Effects of follicle-stimulating hormone on energy balance and tissue metabolic health after loss of ovarian function.

Loss of ovarian function imparts increased susceptibility to obesity and metabolic disease. These effects are largely attributed to decreased estradiol (E2), but the role of increased follicle-stimulating hormone (FSH) in modulating energy balance has not been fully investigated. Previous work that blocked FSH binding to its receptor in mice suggested this hormone may play a part in modulating body weight and energy expenditure after ovariectomy (OVX). We used an alternate approach to isolate the individual and combined contributions of FSH and E2 in mediating energy imbalance and changes in tissue-level metabolic health. Female Wistar rats were ovariectomized and given the gonadotropin releasing hormone (GnRH) antagonist degarelix to suppress FSH production. E2 and FSH were then added back individually and in combination for a period of 3 wk. Energy balance, body mass composition, and transcriptomic profiles of individual tissues were obtained. In contrast to previous studies, suppression and replacement of FSH in our paradigm had no effect on body weight, body composition, food intake, or energy expenditure. We did, however, observe organ-specific effects of FSH that produced unique transcriptomic signatures of FSH in retroperitoneal white adipose tissue. These included reductions in biological processes related to lipogenesis and carbohydrate transport. In addition, rats administered FSH had reduced liver triglyceride concentration (P < 0.001), which correlated with FSH-induced changes at the transcriptomic level. Although not appearing to modulate energy balance after loss of ovarian function in rats, FSH may still impart tissue-specific effects in the liver and white adipose tissue that might affect the metabolic health of those organs.NEW & NOTEWORTHY We find no effect of follicle-stimulating hormone (FSH) on energy balance using a novel model in which rats are ovariectomized, subjected to gonadotropin-releasing hormone antagonism, and systematically given back FSH by osmotic pump. However, tissue-specific effects of FSH on adipose tissue and liver were observed in this study. These include unique transcriptomic signatures induced by the hormone and a stark reduction in hepatic triglyceride accumulation.

Effectiveness of gonadotropin-releasing hormone agonists for ovarian function suppression in premenopausal patients with hormone receptor-positive breast cancer: a retrospective single-center real-world study.

PURPOSE: This study evaluated the effectiveness of ovarian function suppression (OFS) of various gonadotropin-releasing hormone agonists (GnRHa) combined with aromatase inhibitors (AI) in premenopausal patients with hormone receptor-positive (HR-positive) breast cancer. Potential risk factors associated with insufficient OFS were analyzed. PATIENTS AND METHODS: Premenopausal HR-positive breast cancer patients who had received AI with GnRHa were studied retrospectively. Patients were divided into different groups according to monthly or trimonthly GnRHa schedules they received, and the effectiveness of OFS was compared between groups. Insufficient OFS was defined as at least one instance of estradiol ≥ 30 pg/ml. Patient data was gathered from medical records for this comparison. RESULTS: Of the 264 patients enrolled in this study, 117 were administered 3.6 mg of goserelin monthly (goserelin 1 M group), 63 received 3.75 mg of leuprorelin monthly (leuprorelin 1 M group) and 84 were given 11.25 mg of leuprorelin every three months (leuprorelin 3 M group). Overall, 7.20% experienced insufficient OFS. The incidence rates in the three GnRHa depot groups were 7.69%, 6.35%, and 7.14%, respectively, without a significant statistical difference (P = 0.900). Notably, younger patients exhibited a higher likelihood of insufficient OFS [OR = 0.900, 95%CI (0.824-0.982), P = 0.018]. CONCLUSION: Insufficient OFS remains a concern during GnRHa and AI treatment. The effectiveness of the three GnRHa depots commonly used in China seems comparable. Younger patients face a heightened risk of insufficient OFS.

Apelin receptor modulation mitigates letrozole-induced polycystic ovarian pathogenesis in mice.

