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www.nature.com/npjmgrav ARTICLE OPEN Simulated microgravity reduces quality of ovarian follicles and oocytes by disrupting communications of follicle cells 1,3 1,3 1 2 1 1 1 1 1 Kaixin Cheng , Xie’an Feng , Chen Yang , Chiyuan Ma , Shudong Niu , Longzhong Jia , Xuebing Yang , Jing Liang , Yingnan Bo , 1 2 1 2 1 ✉ ✉ ✉ Kaiying Geng , Qin Li , Hua Zhang , Xiaohua Lei and Yan Zhang Ovarian follicles are the fundamental structures that support oocyte development, and communications between oocytes and follicle somatic cells are crucial for oogenesis. However, it is unknown that whether exposure to microgravity inﬂuences cellular communications and ovarian follicle development, which might be harmful for female fertility. By 3D culturing of ovarian follicles under simulated microgravity (SMG) conditions in a rotating cell culture system, we found that SMG treatment did not affect the survival or general growth of follicles but decreased the quality of cultured follicles released oocytes. Ultrastructure detections by high-resolution imaging showed that the development of cellular communicating structures, including granulosa cell transzonal projections and oocyte microvilli, were markedly disrupted. These abnormalities caused chaotic polarity of granulosa cells (GCs) and a decrease in oocyte-secreted factors, such as Growth Differentiation Factor 9 (GDF9), which led to decreased quality of oocytes in these follicles. Therefore, the quality of oocytes was dramatically improved by the supplementations of GDF9 and NADPH-oxidase inhibitor apocynin. Together, our results suggest that exposure to simulated microgravity impairs the ultrastructure of ovarian follicles. Such impairment may affect female fertility in space environment. npj Microgravity (2023) 9:7 ; https://doi.org/10.1038/s41526-023-00248-5 19,20 INTRODUCTION communication . Our recent ﬁndings revealed that an oocyte-derived speciﬁc microvilli (Oo-Mvi) system plays a domi- Gravity is one of the most fundamental physical signals on Earth nant role in integrating communications between oocytes and that regulates creatures development from cell shape to organo- somatic cells by governing the release of oocyte-secreted factors genesis. However, gravity is lacking in space, which leads to a 1–3 (OSFs) . These communicating structures are the convex series of abnormal development in various tissues . The ovary, structures of the membrane and are constructed by the F-actin- which is the organ that supports oocyte development, is crucial to 4,5 based cytoskeletal core on the cellular surface , which should be maintain fertility and female endocrine interactions . In the ovary, sensitive to microgravity . However, few studies have been the follicles, which are composed of an oocyte and surrounding 6–8 conducted to investigate the interaction of GC-TZPs or Oo-Mvi granulosa cells, are the functional units that support oogenesis . related to microgravity and folliculogenesis. Although it was reported that exposure to microgravity might 9–13 In the current study, we established a 3D follicle culture system inﬂuence female reproductive capability in space experiments , under SMG in RCCS, and analyzed the development of cultured the effects of microgravity on follicle development remain elusive. follicles and the quality of its released oocytes under a high- To simulate microgravity, a rotating bioreactor, rotating cell resolution imaging system. Our ﬁndings indicated that SMG culture system (RCCS) that can maintain cells in a controlled rotation markedly disrupted the cytoskeleton-related communicating environment to mimic the impacts of microgravity, was devised . structures on either oocytes or GCs. In addition, the cultured By utilizing the RCCS system, recent studies have examined the 15 follicles presented an abnormal polarity of GCs and a reduced maturation of oocytes and the development of ovarian cortical 16 secretion of oocyte-secreted factors such as GDF9 under SMG. pieces and ovarian follicles in a culture under the SMG condition . These abnormal developments led to a signiﬁcantly reduced In these studies, SMG treatment led to an abnormal meiotic spindle oocyte quality from cultured follicles. Supplementing the SMG organization and induced cytoplasmic blebbing in cultured germinal 15 cultures with GDF9 or the NADPH oxidase inhibitor resulted in the vesicle (GV) oocytes, resulting in a failure of oocyte maturation .At reversal of these effects. In summary, our results provide the tissue level, the SMG condition resulted in a decline in follicle systematic evidences about ultrastructural changes in follicle cells survival in the ovarian cortical pieces and an abnormal morphology under simulated microgravity. These studies shed light on of oocytes in cultured follicles, indicating SMG also disrupted potential mechanisms that can prevent an impairment in the folliculogenesis . However, it is still unknown how SMG disrupts female reproductive system during spaceﬂight. folliculogenesis at the sub-cellular and molecular levels. The ovarian follicle is composed of an oocyte and