Mouse Ovarian Follicles Research Articles

Ex vivo imaging reveals the spatiotemporal control of ovulation.

During ovulation, an egg is released from an ovarian follicle, ready for fertilization. Ovulation occurs inside the body, impeding direct studies of its progression. Therefore, the exact mechanisms that control ovulation have remained unclear. Here we devised live imaging methods to study the entire process of ovulation in isolated mouse ovarian follicles. We show that ovulation proceeds through three distinct phases, follicle expansion (I), contraction (II) and rupture (III), culminating in the release of the egg. Follicle expansion is driven by hyaluronic acid secretion and an osmotic gradient-directed fluid influx into the follicle. Then, smooth muscle cells in the outer follicle drive follicle contraction. Follicle rupture begins with stigma formation, followed by the exit of follicular fluid and cumulus cells and the rapid release of the egg. These results establish a mechanistic framework for ovulation, a process of fundamental importance for reproduction.

Liraglutide improves follicle development in polycystic ovary syndrome by inhibiting CXCL10 secretion

BackgroundAt present, a number of clinical trials have been carried out on GLP-1 receptor agonist liraglutide in the treatment of polycystic ovary syndrome (PCOS). However, the effect of liraglutide on follicle development and its specific mechanism are still unclear.MethodsRNA sequencing was used to explore the molecular characteristics of granulosa cells from patients with PCOS treated with liraglutide. The levels of C-X-C motif chemokine ligand 10 (CXCL10) in follicular fluid were detected by ELISA, the expression levels of ovulation related genes and inflammatory factor genes in follicles and granulosa cells were detected by qPCR and the protein levels of connexin 43 (Cx43), Janus Kinase 2 (JAK2) and phosphorylated JAK2 were detected by Western blot. The mouse ovarian follicles culture system in vitro was used to detect the status of follicle development and ovulation.ResultsIn the present study, we found that liraglutide inhibited the secretion of inflammatory factors in PCOS granulosa cells, among which CXCL10 was the most significant. In addition, CXCL10 was significantly higher in granulosa cells and follicular fluid in PCOS patients than in non-PCOS patients. We applied in vitro follicle culture and other techniques to carry out the mechanism exploration which revealed that CXCL10 disrupted the homeostasis of gap junction protein alpha 1 (GJA1) between oocyte and granulosa cells before physiological ovulation, thus inhibiting follicular development and ovulation. Liraglutide inhibited CXCL10 secretion in PCOS granulosa cells by inhibiting the JAK signaling pathway and can improved dehydroepiandrosterone (DHEA)-induced follicle development disorders, which is reversed by CXCL10 supplementation.ConclusionsThe present study suggests that liraglutide inhibits CXCL10 secretion in granulosa cells through JAK signaling pathway, thereby improving the homeostasis of GJA1 between oocyte and granulosa cells before physiological ovulation and ultimately improving the follicular development and ovulation of PCOS, which provides more supportive evidence for the clinical application of liraglutide in the treatment of ovulatory disorders in PCOS.Trial registrationNot applicable.

Quantitative label-free proteomic analysis of mouse ovarian antral follicles following oral exposure to a human-relevant mixture of three phthalates.

Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are used in personal and medical care products. In the ovary, antral follicles are essential for steroidogenesis and ovulation. DBP, BBP, and DEHP are known to inhibit mouse antral follicle growth and ovulation in vitro, and associate with decreased antral follicle counts in women. Given that the in vivo effects of a three-phthalate mixture on antral follicles are unknown, we evaluated the effects of a human-relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. Adult CD-1 female mice were fed corn oil (vehicle), or two dose levels of a phthalate mixture based on estimated exposures in general (32 µg/kg/d; PHT 32) and occupationally exposed (500 µg/kg/d; PHT 500) populations for 10 d. Antral follicles (>250 µm) were isolated and subjected to proteome profiling via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were detected, of which 194 were differentially abundant between vehicle and PHT 32, and 136 between vehicle and PHT 500. Bioinformatic analysis revealed significantly different responses between the two phthalate doses. Protein abundance differences in the PHT 32 exposure mapped to cytoplasm, mitochondria, and lipid metabolism; whereas those in the PHT 500 exposure mapped to cytoplasm, nucleus, and phosphorylation. When both doses altered proteins mapped to common processes, the associated predicted transcription factors were different. These findings provide novel mechanistic insight into phthalate-associated, ovary-driven reproductive outcomes in women.

