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Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before., (© 2021 Ronchi et al.)

Sepsis is defined as a systemic inflammatory response to infection. This study is aimed to evaluate the effects of experimental sepsis on the proliferation and apoptosis of granulosa and theca cells in the rat ovary. 28-day-old immature Wistar-Albino female rats were treated with pregnant mare serum gonadotrophin to develop the first generation of preovulatory follicles. Sepsis was induced by cecal ligation and puncture (CLP). Following in vivo 5-Bromo-2-deoxyuridine (BrdU) labeling, animals were sacrificed and ovaries were embedded in paraffin and Epon. Besides electron microscopic evaluation, BrdU, cleaved caspase-3, p27 immunostaining, and TUNEL labeling were performed. In CLP-operated animals, cleaved caspase-3 immunoreactivity was significantly increased in Graafian follicles. TUNEL and BrdU labeling in the ovarian follicles were not statistically different between CLP and sham-operated rats. In septic animals, p27 immunoreactivity was increased significantly in the nuclei of oocytes and decreased in the cytoplasm of granulosa and theca cells in multilaminar primary follicles compared to the sham group. In ultrastructural evaluation, increased apoptosis was observed in theca interna and granulosa cells in both the early and late stages of follicles in the CLP group. In conclusion, experimentally-induced sepsis leads to apoptosis in ovarian follicles at advanced stages of development. Our data suggest that although sepsis may not cause a potential threat to developing follicles at least in the short term, more severe damage may occur during advanced stages of follicle development., (Copyright © 2020 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Immunoexpression of 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450c17 (P450c17), androgen receptor (AR), and steroid contents were studied in the ovaries of immature female Wistar rats killed between postnatal days 1 and 30. During days 1-7, ovarian somatic structures lacked AR, 3β-HSD and P450c17, except for the surface epithelium, which featured the presence of these three proteins, suggestive of its androgen responsiveness and steroidogenic function. On day 10, AR appeared in many somatic structures, including the granulosa layers, which coincided with the P450c17 immunoexpression in some theca/interstitial cells, and an increase in ovarian androgen concentration. On the following days a further rise in ovarian androgen and progesterone contents paralleled an increase in 3β-HSD and P450c17 immunoexpression in the theca layer cells and primary interstitial cells. However, the development of the follicles constituting the first follicular wave was aberrant, since they lacked AR expression until the preantral stage and were characterized by a delayed onset and much lower expression of the thecal P450c17. They could not ovulate, since ovarian content of estradiol was too low to evoke the LH surge. The clusters of the secondary interstitial cells found on day 30 exhibited predominant expression of 3β-HSD over P450c17, suggesting more intensive progesterone than androgen synthesis in these structures., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The postnatal mouse ovary is rich in quiescent and early-growing oocytes, each one surrounded by a layer of somatic granulosa cells (GCs) on a basal lamina. As oocytes start to grow the GCs change shape from flattened to cuboidal, increase their proliferation and form multiple layers, providing a unique model for studying the relationship between cell shape, proliferation and multilayering within the context of two different intercommunicating cell types: somatic and germ cells. Proliferation of GCs was quantified using immunohistochemistry for Ki67 and demonstrated that, unusually, cuboidal cells divided more than flat cells. As a second layer of GCs started to appear, cells on the basal lamina reached maximum packing density and the axes of their mitoses became perpendicular to the basal lamina, resulting in cells dividing inwards to form second and subsequent layers. Proliferation of basal GCs was less than that of inner cells. Ultrastructurally, collagen fibrils outside the basal lamina became more numerous as follicles developed. We propose that the basement membrane and/or theca cells that surround the follicle provide an important confinement for rapidly dividing columnar cells so that they attain maximum packing density, which restricts lateral mitosis and promotes inwardly oriented cell divisions and subsequent multilayering.