surrounding GCs. Crosstalk between oocytes and GCs, which relies on the RESULTS communicating structures, is essential for proper folliculogen- 17,18 3D culture of ovarian follicles under SMG conditions esis . Previous studies have shown that transzonal projections (GC-TZPs) are derived from the inner layer GCs that connected To investigate the inﬂuences of microgravity on the development with the oocytes to permit essential germline-somatic of ovarian follicles and oocytes in mice, we modiﬁed a three- 1 2 State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China. Center for Energy Metabolism and Reproduction, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. These authors contributed equally: Kaixin Cheng, Xie’an Feng. email: email@example.com; firstname.lastname@example.org; email@example.com Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; K. Cheng et al. Fig. 1 Tracing the growth of ovarian follicles in a 3D culture system under simulated microgravity condition. a The ﬂowchart of the 3D ovarian follicle culture in RCCS. The follicles were seeded into a Matrigel droplet to support their growth. b, c Tracing the growth of ovarian follicles in the NG and the SMG groups in vitro (b), showing comparable developmental dynamics of follicles in the SMG groups (n = 48) compared to that in the NG group (n = 60) (c). Day 0: p value = 0.13. Day 1: p value = 0.43. Day 2: p value = 0.076. Scale bars: 100 μm. d The ratio of antral formation in the cultured follicles under different conditions, showing no signiﬁcant changes of antral forming proportions of cultured follicles in the SMG group (n = 65) compared to that in the NG group (n = 73). p value = 0.98. Representative images are shown. Data are presented as the mean ± SD. Data were analyzed by two-tailed unpaired Student’s t-test and n.s. P ≥ 0.05. dimensional follicle culture system in a RCCS to support mouse granules (CG) with signiﬁcantly reduced CG density and aberrated 16,23 ovarian follicle development under the SMG condition . This granule aggregation was observed in oocytes of the SMG group was achieved by seeding single follicle (about 200 μm diameter) (Fig. 2b) compared to that in the NG group. Moreover, by treating into liquid Matrigel droplets, which were then transformed to a the cultured follicles with luteinizing hormone (LH) , we detected solid gel in the culture to support follicle growth. The droplets the ratio of ﬁrst polar body (PB1) release in the oocytes from the with follicles were cultured under the SMG condition (15 rotations SMG and the NG groups, and found a signiﬁcant decrease in the per minute, RPM), or normal gravity (NG) with no rotation as the PB1 ratio in oocytes of the SMG group compared to that of the NG control (Fig. 1a). group (24.2% ± 3.6% in SMG v.s. 68.4% ± 2.9% in NG) (Fig. 2c, red With the 3D culture system, the follicles were cultured for 2 days arrowheads), showing that the maturation of oocytes was (Fig. 1b), and the survival ratio of follicles was identical between defected under SMG conditions. According to previous studies, the SMG group and the NG group (75.0% ± 5.0% in SMG v.s. 80.9% microgravity increased ROS production, which represented an ± 1.4% in NG) (Supplementary Fig. 1), showing that SMG enhancement of oxidative stress in various cell types .We treatment had no effect on the survival of follicles in the culture. therefore analyzed the levels of intracellular ROS in live oocytes The diameters of the cultured follicles increased from obtained from the follicles. Consistent with the low ratio of 208.1 ± 4.45 μm to 387.3 ± 10.5 μm during 2 days of the culture maturation in the SMG-treated oocytes, a remarkable increase of in the SMG group, which was consistent with the increase in 2′,7′-dichloroﬂuorescein (DCF) ﬂuorescence intensity, which repre- diameters of the follicles in the NG group (from 210.0 ± 8.23 μmto sents a higher ROS level was also observed in oocytes from the 384.3 ± 5.0 μm) (Fig. 1c). In addition, the ratio of antral follicles in SMG group (Fig. 2d), suggesting that SMG condition reduced the the SMG group was similar to that in the NG group (83.3% ± 19.2% quality of oocytes. in SMG v.s. 83.6% ± 14.4% in NG) (Fig. 1d), showing the robust These results demonstrated reduced quality and maturation of developmental capability of the follicles under SMG condition. oocytes from SMG-treated follicles, suggesting that stimulated These data showed that SMG treatment has no marked effect on microgravity might affect the development of follicles at the the growth of ovarian follicles in a general observation. cellular or molecular levels. Decreased quality of oocytes after follicles developed under SMG treatment disrupted the establishment of GC polarity the SMG conditions and formation of GC projections in follicles Because follicle development was relatively healthy under SMG Because the general growth of follicles is normal, but the quality condition, we next evaluated the quality of oocytes, which were of oocytes decreased in the SMG group, we hypothesized that released from cultured follicles in different groups. The diameter simulated microgravity