AIMS: Polycystic ovarian syndrome (PCOS) is one of the most common (about 5-20%) reproductive disorders in women of reproductive age; it is characterized by polycystic ovaries, hyperandrogenism, and oligo/ anovulation. The levels and expression of ovarian adipokines are deregulated in the PCOS. Apelin is an adipokine that acts through its receptor (APJ) and is known to express in the various tissues including the ovary. It has also been suggested that apelin and APJ could be targeted as therapeutic adjuncts for the management of PCOS. However, no study has been conducted on the management of PCOS by targeting the apelin system. Thus, we aimed to evaluate its impact on combating PCOS-associated ovarian pathogenesis. METHODS: The current work employed a letrozole-induced-hyperandrogenism PCOS-like mice model to investigate the effects of apelin13 and APJ, antagonist ML221. The PCOS model was induced by oral administration of letrozole (1 mg/kg) for 21 days. A total of four experimental groups were made, control, PCOS control, PCOS + aplein13, and PCOS + ML221. The treatment of apelin13 and ML221 was given from day 22 for two weeks. KEY FINDINGS: The letrozole-induced PCOS-like features such as hyperandrogenism, cystic follicle, decreased corpus luteum, elevated levels of LH/FSH ratio, and up-regulation of ovarian AR expression were ameliorated by apelin13 and ML221 treatment. However, the PCOS-augmented oxidative stress and apoptosis were suppressed by apelin 13 treatments only. ML221 treatment still showed elevated oxidative stress and stimulated apoptosis as reflected by decreased antioxidant enzymes and increased active caspase3 and Bax expression. The expression of ERs was elevated in all groups except control. Furthermore, the PCOS model showed elevated expression of APJ and apelin13 treatment down-regulated its own receptor. Overall, observing the ovarian histology, corpus luteum formation, and decreased androgen levels by both apelin13 and ML221 showed ameliorative effects on the cystic ovary. SIGNIFICANCE: Despite the similar morphological observation of ovarian histology, apelin13 and ML221 exhibited opposite effects on oxidative stress and apoptosis. Therefore, apelin13 (which down-regulates APJ) and ML221 (an APJ antagonist) may have suppressed APJ signalling, which would account for our findings on the mitigation of polycystic ovarian syndrome. In conclusion, both apelin13 and ML221 mediated mitigation have different mechanisms, which need further investigation.

Exposure to topiramate and acetazolamide causes endocrine disrupting effects in female rats during estrus.

BACKGROUND: Idiopathic intracranial hypertension (IIH) is a disease characterized by elevated intracranial pressure (ICP) and is a disease of young females. The first line pharmacological treatments include acetazolamide and topiramate and given the nature of IIH patients and the dosing regimen of these drugs, their effect on the endocrine system is important to evaluate. We aimed to assess the effects of acetazolamide and topiramate on steroid profiles in relevant endocrine tissues. METHODS: Female Sprague Dawley rats received chronic clinically equivalent doses of acetazolamide or topiramate by oral gavage and were sacrificed in estrus. Tissue specific steroid profiles of lateral ventricle CP, 4th ventricle CP, CSF, serum, uterine horn and fundus, ovaries, adrenal glands and pituitary glands were assessed by quantitative targeted LC-MS/MS. We determined luteinizing hormone (LH) and follicle stimulating hormones (FSH) levels in paired serum by ELISA. RESULTS: Topiramate increased the concentration of estradiol and decreased the concentration of DHEA in lateral choroid plexus. Moreover, it decreased the concentration of androstenediol in the pituitary gland. Topiramate increased serum LH. Acetazolamide decreased progesterone levels in serum and uterine fundus and increased corticosteroid levels in the adrenal glands. CONCLUSION: These results demonstrate that both acetazolamide and topiramate have endocrine disrupting effects in rats. Topiramate primarily targeted the choroid plexus and the pituitary gland while acetazolamide had broader systemic effects. Furthermore, topiramate predominantly targeted sex hormones, whereas acetazolamide widely affected all classes of hormones. A similar effect in humans has not yet been documented but these concerning findings warrants further investigations.