In the hen ovary, each preovulatory follicle in the hierarchy, irrespective of its size and the level of its maturity is exposed to the preovulatory LH surge in each ovulatory cycle of an egg laying sequence. In the present study, the thecal weight and membrane protein content of theca layers at different stages of hen ovulatory cycle were assessed. Hens were killed 2 h (stage I), 9 h (stage II), 16 h (stage III), and 23 h (stage IV) after oviposition. The first (F1), second (F2), third (F3), fourth (F4) and fifth (F5) largest yellow follicles were utilized. In all follicles except F1, the thecal weight rose considerably between stages I and III (P < 0.05) followed by a slight cessation of the thecal growth at stage IV. The mean content of the theca membrane protein in F1-F5 follicles was lowest at stage III, increasing at stage IV (P < 0.05), although, in the case of individual follicles the difference was significant (P < 0.05) in F3 follicles only. Estradiol-17beta levels in the plasma were lowest (but not significant) at stage III, and a fourfold increase in the plasma progesterone concentration occurred at stage IV. These findings demonstrate for the first time the ovulatory cycle-related alterations in the thecal weight and membrane protein content in the hen preovulatory follicles. Data suggest that the preovulatory rise in ovarian steroid hormones is probably involved in transient termination of the growth and induction of differentiation of the theca in preovulatory follicles as they pass from one category to the next.

We developed a culture system in which two types of ovarian follicular cells were allowed to attach to opposite sides of a collagen membrane. Using this in vitro cell culture system, we studied the effects of granulosa- and theca-cell interaction on the morphology, structure, and function of bovine ovarian follicular cells. In the first part of the study, we explored how the interaction between theca and granulosa cells affects the morphology and structure of the cells. This study was done using follicular cells collected from bovine ovarian follicles at the early developmental stage. Granulosa cells cultured alone were flattened, and formed a monolayer sheet. By contrast, granulosa cells cultured with theca cells were convex, and formed multilayer sheets. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cultured with granulosa cells were also spindle-shaped; however, they appeared convex and more densely packed when compared with theca cells cultured alone. In the second part of the study, the possible role of the cellular interaction in the control of differentiation and growth of granulosa and theca cells was investigated. When follicular cells were isolated from the early stage of follicular development, theca cells reduced progesterone and inhibin production by granulosa cells and augmented the growth of granulosa cells. When the cells were isolated from the late stage of follicular development, by contrast, theca cells augmented hormonal production by granulosa cells, and did not affect the growth of granulosa cells. The growth and androstenedione production by theca cells were increased by the presence of granulosa cells, irrespective of the origin of follicular cells. These results demonstrated that communication between two types of follicular cells results in reciprocal modulation of their morphology, structure, growth, and function. Cellular interactions seem to be one of the major factors controlling the differentiation and growth of the follicular cells during the follicular maturation process. In contrast to granulosa and theca cells cultured alone, cells in the coculture seemed to possess morphological and functional characteristics more similar to those of cells in the growing follicular wall in vivo. Thus, we speculate that the interaction between these two types of follicular cells is essential for the maintenance of original structure and function of the bovine follicular wall.

Objective: In our previous papers, we demonstrated that TCDD and PCBs (both coplanar PCB 126 and non-coplanar PCB 153) caused the decreased estradiol secretion in cultured pig ovarian follicles. However, the mechanism of action is not clearly understood. Moreover, to our knowledge, the expression of AhR in pig follicular cells was not described yet., Methods: In order to elucidate if TCDD, PCB 126, and PCB 153 can disrupt ovarian steroidogenesis via AhR-dependent mechanism, granulosa and theca cells were isolated from pig ovarian small follicles and subsequently treated with 32 pg/ml of TCDD, 100 ng/ml of PCB 153, 100 pg/ml of PCB 126 in an in vitro culture. After 3-hour exposure to the chemicals, cells were prepared for immunohistochemical analysis or collected for immunobloting, and the effects of TCDD and PCBs on the levels of AhR protein were studied., Results: Under basal conditions, AhR staining showed a diffuse cytoplasmic pattern of distribution as well in granulosa and theca cells. However, immunocytochemical labeling of AhR in theca cells was less evident and only few cells displayed cytoplasmic staining. Immunoblotts prepared from granulosa and theca cell lysates detected a strong band of 120 kDa indicating AhR protein expression. The level of AhR expression detected by Western blot analysis was higher in granulosa cells than in theca cells. Intensive cytoplasmic and nuclear labeling corresponding to AhR protein in TCDD and PCB 126 treated granulosa cells was observed. In PCB 153 treated cells, the level of AhR staining was comparable with control. Western blot analysis showed a strong band in TCDD-treated grnulosa cells and only slightly differences compared with control in those exposed to PCB 126. The effect of both PCBs on immunocytochemical AhR staining in theca cells was less evident. Immunoblott analysis did not reveal any substantial differences between the control and TCDD- and PCBs-treated theca cells., Conclusions: This study has shown different AhR expression in porcine theca and granulosa cells, in respect of both its intracellular localization and cellular distribution. Both, dioxin and dioxin-like PCB activated AhRs in granulosa but not in theca cells. Differences in AhR-responsiveness of granulosa and theca cells to TCDD and dioxin-like PCB 126 are probably connected with the differences of these cells in estradiol secretion and different proliferative potential.