might affect the cellular modeling and of the oocytes in the SMG group (65.1 ± 6.8 μm) was not communications of GCs in the follicles. We therefore introduced a T2 signiﬁcantly different from that in the NG group (62.1 ± 4.0 μm) Foxl2-CreER ; mTmG mouse model, which is capable of describing 17,27 (Fig. 2a), indicating that SMG treatment had no effect on oocyte the outline of GCs with membrane-localized GFP (mG) . Tissues size. However, a markedly abnormal distribution of the cortical from this mouse model were visualized with a high-resolution npj Microgravity (2023) 7 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; K. Cheng et al. Fig. 2 SMG treatment decreased the quality of cultured follicle released oocytes. a After follicles developed under SMG or NG conditions, the oocytes were isolated from the cultured follicles. No signiﬁcant changes in the diameters of oocytes were seen when comparing the SMG group (n = 13) with the NG group (n = 13) after 2 days of follicle culture. p value = 0.18. Scale bars, 30 μm. b LCA (Lens Culinaris Agglutinin)- FITC immunostaining showing abnormal cortical granule distribution in the SMG group oocytes (n = 53) compared to that in the NG group (n = 59). p value = 0.000067. Scale bars, 30 μm. c Oocytes obtained from antral follicles after 16 hours of culture with LH in vitro, showing a signiﬁcantly decreased ratio of PB1 (red arrowheads) in the SMG group (n = 74) compared to that in the NG group (n = 63). p value = 0.0000000031. Scale bars, 100 μm. d An increased ﬂuorescence intensity which represented higher ROS level in oocytes of the SMG group (n = 50) compared to that in the NG group (n = 36). p value = 0.00000000000000000000078. Scale bars, 100 μm. Representative images are shown. Data are presented as the mean ± SD. Data were analyzed by two-tailed unpaired Student’s t-test and n.s. P ≥ 0.05, **P < 0.01, ***P < 0.001. imaging system to detect the subcellular structures of GCs in the layers of GCs in the SMG group were kept in non-polarity, the follicles (Fig. 3a). In the follicles of the NG group, we found that number of cellular projections in each cell was signiﬁcantly GCs were clearly separated to three populations with distin- decreased compared with the NG group (Fig. 3c–f, red box). These guished cellular characteristics, e.g. the inner layer GCs (I-GCs), results demonstrated that the simulated microgravity markedly which exhibited cuboidal shape with numerous stretched GC-TZPs affected the cellular polarity and the extension of the cellular polarity towards the oocyte (Fig. 3b, c, green box), the multi- projections of GCs in follicles (Supplementary Fig. 3), which middle layer GCs (M-GCs) with a random outline, many extended disrupts the communications between GCs and the oocyte cellular projections into the surrounding cells (Fig. 3b, c, red box), resulting in reduced quality of oocytes. and the outer layer GCs (O-GCs), which had a few cellular projections toward the M-GCs of the follicle (Fig. 3b, c, blue box). Decline of Oo-Mvi in cultured follicles under SMG condition This high-resolution observation is consistent with the previous The communications between oocytes and GCs are determined observation by TEM , showing a strict cellular arrangement and 17,18 by both the oocyte-speciﬁc Oo-Mvi and the TZPs in GCs .We polarity of GCs in follicles. next investigated whether the formation of Oo-Mvi was affected Next, we detected the GC distribution in the SMG group. under SMG condition. Following our previous study, we crossed Generally, we found a chaotic arrangement of GCs in the SMG- the mTmG mice with Gdf9-Cre mice to label the cell surface of treated follicles compared to that in the NG group. Additionally, oocytes by membrane GFP (Fig. 4a). The Oo-Mvi on oocytes the GCs in all three populations lost their characteristics under the from the follicles after 2 days of culturing under SMG or NG SMG condition. The majority of I-GCs (76.7% ± 9.5%) became conditions were detected (Fig. 4b). As shown in Fig. 4c, our high- round or irregular in appearance (Fig. 3d, e, green box) with a resolution imaging detections showed that a normal density of signiﬁcantly decreased number of GC-TZPs compared to the NG Oo-Mvi with typical mushroom structures was observed on group (0.95 ± 1.05 in SMG v.s. 8.75 ± 1.29 in NG, Fig. 3f). This result oocytes from follicles in the NG group. In sharp contrast, a showed that the communications between GCs and oocytes were marked decrease in Oo-Mvi density was found on oocytes from severely disrupted with SMG treatment. Similar to the I-GCs, more follicles under SMG conditions (Fig. 4c).Thisresultwas conﬁrmed than half of O-GCs (55.4% ± 1.2%) lost their polarity and represented an irregular outline (Fig. 3d, e, blue box), and a by Oo-Mvi counting revealing a signiﬁcantly decreased number reduced number of the cellular projections (1.60 ± 1.23 in SMG v.s. of Oo-Mvi in the SMG group compared to that of the control 3.90 ± 1.48 in NG, Fig. 3f) were also observed in those abnormal oocytes in the NG group (24.5 ± 7.8 in SMG v.s. 40.6 ± 6.3 in NG, O-GCs under SMG condition. In addition, although the