in granulosa cells are still controversial [32]. Our study showed that each of the analyzed cGCs had a cilium lacking a central microtubule. The cilia protrude out of the cell surface in random directions. We hypothesize that they may help the cGCs to perceive the external micro-environment and make adjustment according physiological activity.

Studies have shown that cilia have abundant Ca2+ permeable membrane channels, activation of which can cause Ca2+ influx. Is calcium channel the main sensing channel in granulosa cell cilia? What signals do the cilia sense? What kind of cell activities can they regulate? What is the role of cilia in luteinization of granulosa cells? What is the association between the cilia and infertility? All of these questions deserve further study.