Objective: To localize (by the light and electron microscopy) and partially characterize the antigen recognized by the Mab4E6 in rat ovaries., Methods: Monoclonal antibody (4E6) against a rat ovarian granulosa cell antigen was prepared and identified the 40kDa protein specific for rat steroid producing cells. The localization of this antigen was studied by light and electron microscopic immunocytochemistry., Results: The immunocytochemical observation suggested that the recognized antigen was localized in granulosa and thecal cells in all stages of follicular development. The intensity of immunostaining was found to depend on the developmental stage. In granulosa and thecal stage (health follicle) Mab 4E6 binding molecule was localized on the membranes of rough endoplasmic reticulum (RER) and on the surface of lipid droplets in close association with the RER. In atretic follicles we established that the final destination of the visualized antigen is in structures which we refer as the autophagic vacuoles in close contact with the steroidogenic organelles. In addition, we observed Mab 4E6 binding molecule in the cytoplasm of luteal cells. Leydig cells and adrenocortical cells., Conclusions: The results indicate that the 40kDa antigen may be common to all of rat steroidogenic organs. Our results suggest that the 40kDa protein may be associated with the processes governing steroidogenesis and/or follicular development.

To study ovarian follicular development, we established a procedure to reconstruct a follicular wall in vitro by using three-dimensional collagen gel. Granulosa cells and thecal cells were dissociated from median follicles of porcine ovaries. Isolated thecal cells were embedded in collagen gel and a medium containing isolated granulosa cells was overlaid. A monolayer culture of recombined granulosa and thecal cells in collagen gel was used for the control. To examine the synergism of granulosa cells and thecal cells in this culture system, isolated granulosa and thecal cells were cultured either singly or in combination. The culture media were assayed for steroids by RIA. Cultured granulosa cells in this culture system were epitheloid and grown in multilayer aggregates. In contrast, cells cultured by the conventional method were as flat as a monolayer sheet. Ultrastructural findings for these cells showed abundant organella. Gap junctions were often observed between these cells and the basal lamina-like structure. Estradiol production by both culture methods was significantly higher than the sum of that produced by granulosa and thecal cells cultured separately. Androstenedione accumulation was increased by this culture system, but was suppressed by the conventional culture system. These findings suggest that the constitution of the ovarian follicular wall plays an important role in morphological and functional interaction between granulosa and thecal cells.

Paracrine/autocrine factors like epidermal growth factor (EGF) and transforming growth factor (TGF alpha) produced in the mammalian ovary modulate ovarian functions such as cell proliferation, differentiation, and steroidogenesis. To study similar activities in the avian ovary, we have developed a culture system for chicken granulosa cells using either serum containing or serum-free media. In the defined medium, granulosa cells for the F1, F2, and F3 follicles of the chicken ovary proliferated as indicated by enhanced levels of DNA, synthesised progesterone in similar amounts to those observed in acute experiments, and responded to luteinizing hormone (LH) during and after the culture period. The morphology of the cells and profile of growth in both media (although better in serum than in serum-free medium) are similar to those reported for mammalian cells. EGF stimulated granulosa cell proliferation over 72 hr in culture while inhibiting basal and LH-stimulated progesterone production; these effects were greatest in the less mature cells taken from the F3 follicles. Theca-conditioned medium (TCM) from chicken ovarian theca cell cultures mimicked the effects of EGF when cultured with granulosa cells. Taken together, these results suggest that EGF/TGF alpha-like peptides have a role in avian ovarian function. Moreover, the effects observed for TCM suggest that these peptides may be present in the theca layer with a paracrine function in the adjacent granulosa.