middle Fig. 4d). This data clearly showed that simulated microgravity Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2023) 7 K. Cheng et al. Fig. 3 SMG treatment disrupted GC polarity and the formation of communicating structures in GCs. a Illustration of the strategy to induce T2 labeling of the GCs in Foxl2-CreER ;mTmG follicles. With a low dosage of tamoxifen treatment, membrane-localized red ﬂuorescent protein T2 (mT) switches to green-ﬂuorescent protein (mG) in GCs of Foxl2-CreER ;mTmG follicles, which allows for imaging of the cell outline of GCs under high-resolution imaging system. b The standard to separate the GCs in growing follicles. The inner layer of GCs was deﬁned as the layer which directly connected to the oocyte by GC-TZPs (I-GCs, green); the middle layers of GCs was distributed in the middle multilayers position of follicle (M-GCs, pink); The outer layer of GCs was deﬁned as the layer adjacent to the theca cells (O-GCs, blue). c Images of Foxl2- T2 CreER ;mTmG follicles showing the morphology of GCs in different regions in the NG group. I-GCs (green box) exhibited cuboidal shape with tree root-like GC-TZPs oriented toward the oocyte. Rounded M-GCs (red box) exhibited extended random cellular projections. Similar shaped cell with I-GCs, cuboidal O-GCs (blue box) extended a few cellular projections toward to M-GCs. Scale bars, 15 μm. d In the SMG group, both the polarity and the communicating structures on GCs were abnormal, showing a failure of GC-TZPs on I-GCs (green boxes), and dramatically reduced cellular projections on M-GCs (red boxes) and O-GCs (blue boxes). I-GCs and O-GCs exhibited a loss of polarity shape under SMG condition compared to that in the NG group. Scale bars, 15 μm. The cartoon model as shown in Supplementary Fig. 3. e Statistical analysis of GCs showing a signiﬁcant increase in the proportion of non-polarity in I-GCs and O-GCs under SMG conditions (n = 10) compared to the NG group (n = 10). I-GCs: p value = 0.0000000010, M-GCs: p value = 0.64, O-GCs: p value = 0.00000082. f Loss of cellular projections in all layers of GCs under SMG conditions (n = 20) compared to that in NG conditions (n = 20). I-GCs: p value = 0.00000000000000000000064, M-GCs: p value = 0.00000000059, O-GCs: p value = 0.0000051. Representative images are shown. Data are presented as the mean ± SD. Data were analyzed by two-tailed unpaired Student’s t-test and n.s. P ≥ 0.05, ***P < 0.001. The colors were inverted to black/white (b/w) to highlight GCs in (c, d). also disrupts the formation of oocyte communicating structures SMG only affects the formation of communicating structures, but Oo-Mvi, which could account for the low quality of oocytes seen has no marked effect on the function of these structures such as in the SMG group. the enrichment of OSFs in Oo-Mvi. Oo-Mvi plays a crucial regulating role for the orderly release of OSFs, and the efﬁciency of OSF release for each Oo-Mvi is Supplying OSFs or Apocynin to cultured follicles improved the determined by the length and the size of vesicles on the tip of Oo- oocyte quality under SMG conditions Mvi . We therefore measured and compared the length of Oo- Mvi and the size of Oo-Mvi vesicles after culturing in SMG versus Our ﬁndings demonstrate that SMG disrupts the formation of Oo- Mvi, therefore it may affect the release of OSFs in the follicles. We NG. We found that although the average length of Oo-Mvi on oocytes of the SMG group was shorter than that on oocytes in the next detected the expression levels of several well-studied OSFs, 29,30 30,31 32 including Gdf9 , Bmp15 and Fgf8 in the cultured follicles. NG group (Fig. 4e, f), the average diameter of vesicles on the tip of Oo-Mvi was comparable on oocytes in the SMG and the NG We found that the mRNA levels of all detected OSFs were groups (Supplementary Fig. 3a–b). These results suggest that the signiﬁcantly reduced in follicles of the SMG group compared to npj Microgravity (2023) 7 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA K. Cheng et al. Fig. 4 SMG treatment decreased the formation of Oo-Mvi in cultured follicles. a Illustration of the strategy to label the Oo-Mvi by Gdf9- Cre;mTmG mouse model. The membrane-localized red ﬂuorescent protein (mT) switches to green-ﬂuorescent protein (mG) in oocytes of Gdf9- Cre;mTmG mouse to label oocyte membrane morphology. b Images of Gdf9-Cre;mTmG oocytes, showing the mushroom-like Oo-Mvi with vesicle tips distributed in the zona pellucida of oocytes in both the SMG and the NG groups. Scale bars, 30 μm. c 3D high-resolution images showing a decreased density of Oo-Mvi on the oocytes’ surface under SMG. Scale bars, 10 μm. d Numbers of Oo-Mvi reduced in SMG oocytes (n = 8) compared to that in NG (n = 8), showing a signiﬁcantly reduced number of Oo-Mvi on oocytes in follicles after SMG treatment. p value = 0.00052. e High magniﬁcation showing that the length of Oo-Mvi in the SMG group was shorter than that in the NG group. Scale bars, 5 μm. f Quantiﬁcation of the length of Oo-Mvi conﬁrmed a dramatic decrease in the SMG group (n = 30) compared to that in the NG group (n= 30). p value = 0.00000000000000000052. The