P-349 Impact of an Aurora kinase inhibitor on AML cells and mouse ovarian follicles in vitro

Abstract Study question Can an aurora kinase B/C inhibitor suppress AML cell line proliferation while not impacting on mouse follicles in vitro? Summary answer Exposure to an aurora kinase inhibitor at a predetermined cytotoxic concentration suppressed AML cell proliferation but had no impact on subsequent follicle health in vitro. What is known already There is a risk that cryopreserved ovarian tissue from young women with a leukaemia diagnosis harbours leukemic cells, which on grafting could transfer disease. Novel chemotherapy agents, specifically aurora kinase inhibitors [ e.g. GSK 1070916 (GSK) ], target proteins involved in chromosomal alignment and segregation during mitosis and meiosis. It is anticipated that aurora kinase inhibitors would have an impact on the development of all somatic cells, including granulosa cells proliferating during follicle growth, however a previous study using ovarian tissue pieces indicated no impact on follicles. Study design, size, duration Primary and secondary follicles (diameter ≤ 100 µm) were manually dissected from adult mouse ovaries and embedded in an extracellular scaffold together with >1,000 AML cells. Individual follicles and AML cells were cultured for 7 days followed by exposure to GSK for 24 hours. GSK was removed by washing and the culture continued for another two days at which time cells were assessed for normal morphology and survival with live/dead staining (Calcein AM/ Ethidium Homodimer-1). Participants/materials, setting, methods The leukemic cell line OC1-AML-3 (DSMZ) was cultured in RPMI-1640 +10% FCS and routinely passaged as recommended. Cell proliferation and cytotoxicity were assessed using the Alamar Blue assay. 20% DMSO was used as a positive control in the cytotoxicity assays and live/dead evaluation. Follicles were cultured in αMEM, ITS, FSH, ascorbic acid and 10% FCS under oil in 5%CO2 : 95% air. Survival and growth were evaluated in two extracellular scaffolds; UltiMatrix and Hystem™-HP. Main results and the role of chance Follicle survival and growth was better (p < 0.05) in UltiMatrix (84.7%; 61/72) compared to Hystem™-HP (72.0%;77/107) with an average increase of 20 µm in diameter at day 7. When OCI-AML-3 cells (0.7 x105/ml) were exposed to a range of concentrations of GSK (10nM to 10µM) for 24hr in an in vitro cytotoxicity assessment, survival was reduced to 0% at a concentration of 10µM. The 20% DMSO positive control also resulted in 0% survival. However, in the 3- dimensional scaffold culture, exposure to 10 µM GSK for 24hr resulted in a mean survival of OCI-AML-3 cells of 27% (100 cells counted/ well) which was similar to the 20% DMSO positive control (32%) and significantly different to the negative (no GSK) control (95%; p < 0.01). Follicles exposed to GSK appeared alive, emitting a strong green fluorescence for both oocyte and granulosa cells, although some of the surface follicular cells were dead. Live/dead staining of GSK exposed follicles was similar to follicles not exposed to GSK. In contrast, the majority of the follicular cells were dead following exposure to 20% DMSO. Limitations, reasons for caution Due to whole mount assessment of the follicles for live/dead staining, it is difficult to determine whether the oocyte is alive. In scaffold culture some of the free cells present may be of follicular origin, potentially resulting in overestimation of the proportion of AML cells alive after GSK treatment. Wider implications of the findings This 3- dimensional culture system, incorporating a follicle and a leukemic cell line within a scaffold provides a good model to assess the impact of chemotherapy on both cell types and could be applicable for human follicles. Trial registration number Not applicable