Exposure to 7,12-dimethylbenz[a]anthracene impacts ovarian DNA damage sensing and repair proteins differently in lean and obese female mice and weight loss may mitigate obesity-induced ovarian dysfunction.

Obesity impairs oocyte quality, fertility, pregnancy maintenance, and is associated with offspring birth defects. The model ovotoxicant, 7,12-dimethylbenz[a]anthracene (DMBA), causes ovarian DNA damage and follicle loss. Both DMBA-induced chemical biotransformation and the DNA damage response are partially attenuated in obese relative to lean female mice but whether weight loss could improve the DNA damage response to DMBA exposure has not been explored. Thus, at six weeks of age, C57BL/6 J female mice were divided in three groups: 1) Lean (L; n = 20) fed a chow diet for 12 weeks, 2) obese (O; n = 20) fed a high fat high sugar (HFHS) diet for 12 weeks and, 3) slim-down (S; n = 20). The S group was fed with HFHS diet for 7 weeks until attaining a higher body relative to L mice on week 7.5 and switched to a chow diet for 5 weeks to achieve weight loss. Mice then received either corn oil (CT) or DMBA (D; 1 mg/kg) for 7 d via intraperitoneal injection (n = 10/treatment). Obesity increased (P < 0.05) kidney and spleen weight, and DMBA decreased uterine weight (P < 0.05). Ovarian weight was reduced (P < 0.05) in S mice, but DMBA exposure increased ovary weight in the S mice. LC-MS/MS identified 18, 64, and 7 ovarian proteins as altered (P < 0.05) by DMBA in the L, S and O groups, respectively. In S and O mice, 24 and 8 proteins differed, respectively, from L mice. These findings support weight loss as a strategy to modulate the ovarian genotoxicant response.

Donepezil protects against cyclophosphamide-induced premature ovarian failure in mice: A focus on proinflammatory cytokines and NLRP3/TLR-4/NF-κB interplay.

BACKGROUND AND AIM: Cyclophosphamide (CP) chemotherapy is a significant iatrogenic component of premature ovarian failure (POF). The aim of this work was to evaluate the potential protective effects of donepezil, a centrally acting acetylcholinesterase (AChE) inhibitor, on CP-induced POF in mice. METHODS: 40 female Swiss albino mice were split into 5 equal groups: group 1 (control), group 2 (CP-POF); induced by intraperitoneal injection of CP on 8th day of the experiment, and group (3-5); mice received oral donepezil daily (1, 2, or 4 mg/kg, respectively) 8 days before CP injection. Mice were euthanized after 24 h of CP injection, and blood samples were collected to assay serum anti-Mullerian hormone (AMH) levels. Ovarian tissues were dissected, and the right ovary was processed for further assays of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interlukin-6 (IL-6), nucleotide-binding domain-like receptor family, the Pyrin domain-containing 3 (NLRP3) inflammasome, and Toll-like receptor 4 (TLR-4), while the left one was processed for histopathological and immunohistochemical examination of nuclear factor-Kappa beta (NF-κB) and caspase-3. RESULTS: Donepezil, in a dose-dependent manner particularly (4 mg/kg), has an inhibitory action on NO (40 ± 2.85 vs. 28.20 ± 2.23, P < 0.001), proinflammatory cytokines (P < 0.001), the TLR-4/ NF-κB / NLRP3 inflammasome pathway (P < 0.001), and apoptosis (P < 0.001), with a significant elevation in the AMH levels (4.57 ± 1.08 vs. 8.57 ± 0.97, P < 0.001) versus CP-POF group. CONCLUSION: Donepezil may be a potential protective agent against CP-induced POF in mice, but further research is needed to fully understand its therapeutic function experimentally and clinically.

The value of low-intensity pulsed ultrasound in reducing ovarian injury caused by chemotherapy in mice.