colors were inverted to black/white (b/w) to highlight Oo-Mvi in (e). Representative images are shown. Data is presented as the mean ± SD. Data were analyzed by two-tailed unpaired Student’s t-test and ***P < 0.001. that in the NG group (Fig. 5a, Gdf9: 0.54 ± 0.15 times, Bmp15: Our experimental ﬁndings also showed an increased ROS level 0.53 ± 0.11 times and Fgf8: 0.58 ± 0.19 times), showing damage to in oocytes from follicles under SMG condition, and the NADPH- oocytes in the cultured follicles under the SMG condition. oxidase inhibitor Apocynin was reported to be functional to Furthermore, we detected the cytoskeleton-related genes improve the general cell viability under microgravity . Therefore, 33 34 Myo10 and Fscn1 , which were reported to control the formation we also cultured follicles with or without Apocynin (15 μg/mL) of GC-TZPs through the regulation of OSFs in GCs . As expected, under SMG conditions to test the oocyte quality after culture. After we found a dramatic decrease in the expression of both Myo10 2 days of culturing, we found a signiﬁcantly reduced ﬂuorescent (0.41 ± 0.26 times) and Fscn1 (0.45 ± 0.26 times) in the follicles of intensity of DCF in oocytes of the SMG group with Apocynin, the SMG group compared to that in the NG group (Fig. 5b). These showing a decreased ROS level in oocytes compared to that in the results indicated that simulated microgravity caused the decline in SMG group without Apocynin treatment (Fig. 6a, b). Moreover, the the expression of two genes regulating connecting structures of ratio of PB1 in the oocytes of the Apocynin group was signiﬁcantly the cytoskeleton. These abnormalities could lead to an insufﬁcient increased from 22.8% ± 2.7% in the SMG group to 50.7% ± 2.8% release of OSFs to support follicluogenesis. (Fig. 6c, d, red arrowheads), showing an efﬁcient rescue of oocyte To conﬁrm our hypothesis, we tested whether supplying OSFs quality by Apocynin treatment during follicle development. to the cultured follicles could rescue the damage of follicles and oocytes under SMG condition. Based on our previous ﬁnding, supplying GDF9 stimulates the formation of GC-TZPs and 17,19,35 DISCUSSION improves follicle development . We therefore cultured With the advances in aerospace technology, the biological effect ovarian follicles with or without GDF9 under SMG conditions of weightlessness on the reproductive health has received and examined the ratio of oocyte maturation in different groups. 37,38 widespread attention . Unlike males, who continuously pro- As expected, the ratio of PB1 in oocytes signiﬁcantly increased duce sperm from spermatogonial stem cells , the ovarian follicles from 22.8% ± 2.7% in follicles in the SMG group to 42.3% ± 4.1% of the female reproductive reserve cannot be renewed in adult after 2 days of GDF9 treatment (500 ng/mL) (Fig. 5c, d, red 40,41 life . Therefore, any factors that cause ovarian follicle damage arrowheads). These results demonstrate that supplying OSF is efﬁcient to rescue the defect of oocyte quality in cultured follicles at any stage of follicular development lead to the irreversible under SMG. reduction in female reproductive ability. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2023) 7 K. Cheng et al. Fig. 5 Supplying OSFs rescued the damage of oocytes by SMG treatment. a Relative mRNA levels of Gdf9, Bmp15, and Fgf8 under the SMG or the NG group, showing a decreased expression of OSFs in the SMG group (n = 4). Gdf9: p value = 0.0081, Bmp15: p value = 0.0032, Fgf8: p value = 0.021. b Relative mRNA levels of Fscn1 and Myo10 under the SMG or the NG group, showing that the expression of GC-TZP forming related genes was downregulated after SMG treatment (n = 4). Fscn1: p value = 0.025, Myo10: p value = 0.021. c Supplying GDF9 increased the PB1 ratio of oocytes (red arrowheads) from SMG treated follicles. Scale bars, 100 μm. d The ratio of PB1 in different groups, showing that the GDF9 supplement signiﬁcantly increased the maturation of oocytes (n= 38 in NG, n = 35 in SMG and n = 61 in SMG + GDF9). NG v.s. SMG: p value = 0.00021, SMG v.s. SMG + GDF9: p value = 0.021. Representative images of oocytes are shown. Data are presented as the mean ± SD. Data were analyzed by two-tailed unpaired Student’s t-test in (a, b) and two-way ANOVA in (d). n.s. P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. As the fundamental units of female reproduction, ovarian guaranteeing both the development of follicles and the maturing 19,28 follicles are composed of a single oocyte and surrounding somatic of oocytes . In the current study, by utilizing a Foxl2- 4 T2 cells . Here, we investigated the development of follicles under CreER ;mTmG reporter mouse model to detect the membrane SMG conditions with a focus on the cellular communication outline of GCs in different layers, we identiﬁed the cellular structures in follicle cells. We found the simulated microgravity morphology of GCs with different localizations, and identiﬁed disrupted the construction of GC polarity and affected the these three areas of GCs with distinguishable cellular features: formation of cellular communicating