The influence of carbofuran insecticide exposure on the number of ovarian follicles in mice (Mus musculus)

Objective: The aim of this research was to investigate the histopathological damage to the ovaries of mice (Mus musculus) caused by exposure to carbofuran. Method: Twenty female mice aged 10 weeks with a body weight ranging from 25-30 mg were used in this study. The mice were divided into four groups, with each group consisting of five replicates. The control group (P0) received 0.5% physiological NaCl, while the treatment groups (P1, P2, and P3) were exposed to carbofuran at doses of 0.0833 mg/kg BW, 0.0417 mg/kg BW, and 0.0208 mg/kg BW, respectively, for ten days. On the 12th day, ovarian necropsy and HE staining were performed to observe the number of primary, secondary, tertiary, and Graafian follicles. Data analysis was conducted using ANOVA and BNJ tests. Result: The results of this research showed that oral administration of carbofuran leads to a decrease in the number of secondary, tertiary, and Graafian follicles. High doses, such as 0.0833 mg/kg body weight, resulted in a more pronounced reduction in the number of secondary, tertiary, and Graafian follicles compared to the control group. Conclusion: A reduction in the number of secondary, tertiary, and Graafian follicles with increasing doses of carbofuran.

A spatiotemporal molecular atlas of the ovulating mouse ovary

Ovulation is essential for reproductive success, yet the underlying cellular and molecular mechanisms are far from clear. Here, we applied high-resolution spatiotemporal transcriptomics to map out cell type- and ovulation stage-specific molecular programs as function of time during follicle maturation and ovulation in mice. Our analysis revealed dynamic molecular transitions within granulosa cell types that occur in tight coordination with mesenchymal cell proliferation. We identified molecular markers for the emerging cumulus cell fate during the preantral-to-antral transition. We describe transcriptional programs that respond rapidly to ovulation stimulation and those associated with follicle rupture, highlighting the prominent roles of apoptotic and metabolic pathways during the final stages of follicle maturation. We further report stage-specific oocyte-cumulus cell interactions and diverging molecular differentiation in follicles approaching ovulation. Collectively, this study provides insights into the cellular and molecular processes that regulate mouse ovarian follicle maturation and ovulation with important implications for advancing therapeutic strategies in reproductive medicine.

Evaluation of the morphology and development of preantral ovarian follicles in mice submitted to a chronic diet of dietary supplementation with Pereskia aculeata Miller leaves.

This study aimed to investigate the effect of including mouse feed with different concentrations (5, 10, or 20%) of Pereskia aculeata Miller (PAM) leaves on the morphology and development of preantral ovarian follicles and ovarian stromal cell density. The oral toxicity was performed using repeated dose toxicity assays subdivided into experiments of 30 days and 90 days of treatment. After the experiments, the ovaries of each animal were collected and submitted to classical histology. At 30 and 90 days, there was an equivalent percentage of normal, primordial, and developing follicles (P > 0.05) between PAM treatments compared to the control. Regarding the different stages of follicular development, after 90 days, there was a higher percentage (P < 0.05) of developing follicles only in the control group compared to day 30. The PAM 5% treatment was the only one that affected the cell density in the stroma after 90 days of treatment. Thus, we observed that supplementing the diet with P. aculeata did not pose any risk concerning animal consumption; specifically, there were no toxic reproductive effects observed from adding Pereskia aculeata Miller to the mouse diet.

Participation of preovulatory follicles in the activation of primordial follicles in mouse ovaries.