BACKGROUND: Ovarian damage and follicle loss are major side effects of chemotherapy in young female patients with cancer. However, effective strategies to prevent these injuries are still lacking. The purpose of this study was to verify low-intensity pulsed ultrasound (LIPUS) can reduce ovarian injury caused by chemotherapy and to explore its underlying mechanisms in mice model. METHODS: The mice were randomly divided into the Control group, Cisplatin group, and Cisplatin + LIPUS group. The Cisplatin group and Cisplatin + LIPUS group were intraperitoneally injected with cisplatin every other day for a total of 10 injections, and the Control group was injected with saline. On the second day of each injection, the Cisplatin + LIPUS group received irradiation, whereas the other two groups received sham irradiation. We used a variety of biotechnologies to detect the differences in follicle count, granulosa cell apoptosis, fibrosis, transcriptome level, oxidative damage, and inflammation in differently treated mice. RESULT: LIPUS was able to reduce primordial follicle pool depletion induced by cisplatin and inhibit the apoptosis of granulosa cells. Transcriptomic results confirmed that LIPUS can reduce ovarian tissue injury. We demonstrated that LIPUS can relieve ovarian fibrosis by inhibiting TGF-ß1/Smads pathway. Meanwhile, it can reduce the oxidative damage and reduced the mRNA levels of proinflammatory cytokines caused by chemotherapy. CONCLUSION: LIPUS can reduce the toxic effects of chemotherapy drugs on ovaries, inhibit ovarian fibrosis, reduce the inflammatory response, and redcue the oxidative damage, reduce follicle depletion and to maintain the number of follicle pools.

Repeated exposure to physiologically effective doses of contraceptive hormones ethinyl estradiol or levonorgestrel do not alter the reinforcing effects of a brief visual stimulus in ovary-intact rats.

Estradiol and progesterone potentiate and attenuate reward processes, respectively. Despite these well-characterized effects, there is minimal research on the effects of synthetic estrogens (e.g., ethinyl estradiol, or EE) and progestins (e.g., levonorgestrel, or LEVO) contained in clinically-utilized hormonal contraceptives. The present study characterized the separate effects of repeated exposure to EE or LEVO on responding maintained by a reinforcing visual stimulus. Forty ovary-intact female Sprague-Dawley rats received either sesame oil vehicle (n = 16), 0.18 µg/day EE (n = 16), or 0.6 µg/day LEVO (n = 8) subcutaneous injections 30-min before daily one-hour sessions. Rats' responding was maintained by a 30-sec visual stimulus on a Variable Ratio-3 schedule of reinforcement. The day after rats' last session, we determined rats estrous cycle phase via vaginal cytology before sacrifice and subsequently weighing each rat's uterus to further verify the contraceptive hormone manipulation. The visual stimulus functioned as a reinforcer, but neither EE nor LEVO enhanced visual stimulus maintained responding. Estrous cytology was consistent with normal cycling in vehicle rats and halting of normal cycling in EE and LEVO rats. EE increased uterine weights consistent with typical uterotrophic effects observed with estrogens, further confirming the physiological impacts of our EE and LEVO doses. In conclusion, a physiologically effective dose of neither EE nor LEVO did not alter the reinforcing efficacy of a visual stimulus reinforcer. Future research should characterize the effects of hormonal contraceptives on responding maintained by other reinforcer types to determine the generality of the present findings.

Effect of dexpanthenol on cyclophosphamide-induced ovarian toxicity: a histological and molecular study in rats.