structures in both the oocyte cuboidal shape I-GCs whose GC-TZPs exhibited tree root-like and GCs. With high-resolution single-cell imaging, our 3D imaging structure polarity oriented and connected with oocytes; M-GCs results clearly illustrated that the bidirectional communications which extended random cellular projections chaotically arranged between oocytes and GCs, including both Oo-Mvi and GC-TZPs in the middle position of a follicle; O-GCs adjacent to theca cells were severely damaged by simulated microgravity, leading to the had few cellular projections toward M-GCs. These observations decline in oocyte quality. Previous studies reported that micro- suggested that the GCs in various areas of the follicle had complex gravity disrupted the polymerization and depolymerization functional divisions to help orchestrate folliculogenesis. Moreover, processes of the main cytoskeleton polymers, especially the we found that the cellular structures of GCs in all layers were cortical actin, which causes the circularity of the cells by reducing disrupted by SMG. Such ovarian follicles are the typical structures 42,43 cytoskeleton-generated tensions . In addition, it has been that play their function by an integrative unit. reported that microﬁlament-based membrane structure ﬁlopodia The effects of microgravity on mitochondrial stress, leading to 22,44 tend to not form under microgravity . This is consistent with increased ROS levels in several cell types, have been widely 25,46–48 our imaging observation that some I-GCs’ cuboidal shape changes reported . Our data showed the downregulation of actin- to a circular shape, and there is a reduced length of the Oo-Mvi associated genes and an increase of ROS level in oocytes and a reduced number of GC-TZPs under SMG conditions, which exposed to SMG, which resulted in the reduction of oocyte was conﬁrmed by the down-expression of F-actin regulator Fscn1 quality. Our results are consistent with previous studies that and Myo10 under SMG. Therefore, our results strongly suggest reported cytoskeletal disruption under SMG conditions induces that the microgravity-related disorder of the cytoskeleton ROS production and NADPH oxidase gene up-regulation by construction of follicle cells could be of major risk causing female affecting mitochondrial membrane structure, oxygen consump- 25,46,49,50 reproductive disorders in space. tion, and respiratory capacity .Ofnote, ourstudy also The main function of GCs is to nourish and support the showed that adding OSFs (GDF9) or the NADPH-oxidase inhibitor development of oocytes . With follicle development, the GCs Apocynin to the follicle of SMG cultures was able to restore proliferate to construct multi-layer structures . It is well-known oocyte quality, which provided preliminary results regarding that the different layers of GCs exhibit distinguished molecular the protective strategy to reduce the impairment of female characteristics which construct a systematic regulating network, reproduction in microgravity conditions. More importantly, this npj Microgravity (2023) 7 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA K. Cheng et al. Fig. 6 Apocynin rescued SMG-related oocyte damage by decreasing the ROS level. a After follicle culturing with Apocynin under SMG condition, the follicle-released oocytes showed a dramatically decreased ROS level compared to the oocytes without Apocynin. Scale bars, 100 μm. b The statistical analysis of DCF ﬂuorescence intensity, showing a decreased ROS level in oocytes of the Apocynin group (n = 22 in NG, n = 26 in SMG and n = 41 in SMG + Apocynin). NG v.s. SMG: p value = 0.00000000000010, SMG v.s. SMG + Apocynin: p value = 0.000000028. c The ratio of PB1 (arrowheads) was markedly increased in the Apocynin-treated group compared to that in the SMG group. Scale bars, 100 μm. d Statistic analysis showing a signiﬁcantly increased ratio of PB1 in the SMG + Apocynin group compared to that in the SMG group (n = 38 in NG, n = 35 in SMG and n = 43 in SMG + Apocynin). NG vs SMG: p value = 0.00021, SMG vs SMG + Apocynin: p value = 0.0019. Representative images of oocytes are shown. Data are presented as the mean ± SD. Data were analyzed by two-way ANOVA and n.s. P ≥ 0.05, **P < 0.01, ***P < 0.001. ﬁnding also suggests that the damage of follicles in microgravity 26 °C with access to chow and water ad libitum. The methods can be prevented. were performed in accordance with relevant guidelines and Because gravity is an acceleration, not a force, it depends on the regulations and approved by the Institutional Animal Care and Use Committee of China Agricultural University, No. AW80211202-3-1. mass of the whole organism to create the mechanical stimulation of cells on Earth that is then diminished during spaceﬂight . Although various simulation devices have been developed and In vitro culture of ovarian follicles optimized, it remains difﬁcult to realistically simulate the The follicles with the diameter of around 200 μm (average microgravity environment on the ground. This study shows the diameter: 210.0 ± 5.78 μm) were separated by tearing postnatal possibility of creating