The mechanisms behind the selection and initial recruitment of primordial follicles (PmFs) from the non-growing PmF pool during each estrous cycle in females remain largely unknown. This study demonstrates that PmFs closest to the ovulatory follicle are preferentially activated in mouse ovaries under physiological conditions. PmFs located within 40 μm of the ovulatory follicles were more likely to be activated compared to those situated further away during the peri-ovulation period. Repeated superovulation treatments accelerated the depletion of the PmF reserve, whereas continuous suppression of ovulation delayed PmF reserve consumption. Spatial transcriptome sequencing of peri-ovulatory follicles revealed that ovulation primarily induces the degradation and remodeling of the extracellular matrix (ECM). This ECM degradation reduces mechanical stress around PmFs, thereby triggering their activation. Specifically, Cathepsin L (CTSL), a cysteine proteinase and lysosomal enzyme involved in ECM degradation, initiates the activation of PmFs adjacent to ovulatory follicles in a distance-dependent manner. These findings highlight the link between ovulation and selective PmF activation, and underscore the role of CTSL in this process under physiological conditions.

Withanolide derivatives: natural compounds with anticancer potential offer low toxicity to fertility and ovarian follicles in mice

Withanolide derivatives: natural compounds with anticancer potential offer low toxicity to fertility and ovarian follicles in mice

The role of the aryl hydrocarbon receptor in mediating the effects of mono(2-ethylhexyl) phthalate in mouse ovarian antral follicles†.

Di(2-ethylhexyl) phthalate (DEHP) is a pervasive environmental toxicant used in the manufacturing of numerous consumer products, medical supplies, and building materials. DEHP is metabolized to mono(2-ethylhexyl) phthalate (MEHP). MEHP is an endocrine disruptor that adversely affects folliculogenesis and steroidogenesis in the ovary, but its mechanism of action is not fully understood. Thus, we tested the hypothesis that the aryl hydrocarbon receptor (AHR) plays a functional role in MEHP-mediated disruption of folliculogenesis and steroidogenesis. CD-1 mouse antral follicles were isolated and cultured with MEHP (0-400μM) in the presence or absence of the AHR antagonist CH223191 (1μM). MEHP treatment reduced follicle growth over a 96-h period, and this effect was partially rescued by co-culture with CH223191. MEHP exposure alone increased expression of known AHR targets, cytochrome P450 (CYP) enzymes Cyp1a1 and Cyp1b1, and this induction was blocked by CH223191. MEHP reduced media concentrations of estrone and estradiol compared to control. This effect was mitigated by co-culture with CH223191. Moreover, MEHP reduced the expression of the estrogen-sensitive genes progesterone receptor (Pgr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr) and co-treatment with CH223191 blocked this effect. Collectively, these data indicate that MEHP activates the AHR to impair follicle growth and reduce estrogen production and signaling in ovarian antral follicles.

The effect of Coenzyme Q10 on mitochondrial biogenesis in mouse ovarian follicles during in vitro culture.

The aim of this research was to investigate the effect of Coenzyme Q10 (CoQ10) on the expression of the Transcription Factor A Mitochondrial (Tfam) gene and mtDNA copy number in preantral follicles (PFs) of mice during in vitro culture. To conduct this experimental study, PFs were isolated from 14-day-old National Medical Research Institute mice and cultured in the presence of 50 µm CoQ10 for 12 days. On the 12th day, human chorionic gonadotropin was added to stimulate ovulation. The fundamental parameters, including preantral follicle developmental rate and oocyte maturation, were evaluated. Additionally, the Tfam gene expression and mtDNA copy number of granulosa cells and oocytes were assessed using the real-time polymerase chain reaction. The results revealed that CoQ10 significantly increased the diameter of PFs, survival rate, antrum formation, and metaphase II (MII) oocytes (P < 0.05). Moreover, in the CoQ10-treated groups, the Tfam gene expression in granulosa cells and oocytes increased considerably compared with the control group. The mtDNA copy number of granulosa cells and oocytes cultured in the presence of CoQ10 was substantially higher compared with the control groups (P < 0.05). The addition of CoQ10 to the culture medium enhances the developmental competence of PFs during in vitro culture by upregulating Tfam gene expression and increasing mtDNA copy number in oocyte and granulosa cells.