We have developed a culture system in which bovine granulosa and theca cells are allowed to attach to opposite sides of a collagen membrane. We studied the interaction between theca and granulosa cells by investigating the morphology, proliferation, and steroidogenesis of the cells. Granulosa cells cultured alone were flattened and polygonal and formed monolayer sheets. Granulosa cells cocultured with theca cells formed multilayer sheets. The apical surface of each cell appeared convex. Numerous filopodia spread over the cellular surface connecting cells. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cocultured with granulosa cells were also spindle-shaped; however, the apical surface appeared convex. Cocultured cells were more densely packed than theca cells cultured alone. The number of both granulosa and theca cells in the cocultured group increased approximately twofold compared to control cells cultured alone. Progesterone content per 1 x 10(5) granulosa cells in 24-h culture medium of the cocultured group was reduced to 40% of that of the control group. In contrast, androstenedione content per 1 x 10(5) theca cells of the cocultured group increased approximately threefold compared to androstenedione content of control group. These results indicate that communication between these two types of follicular cells results in reciprocal modulation of their proliferation, morphology, and function.

In previous investigations we could demonstrate that angiitis or angiolytic changes of the vascular wall with loosening or duplication of the basement membrane may be caused by a low calcium diet. In this paper we present experiments using the same experimental procedures which lead to massive necrotic changes of granulosa cells in the ovary. These phenomena are thought to be induced by loosening and swelling of the basement membrane which delimits the granulosa layers from the outermost tissue.

Homocellular gap junctions between granulosa cells and between theca interna cells, and heterocellular gap junctions between granulosa cells and oocytes persist in rat ovarian follicles for as long as 90 days following hypophysectomy. Gonadotrophic and/or steroid hormones are therefore not required for the maintenance of gap junctions between these cells during early follicular growth. However, replacement therapy with estrogen and human chorionic gonadotrophin results in amplification of gap junctions in granulosa and theca interna cells respectively. Within 24h following hormonal stimulation, growth of gap junctions is characterized by the appearance of formation plaques as observed in freeze-fracture replicas and by the association of microfilamentous material located subadjacent to gap junction membrane observable in thin-sectioned cells.

No structural changes could be observed in the stigma until 2 h before ovulation. Within a few minutes of ovulation, the stratum granulosum and theca interna disappear completely in the middle part of the stigma. By 30 min before ovulation, the rough endoplasmic reticulum in the fibroblasts of the theca externa develops conspicuously. Then a few minutes before ovulation, the theca externa becomes much thinner with a conspicuous disintegration of the collagenous fibers into individual fibrils. These structural changes may contribute to the fragility of the stigma tissue and induce rupture of the follicle.

Gap junctions were identified in the membrana granulosa and cumulus oophorus, and between cells of the internal theca, of the preovulatory rat follicle. In replicas of freeze fractured follicles, the A face presented clusters of closely packed intramembrane particles, 7--9 nm in diameter, forming a mosaic pattern, while the B face showed a similar pattern of small pits. Optical diffraction analysis of these electron micrographs revealed that both the intramembrane particles and the corresponding pits were organized in hexagonal lattices with centre-to-centre spacing of 9-10 nm. In small junctions (up to 0.4 mum in diameter), both A and B faces generally consisted of a single lattice, while large junctions (0.5-2 mum) contained multiple lattices differing in orientation. Occasionally intramembrane particles and pits were more loosely arranged throughout the junctional area and failed to show a hexagonal pattern. Both granulosa and thecal cells often contained intracellular vesicles resembling annular junctions. These contained intramembrane particles whose assembly resembled that of the gap junctions with regard to periodicity and lattice organization. Examination of thin section of thin sections suggested that small gap junctions occur also between cytoplasmic processes of coronal cells and the oolemma. No tight junctions were detected between granulosa cells and between thecal cells.