countermeasures to the possible harmful day (PD) 23 ovaries of C57BL/6 (for Fig. 1/ Fig. 2/ Fig. 5/ Fig. 6)or effects of spaceﬂight on the reproductive system of female T2 Foxl2-CreER ;mTmG (for Fig. 3) or Gdf9-Cre;mTmG mice (for astronauts. If the opportunities arise, it is preferable to carry out Fig. 4). To observe the growth of follicles, the follicles were experiments in space and to compare the results using ground- cultured in Matrigel (BD, 354234) culture system as previously 23,55–57 based simulations. However, microgravity possibly works with reported . The culture medium consisted of 10 mL MEMα space radiation to affect female reproduction during spaceﬂight. (Gibco, 32-571-036), 2.1 mg/mL NaHCO , 5% FBS (Gibco, 10-099- 141), 100 IU/mL penicillin-streptomycin (15140122, Invitrogen), 1% ITS (Sigma-Aldrich, 13146) and 10 ng/mL FSH (ovine Follicle METHODS Stimulating Hormone, NHPP). In the experimental group, a Mice rotating cell culture system (RCCS-4D, SYNTHECON) was used to C57BL/6 mice were from the Laboratory Animal Center of the mimic some aspects of microgravity on the encapsulated T2 Institute of Genetics (Beijing, China). Gdf9-Cre, Foxl2-CreER and follicles . The underlying principle of the RCCS is that an 27,52,53 mTmG mice were generated as previously reported . Foxl2- increase in the rate of rotation will result in a decrease in the T2 58 CreER mice were a gift from Dr. Liu Kui. To partly label GCs, a perimeter of the circular path . The microgravity condition is single intraperitoneal injection of tamoxifen (75648, Sigma- simulated when the Matrigel/follicles eventually begin to rotate Aldrich) at a dosage of 5 mg/kg body weight (BW) were given around their own axis. Previous studies have calculated this T2 54 59,60 to Foxl2-CreER ;mTmG females at postnatal day (PD) 21 . All mice rate to be 15 rotations per minute . Matrigel containing were housed in mouse facilities under 16/8-h light/dark cycles at single follicles and not rotated was used as controls. In the Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2023) 7 K. Cheng et al. GDF9-supplying experiment, we added GDF9 protein (500 ng/mL, polarity GCs. For every group, we counted the polarity of 50-100 T2 739-G9-010/CF, R&D) into follicle cultured medium for 2 days. In cells per layers of Foxl2-Cre ;mTmG secondary follicle (n = 10 the Apocynin-treatment experiment, we added Apocynin follicles) as shown in Fig. 3e. (15 μg/mL, NSC 2146, Selleck) to the medium for the inhibition To evaluate the loss of cellular projections or TZPs of single GC of ROS levels for 2 days. During culturing, the medium was half- of follicles exposed to SMG, we counted the number of cellular changed every day, and the diameter of follicles was recorded projections or TZPs of single GC from 8 μm tissue light-sections of T2 every 24 h for 2 continuous days. Cultures were visualized Foxl2-Cre ;mTmG follicles (n = 20 GCs of per layer from 10 sec- using a Nikon Eclipse Ti digital ﬂuorescence microscope in a ondary follicles of each groups) as shown in Fig. 3f. bright ﬁeld channel. To observe the Oo-Mvi in oocytes, denuded oocytes at the GV stages of antral follicles were collected from the NG or the SMG groups after 2 days culture by tearing the follicles of GDF9- Detection of Intracellular ROS in Oocytes Cre;mTmG females with a syringe needle. The oocytes were To analyze the levels of intracellular reactive oxygen species (ROS), transferred via mouth pipetting ~20 μL minimum MEMα-FBS-ITS a reactive oxygen species DCFH diacetate kit (E004-1-1, DCFH-DA, medium: MEMα with 10% FBS and 1% ITS, covered with mineral Nanjing Jiancheng Bioengineering Institute) was used to deter- oil (Sigma-Aldrich, M8410) and photographed in a living cell mine the ROS levels in living oocytes . Denuded oocytes were workstation (Okolab) at 37 °C, 5% CO . Images were typically incubated with DCFH-DA (1:1000) in PBS for 30 minutes at 37 °C in acquired with an optical slice thickness of 0.5 μm and covered a5% CO incubator. In the presence of ROS, H DCF is rapidly 2 2 ~40 μm of oocytes. oxidized to 2′,7′-dichloroﬂuorescein (DCF), of which ﬂuorescent intensity represented ROS level in oocytes . The oocytes were The Cortical Granules Staining by LCA (Lens Culinaris then washed three times and examined immediately for green Agglutinin)-FITC ﬂuorescent signals using a Nikon Eclipse Ti digital ﬂuorescence To detect the cortical granules, oocytes of the NG and the SMG microscope. The ﬂuorescence intensity of the oocytes was groups at the GV stage were ﬁxedin4%PFA in PBS for 15 minat measured using ImageJ 1.42q software (NIH). room temperature (25 °C), followed by treatment with 0.5% Triton X-100 for 20 min. Oocytes were subsequently incubated in Oocyte maturation in vitro PBS supplemented with 1 mg/mL BSA (Sigma-Aldrich, V900933) For the maturation of oocytes, the follicle culture medium was for 1 h. After staining with LCA (Lens Culinaris Agglutinin)-FITC for changed into maturation media composed of 10 mL MEMα, 2 h (1:100 dissolved in PBS, L32475, ThermoFisher Scientiﬁc) at 2.1 mg/mL NaHCO , 5% FBS, 100 IU/mL penicillin-streptomycin, room temperature, the oocytes