Successful 3D culture and transplantation of mouse isolated preantral follicles in hydrogel of bioengineered Wharton's jelly.

Due to Human Wharton's Jelly (HWJ) could be applied in tissue engineering as a bio scaffold, the present study was conducted to investigate the effects of HWJ hydrogel on in vitro culture and auto-transplantation of mouse ovarian follicles. HWJ was isolated from umbilical cord and decellularized with SDS/Tris/EDTA. DNA, Collagen and Glycosaminoglycans (GAGs) were measured. Decellularized Wharton's Jelly (DWJ) was dissolved to make Wharton's Jelly Hydrogel (WJH), and composited with Alginate (ALG) (1.5%) in equal ratio (WJH+ALG). Then, mouse preantral follicles were isolated and encapsulated in 10μL droplets of WJH and randomly considered for both 14 days culture and auto-transplantation. Collagen, GAGs and DNA evaluations showed majority of WJ cells have been removed and MTT approved no toxicity. Degradation rate and rheological analysis represented optimal hydrogel compatibility. The data from in vitro culture revealed significant antral formation in WJH+ALG (P≤0.05). In transplantation, follicles failed to survive in ALG; however, survived in WJH+ALG to antral stage (P<0.05). VEGF and CD34 had greater expression in WJH+ALG than ALG (P< 0.05). Wharton's jelly hydrogel and Alginate compound is interesting composite for successful development of mouse preantral follicles in both 3D in vitro culture and transplantation.

Abstract 019: Perimenopause Exacerbates Hypertension In Doca-salt Rats

Blood pressure (BP) is lower in premenopausal women than in age-matched men, but after menopause BP rates and the incidence of hypertension (HTN) show opposite tendencies. The mechanisms by which fluctuating ovarian hormone environment during that period impacts the onset and development of HTN are poorly understood, and strategies to reduce HTN risk in peri- and postmenopausal females are lacking. An ovary-intact rat model of perimenopause using the chemical, 4-vinylcyclohexene diepoxide (VCD), has been developed to accelerate the natural process of slow follicular loss. Daily dosing with VCD for 15 days selectively destroys primordial and primary follicles in ovaries of mice and rats by accelerating atresia processes, thus inducing precocious perimenopause/menopause. Hypothesis: The induction of perimenopause by VCD will increase susceptibility to the development of HTN in DOCA-salt rats. Female Sprague-Dawley rats at post-natal day 28 received subcutaneous injection of VCD (160mg/kg) or corn oil (control) for 15 days. 57 days after the first injections, nephrectomy and telemetric pressure transducer implantation were performed. After 2 weeks of recovery, a 100 mg/kg DOCA silicone or placebo pellet was implanted subcutaneously. The rats that received DOCA were provided 0.9% saline during the 21 days of DOCA protocol and mean arterial pressure (MAP) was continuously evaluated. Rats were housed weekly in metabolic cages for 24 hours to measure saline intake and urine volume. After protocol completion the animals were decapitated, and blood was collected for the measurement of anti-Mullerian hormone (AMH) and estradiol (E2) by ELISA. The AMH plasma levels were reduced in the periestropause rats (VCD + DOCA, n=9) compared to oil (Oil + DOCA, n=6) confirming the VCD-induced follicular depletion. The steady state increase in MAP (day 21 of DOCA-salt) was higher in periestropause (±45.7mmHg) compared to oil (±28.6 mmHg). Urine volume was higher in periestropause rats compared to oil. No differences were observed in the saline intake between the groups. Our data demonstrate that periestropause rats showed greater susceptibility to the development of HTN in a DOCA-salt rat model.

Ovarian antral follicles metabolize imidacloprid in vitro.