RESEARCH QUESTION: Does dexpanthenol work as an effective therapeutic agent against cyclophosphamide (CYC)-induced premature ovarian failure (POF) in rats? DESIGN: A total of 28 female Wistar Albino rats were randomly divided into four groups (n = 7 per group). The POF and POF plus dexpanthenol groups were intraperitoneally administered CYC at an initial dose of 50 mg/kg, followed by 8 mg/kg for 14 days. The dexpanthenol and POF plus dexpanthenol groups were both intraperitoneally administered dexpanthenol at a dose of 500 mg/kg/day for 15 days. RESULTS: In the group administered CYC, the following was observed: a decrease in the ovarian index; a decrease in the numbers of primordial, primary, secondary and antral follicles; an increase in the number of corpus luteum and atretic follicles; a decrease in proliferation cell nuclear antigen immunoreactivity; a significant reduction in anti-Müllerian hormone and oestradiol levels; and an increase in serum FSH levels compared with controls. Dexpanthenol, on the other hand, reversed these effects. Quantitative reverse transcription polymerase chain reaction analyses showed that dexpanthenol increased Bcl-2, Akt1, mTOR, Nrf2 and HO-1 in CYC-induced ovarian tissues, but decreased Bax, Cas3, Hsp27, Hsp70, and Hsp90. Dexpanthenol treatment has a potential for inhibiting the intrinsic apoptotic pathway and oxidative stress levels in ovarian tissues via the downregulation of the mRNA expression of heat shock proteins and the activation of Nrf2/HO-1 pathways. CONCLUSIONS: Our findings demonstrated that dexpanthenol is an effective agent against POF caused by CYC; however, further experimental and clinical data are needed to use it effectively.

Fertility protection: a novel approach using pretreatment with mesenchymal stem cell exosomes to prevent chemotherapy-induced ovarian damage in a mouse model.

BACKGROUND: Primary ovarian insufficiency refers to the loss of ovarian function before the age of 40 years and leads to amenorrhea and infertility. Primary ovarian insufficiency has diverse causes, but a common cause is exposure to gonadotoxic chemotherapy used in cancer treatment. Because of the risk for developing primary ovarian insufficiency, patients who want to preserve their fertility may consider various procedures for fertility preservation. However, current fertility preservation options are highly invasive, carry substantial risks, and have uncertain success rates. Recent studies from our group and others reported that mesenchymal stem cells and mesenchymal stem cell-derived exosomes can restore ovarian function in preclinical models of primary ovarian insufficiency by restoring damaged cells and inhibiting apoptosis. Although the restorative effect of mesenchymal stem cell-derived exosomes has been well reported in previous studies, the potential of mesenchymal stem cell-derived exosomes in preventing ovarian damage has not been fully elucidated. OBJECTIVE: This study hypothesized that the antiapoptotic potential of mesenchymal stem cell-derived exosomes may protect ovarian tissue from chemotherapy-induced damage. STUDY DESIGN: In this study, we delivered mesenchymal stem cell-derived exosomes directly into the ovaries of mice before administration of chemotherapy. A total of 60 mice were divided into 3 groups (20 per group), which were labeled the control, chemotherapy, and fertility protection groups. Only the fertility protection group mice received exosomes, whereas the control and chemotherapy group mice received saline. After exosome injection, the chemotherapy and fertility protection groups of mice were subjected to chemotherapy to induce ovarian damage. After chemotherapy, we evaluated the protective effects of exosome treatment on ovarian function, such as estrous cyclicity, serum hormone levels, and the fertility rate, by comparing these outcomes between the chemotherapy and fertility protection groups. These outcomes were also compared with those of the control group for comparison with outcomes under healthy conditions. RESULTS: After intraovarian injection of exosomes before chemotherapy, the mice were able to maintain their estrous cycle (4- to 5-day cyclicity), serum anti-müllerian hormone level (66.06±26.40 ng/mL, not significantly different from that of the healthy controls), folliculogenesis (32.2±11.3 in the chemotherapy group vs 46.4±14.1 in the fertility protection group; P<.05), expression of the steroidogenic acute regulatory protein gene (a the steroidogenesis marker) (0.44±0.11-fold expression in the chemotherapy group and 0.88±0.31-fold expression in the fertility protection group; P<.05), and fertility (2 of 8 in the chemotherapy group and 5 of 8 in the fertility protection group), thereby showing prevention of chemotherapy-induced damage. We found that exosome treatment before chemotherapy can preserve ovarian function and protect fertility through the overexpression of ATP synthase-binding cassette transporters, such as ABCB1b (10.17±17.75-fold expression in the chemotherapy group and 44.14±33.25-fold expression in the fertility protection group; P<.05) and ABCC10 (3.25±0.59-fold expression in the chemotherapy group and 5.36±1.86-fold expression in the fertility protection group; P<.05). CONCLUSION: In this study, we present a novel fertility protection method using mesenchymal stem cell-derived exosomes. We concluded that mesenchymal stem cell-derived exosomes are a promising and simple treatment option for fertility protection in reproductive-aged patients who are receiving gonadotoxic chemotherapy.