were washed three times in PBS 1% ITS, and 1 μg/mL LH (ovine luteinizing hormone, NHPP) after and imaged using an Andor Dragonﬂy spinning-disc confocal 2 days in culture. After LH treatment for 16 h at 37 °C, 5% CO , 64 microscope as previously described . All steps were at room oocytes were gently removed from the follicles by a syringe temperature (25 °C). needle. The polar body 1 stage of oocytes was counted as an indicator of oocyte maturation . Gene expression analysis To detect the gene expression of follicles after culturing, the mRNA High-resolution imaging of follicles and isolated oocytes to of follicles from the NG and the SMG groups was extracted by detect subcellular structure TRIZOL Reagent (Thermo-Ambion, 15596018) according to the All high-resolution images of follicles and isolated oocytes were manufacturer’s protocol. The quantity and quality of the total RNA acquired using an Andor Dragonﬂy spinning-disc confocal were determined using a Nanodrop (Thermo Scientiﬁc). Reverse microscope equipped with a ×40 or ×63 objectives, a scientiﬁc transcription (TAKARA, RR047Q) was performed using 0.5 μg total complementary metal-oxide semiconductor (sCMOS) camera RNA per sample. QRT-PCR reactions were performed in 96-well (Andor Zyla 4.2), and the 488-nm (mG) and 568-nm (mT) lines of plates (Applied Biosystems, 4316813) in 10 μL reaction volumes the Andor Integrated Laser Engine (ILE) system with a spinning- and analyzed by an Applied Biosystems 7500 Real-Time PCR disc confocal scan head (Andor Dragonﬂy 500). Images were System (Applied Biosystems, 4472908) using the following acquired by Fusion 2.1 software. parameters: 10 minutes at 95 °C, followed by 40 cycles of To identify GCs polarity, follicles were isolated from the 15 seconds at 95 °C and 20 seconds at 50 °C and 30 seconds at T2 ovaries of Foxl2-CreER ;mTmG females following 48 h of 72 °C. Data were normalized to β-actin. The primer list is provided tamoxifen treatment. After 2 days in culture, the follicles were in Supplementary Table 1. collected from Matrigel and ﬁxed in 4% PFA (Paraformaldehyde) in PBS for 1 h. The follicles were then transferred to clearing Statistical analysis medium C 3D via mouth pipetting and incubated in the dark at All experiments were repeated at least three times and representa- room temperature on a rotor for 24 hours, as described tive results are shown. Data are presented as the mean ± standard previously . The cleared follicles were embedded in a 35 mm deviation (SD) of each experiment. Data were analyzed by Student’s dish with a 14 mm glass bottom (Cellvis, D35-14-517 1-N) t-testortwo-way ANOVAand were considered statistically signiﬁcant containing fresh clearing solution and tightly covered by a at P < 0.05. P is indicated as follows: *(P < 0.05), **(P <0.01), coverslip. Confocal imaging was acquired with Z-step 0.6 μmfor ***(P < 0.001), and n.s. (not signiﬁcant, P ≥ 0.05). Statistics and graphs 150 μm(63×objective). were obtained using Prism 5 (GraphPad Software, La Jolla). In order to quantify the polarity of granulosa cells under SMG conditions, we deﬁned polarity GCs and non-polarity GCs according to cell morphology and localization. Polarity GCs in Reporting Summary the inner layer of secondary follicles showed cuboidal cell shape Further information on research design is available in the Nature with TZPs projecting into the zona pellucida. The outer layer of Research Reporting Summary linked to this article. GCs adjacent to the theca cells showed cuboidal cell shape with few cellular projections towards the middle layers of GCs. Non- polarity GCs refer to those GCs that exhibit round or irregular DATA AVAILABILITY shape. 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Open Access This article is licensed under a Creative Commons ACKNOWLEDGEMENTS Attribution 4.0 International License, which permits use, sharing, This study was supported by the National Key Research and Development Program of adaptation, distribution and reproduction in any medium or format, as long as you give China to Y.Z., H.Z. and X.L. (2021YFA0719303; 2018YFC1003800; 2018YFC1003700; appropriate credit to the original author(s) and the source, provide a link to the Creative 2022YFC2703800), the 2115 Talent Development Program of China Agricultural Commons license, and indicate if changes were made. The images or other third party University (1021-109022). material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory AUTHOR CONTRIBUTIONS regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// K.C., X.F., H.Z., X.L. and Y.Z. designed the research; K.C., X.F., C.Y., C.M., S.N., L.J., X.Y., creativecommons.org/licenses/by/4.0/. J.L., Y.B., K.G., Q.L. and Y.Z. performed the experiments; K.C., X.F., C.Y., C.M., S.N., L.J., X.Y., J.L., Y.B., K.G., Q.L., H.Z. and Y.Z. analyzed the data; K.C., X.F., X.L., H.Z. and Y.Z. wrote the paper. All authors have seen and approved the ﬁnal version. K.C. and X.F. © The Author(s) 2023 contributed equally as co-ﬁrst author. npj Microgravity (2023) 7 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA
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