Neonicotinoid insecticides are synthetic nicotine derivatives that have high affinity for invertebrate nicotine receptors and low affinity for mammalian nicotine receptors. However, imidacloprid (IMI), the most commonly used neonicotinoid, can be bioactivated by the liver in mammals to desnitro-imidacloprid, an intermediate metabolite that effectively binds and activates mammalian receptors. However, it is not known if other tissues such as the ovaries can metabolize IMI. Thus, the present study tested the hypothesis that ovarian antral follicles metabolize and bioactivate IMI. Antral follicles were dissected from the ovaries of CD-1 mice and cultured in media containing dimethyl sulfoxide or IMI (0.2-200 µg/ml) for 48 and 96 h. Media were subjected to liquid chromatography-mass spectrometry for detection of phase I IMI metabolites. Follicles from the cultures were used for gene expression analysis of metabolic enzymes associated with IMI metabolism. All IMI metabolites were detected at 48 and 96 h. Oxidized IMI intermediates were detected in media from cultured follicles, but not environmental controls. Reduced IMI intermediates were detected in media from cultured follicles and the environmental controls. At 48 h, IMI did not affect expression of any metabolic enzymes compared with control. At 96 h, IMI induced Cyp2e1 and Cyp4f18 compared with control. These data indicate that mouse ovarian follicles metabolize IMI and that IMI induces ovarian Cyp expression over time.

P-408 The ovarian tissues outside of the radiation field could be damaged by radiation-induced abscopal effects in mice: an experimental study.

Abstract Study question Could irradiation-induced abscopal effects (RIAEs) induce structural and functional damage in the distal ovary in female mice, and what is its mechanism? Summary answer The RIAEs can induce an inflammatory reaction and inhibit the growth and development of ovarian follicles in mice leading to a decrease in ovarian reserve. What is known already The irradiated tumor could induce cell and tissue injury in the organs distant from the radiation site, referred to as the radiation-induced abscopal effects (RIAEs). Many pieces of research have shown out-of-field tumor regression effects of radiation therapy. However, RIAEs is a double-edged sword and may cause serious side effects on normal tissues. Premature ovarian insufficiency (POI) is one of the main side effects of radiotherapy, but the relationship between RIAE and POI has not been clarified. Study design, size, duration C57BL/6J mice were used to establish a RIAEs model by irradiating the thorax and the abdomens were shielded. Sixteen C57BL/6J female mice were randomly allocated to sham and experimental groups (n = 8/group) according to the presence or absence of irradiation with an 8 Gy X-ray on the local area of the chest every day for three days. After irradiation for twenty-one days, The effect and possible mechanism of RIAEs on non-irradiated ovarian were discussed. Participants/materials, setting, methods After irradiation, the estrous cyclicity, serum steroid hormones, and pro-inflammatory factors were compared between groups. Furthermore, RNA-seq was used to detect the expression of transcriptional levels in ovarian tissues in both groups. The differentially expressed genes (DEGs) were screened and analyzed by gene ontology-biological process (GO_BP) between irradiated and sham groups. The expression and localization of spermatogenesis-and oogenesis-specific basic helix-loop-helix-containing protein 1(SOHLH1) and neutrophil elastase (NE) in ovarian tissues were detected by immunohistochemistry(IHC). Main results and the role of chance Compared with mice in the sham group, the irradiation group had disordered estrous cycles, reduced primordial follicles (P&amp;lt;0.001)] and growing follicles (P&amp;lt;0.001), significantly increased atretic follicles (P&amp;lt;0.001). Levels of serum estradiol [(70.28 ± 5.27) pmol/L] and AMH [(104.00±6.98) ng/L] in the irradiation group were significantly lower than those in the sham group [estradiol (97.58±7.25) pmol/L, P=0.016; AMH [(129.70±8.39) ng/mL, P=0.046], but FSH levels in the irradiation group were not significantly different from those in the sham group (P=0.996). Compared with the sham group, Serum levels of TNF-α [(488.30±36.20) ng/L vs. (31.61±12.89) ng/L, P&amp;lt;0.001] and IL-1β [(62.37±2.50) ng/L vs. (52.75±2.06) ng/L, P=0.018)] in the irradiation group were significantly increased. Serum levels of IL-6 in the irradiation group were also increased compared with the sham group, but the difference was not statistically significant (P=0.301). The results of GO_BP analysis showed that down-regulated DGEs were mainly involved in the process of follicular development, and up-regulated DGEs were involved in the inflammation process. The IHC results showed that the positive expression area of SOHLH1 in the irradiation group was significantly lower than in the sham group (P=0.005). In comparison, the positive expression area of NE was significantly higher than that of the Sham group(P=0.024). Limitations, reasons for caution This study was performed using a short, 3-day irradiation treatment condition. Therefore, the results need to be cautiously interpreted concerning radiation-associated chronic toxicity. Moreover, further studies in mouse tumor models are required to determine the clinical relevance of the role of RIAE. Wider implications of the findings Although the precise molecular mechanisms of these distal effects on the genital system are still unclear, the current discovery might pave the way for establishing novel fertility preservation protocols. The research offers hope to women with POI related to cancer therapy through the modulation of abscopal effects associated with radiotherapy. Trial registration number not applicable