Plasma estradiol-17 beta and progesterone profiles were correlated with morphological changes in ovarian follicles during the preovulatory and postovulatory periods in the white-spotted char, Salvelinus leucomaenis, Plasma estradiol levels were highest in September, and were followed by a sharp drop in October; they remained very low throughout the postovulatory period. There was a good correlation between plasma estradiol levels and the gonadosomatic index, thus suggesting that estradiol is responsible for the synthesis of vitellogenic proteins. Plasma progesterone levels were very low in August, began to rise in September and reached a peak soon after ovulation; progesterone remained high for several days after ovulation. A preovulatory rise in plasma progesterone levels was recorded, and this is discussed in relation to the induction of oocyte maturation. In the preovulatory follicles, neither granulosa cells nor special thecal cells (ST cells) showed delta 5, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. In the young postovulatory follicles, in contrast, the ST cells showed intense 3 beta-HSD activity with extensive agranular endoplasmic reticulum and numerous large mitochondria, while granulosa cells did not show 3 beta-HSD activity. These results strongly suggest that the ST cells are the major sites of progesterone synthesis during the postovulatory period.

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.

Using scanning electron microscopy, I examined the granulosa cells of the hen (Gallus domesticus) before and after ovulation. Although the granulosa cells were separated during the final 7-8 days of oocyte maturation, they maintained cell-to-cell contact via cytoplasmic processes. The granulosa cells were cuboidal except at the animal pole (the site of the germinal vesicle) where they were columnar and had surface folds. These morphological characteristics were retained until germinal vesicle breakdown, 1-2 hr before ovulation. At that time the surface of granulosa cells at the animal pole was smoother and their cytoplasmic processes were less numerous and appeared atrophic. Similar changes occurred on the granulosa cells overlying the vegetal pole. About 30 min after ovulation, the granulosa, which remained adherent to its basal lamina, consisted of irregularly shaped columnar cells that lacked surface modifications. These smooth surface cells had rounded apical faces and were generally wider at the apical half. Postovulatory granulosa cells do not transform into luteal cells but undergo fatty degeneration and desquamation during the 4 days after ovulation.

This paper reviews current knowledge of the light and electron microscopic structure of the three layers of the mammalian follicular wall-follicular epithelium (membrana granulosa), theca interna and theca externa--and discusses correlations between structure and function. The ultrastructure of follicular epithelial cells in growing follicles emphasized their protein synthetic and secretory functions; features suggestive of a major steroidogenic function appear only at later stages. Regional differences in follicular epithelial cell function are probably important, although structurally these cells are relatively homogeneous. Structural diversity is more marked in the thecal layers: differentiation in the theca interna is towards fibrocytic and steroidogenic cell types, while that in the theca externa is towards fibrocytic and myoid types. Adherens and gap junctions are present between cells in all layers; however, tight (occludens) junctions have not been convincingly demonstrated between the cells in any of the three layers. Blood and lymph vessels are confined to the thecal layers. However, follicles possess no structural barrier comparable to that associated with the "blood-testis barrier', and show a correspondingly greater permeability to large molecules than seminiferous tubules. Interactions between the layers of the follicular function. To date, the best-documented interaction between layers is that described in the "two-cell hypothesis' of oestrogen production. some potentially useful directions for future research are proposed.

The wall of hamster ovarian follicles is composed of the following cell layers: granulosa, theca interna, theca externa, and the surface epithelium. To determine the morphological changes occurring in the follicle during formation of the rupture site, we measured: the thickness of each layer, the number of cells in each layer, and size of cells in each layer, at the apex and base of follicles at specific times during the final 13 hr before ovulation. Changes in the wall occur in 3 stages. During stage 1 (T0-T8), which includes the first 8 hr following the surge in luteinizing hormone, the apical and basal walls thin at the same rate and the antrum increases in size. During stage 2 (T9-T12), there is no change in the thickness of the apical and basal follicle wall nor in the size of the antrum. During the third stage (T12-T13), the size of the antrum decreases slightly and there is an increase in the thickness of the basal wall, which is correlated with its constriction caused by the contraction of smooth muscle cells. The apical wall rapidly thins to the point of rupture. The morphology of cells from each layer is described. Theca interna cells form the final tissue preventing escape of the oocyte-cumulus complex. The roles of cell migration, stretching, and death in thinning of the apical wall are described, and the mechanisms involved in follicle rupture are discussed.