Three-dimensional imaging and quantification of mouse ovarian follicles via optical coherence tomography.

Ovarian tissue cryopreservation has been successfully applied worldwide for fertility preservation. Correctly selecting the ovarian tissue with high follicle loading for freezing and reimplantation increases the likelihood of restoring ovarian function, but it is a challenging process. In this work, we explore the use of three-dimensional spectral-domain optical coherence tomography (SD-OCT) to identify different follicular stages, compare the identifications with H&E images, and measure the size and age-related follicular density distribution differences in mice ovaries. We use the thickness of the layers of granulosa cells to differentiate primordial and primary follicles from secondary follicles. The measured dimensions and age-related follicular distribution agree well with histological images and physiological aging. Finally, we apply attenuation coefficient map analyses to significantly improve the image contrast and the contrast-to-noise ratio (p < 0.001), facilitating follicle identification and quantification. We conclude that SD-OCT is a promising method to noninvasively evaluate ovarian follicles for ovarian tissue cryopreservation.

Inhibition of phosphodiesterase PDE8B reduces activation of primordial follicles in mouse ovaries.

In the ovaries, cyclic adenosine 3',5'-monophosphate (cAMP) is a second messenger supporting the generation of steroids. Phosphodiesterases (PDEs) are regulators of intracellular cAMP, and therefore, potential regulators of ovarian function. Interestingly, the family of PDE genes are differentially expressed in human oocytes and granulosa cells from primordial and primary follicles, suggesting diverse roles. In this study, we addressed the functions of PDE3B and PDE8B in primordial follicle regulation using inhibitors of PDE3B and PDE8B in murine ovary primary in vitro cultures. Inhibition of PDE8B in ovarian cultures prevented primordial follicle activation, while inhibition of PDE3B had no effect on follicle distribution in the ovary, under the tested conditions. As cAMP levels may increase steroid levels, we assessed the protein levels of the steroidogenic acute regulatory protein (StAR) and aromatase enzymes, and found that inhibition of PDE3B reduced StAR protein levels, whereas inhibition of PDE8 did not alter StAR expression in our murine ovary culture system conditions. Our results showed that ketotifen-induced inhibition of PDE8B can decrease primordial follicle activation, whereas we observed no effect of follicle distribution, when PDE3B was inhibited. Expression of the StaR enzyme was not altered when PDE8B was inhibited, which might reflect not sufficient inhibition by ketotifen to induce StAR alterations, or redundant mechanisms.

CAMP controls the balance between dormancy and activation of primordial follicles in mouse ovaries.

In mammalian ovaries, the balance between dormancy and activation of primordial follicles determines the female fecundity and endocrine homeostasis. Recently, several functional molecules and pathways have been reported to be involved in the activation of primordial follicles. However, the homeostasis regulatory mechanisms of primordial follicle activation are still scant. Our previous study has proved that a relatively higher concentration of cyclic AMP (cAMP) is required for primordial follicle formation. Here, we identified that cAMP also plays a vital role in the balance between dormancy and activation of primordial follicles. Our results showed that the concentration of cAMP remained stable in neonatal mouse ovaries, which is due to ADCY3, the synthetase of cAMP, and PDE3A, the hydrolytic enzyme of cAMP, were synchronously increased during the activation of primordial follicles in mouse ovaries. Once the concentration of cAMP in neonatal ovaries was either elevated or reduced in vitro, the activation of primordial follicles was either accelerated or decelerated accordingly. In addition, a higher concentration of cAMP in the ovaries of puberty mice improved primordial follicle activation in vivo. Finally, cAMP promoted primordial follicle activation via canonical mTORC1-PI3K signaling cascades and PKA signaling. In conclusion, our findings reveal that the concentration of cAMP acts as a key regulator in balancing the dormancy and activation of primordial follicles in the mouse ovary.

Lipid droplet synthesis is associated with angiogenesis in mouse ovarian follicles†.

Lipid droplets (LDs) are endoplasmic reticulum (ER)-derived organelles comprising a core of neutral lipids surrounded by a phospholipid monolayer. Lipid droplets play important roles in lipid metabolism and energy homeostasis. Mammalian ovaries have been hypothesized to use neutral lipids stored in LDs to produce the hormones and nutrients necessary for rapid follicular development; however, our understanding of LD synthesis remains incomplete. In this study, we generated transgenic reporter mice that express mCherry fused to HPos, a minimal peptide that localizes specifically to nascent LDs synthesized at the ER. With this tool for visualizing initial LD synthesis in ovaries, we found that LDs are synthesized continuously in theca cells but rarely in inner granulosa cells (Gc) during early follicular development. Administration of exogenous gonadotropin enhances LD synthesis in the Gc, suggesting that LD synthesis is hormonally regulated. In contrast, we observed copious LD synthesis in the corpus luteum, and excessive LDs accumulation in atretic follicles. Furthermore, we demonstrated that LD synthesis is synchronized with angiogenesis around the follicle and that suppressing angiogenesis caused defective LD biosynthesis in developing follicles. Overall, our study is the first to demonstrate a spatiotemporally regulated interplay between LD synthesis and neovascularization during mammalian follicular development.

Endometrial cell-derived conditioned medium in combination with platelet-rich plasma promotes the development of mouse ovarian follicles.

Fertility preservation is one of the most important issues in assisted reproductive technology. Previous studies have shown that cytokines and growth factors can improve follicle growth. The endometrial stromal cells secrete various factors that are involved in maintaining the integrity of uterine and epithelial secretory function. The platelet-rich plasma contains a large assembly of platelets suspended in plasma that successfully improves the viability and growth of various cell lines. This work aimed to investigate the influences of conditioned medium (CM) and platelet-rich plasma (PRP) on the development of ovarian follicles in infertile mice due to cyclophosphamide (CYC) exposure. In this study, 65 healthy BALB/c female mice (∼28-30 g and 6-8 weeks old) in five groups were studied. Immunohistochemistry (IHC) was used to detect growth differentiation factor 9 (GDF9)-positive cells. The mRNA expression levels of SMAD1, SMAD2, and BMP15 was assessed using reverse transcription-polymerase chain reaction (RT-PCR) method. The expression levels of SMAD1, GDF9, BMP15, and SMAD2 in the CM+PRP group was significantly more than in the CM and PRP groups. In addition, live birth occurred in the CM+PRP group. Treatment with CM+PRP in infertile mice due to Cy exposure increased fertility and live-birth rate. In general, our study suggested that the CM and PRP combination could improve the growth of mice ovarian follicles in vivo.