Identifying suitable methods for evaluating the sterilizing effects of pyriproxyfen on adult malaria vectors: a comparison of the oviposition and ovary dissection methods.

BACKGROUND: Nets containing pyriproxyfen, an insect growth regulator that sterilizes adult mosquitoes, have become available for malaria control. Suitable methods for investigating vector susceptibility to pyriproxyfen and evaluating its efficacy on nets need to be identified. The sterilizing effects of pyriproxyfen on adult malaria vectors can be assessed by measuring oviposition or by dissecting mosquito ovaries to determine damage by pyriproxyfen (ovary dissection). METHOD: Laboratory bioassays were performed to compare the oviposition and ovary dissection methods for monitoring susceptibility to pyriproxyfen in wild malaria vectors using WHO bottle bioassays and for evaluating its efficacy on nets in cone bioassays. Blood-fed mosquitoes of susceptible and pyrethroid-resistant strains of Anopheles gambiae sensu lato were exposed to pyriproxyfen-treated bottles (100 µg and 200 µg) and to unwashed and washed pieces of a pyriproxyfen long-lasting net in cone bioassays. Survivors were assessed for the sterilizing effects of pyriproxyfen using both methods. The methods were compared in terms of their reliability, sensitivity, specificity, resources (cost and time) required and perceived difficulties by trained laboratory technicians. RESULTS: The total number of An. gambiae s.l. mosquitoes assessed for the sterilizing effects of pyriproxyfen were 1745 for the oviposition method and 1698 for the ovary dissection method. Fertility rates of control unexposed mosquitoes were significantly higher with ovary dissection compared to oviposition in both bottle bioassays (99-100% vs. 34-59%, P < 0.05) and cone bioassays (99-100% vs. 18-33%, P < 0.001). Oviposition rates of control unexposed mosquitoes were lower with wild pyrethroid-resistant An. gambiae s.l. Cové, compared to the laboratory-maintained reference susceptible An gambiae sensu stricto Kisumu (18-34% vs. 58-76%, P < 0.05). Sterilization rates of the Kisumu strain in bottle bioassays with the pyriproxyfen diagnostic dose (100 µg) were suboptimal with the oviposition method (90%) but showed full susceptibility with ovary dissection (99%). Wild pyrethroid-resistant Cové mosquitoes were fully susceptible to pyriproxyfen in bottle bioassays using ovary dissection (> 99%), but not with the oviposition method (69%). Both methods showed similar levels of sensitivity (89-98% vs. 89-100%). Specificity was substantially higher with ovary dissection compared to the oviposition method in both bottle bioassays (99-100% vs. 34-48%) and cone tests (100% vs.18-76%). Ovary dissection was also more sensitive for detecting the residual activity of pyriproxyfen in a washed net compared to oviposition. The oviposition method though cheaper, was less reliable and more time-consuming. Laboratory technicians preferred ovary dissection mostly due to its reliability. CONCLUSION: The ovary dissection method was more accurate, more reliable and more efficient compared to the oviposition method for evaluating the sterilizing effects of pyriproxyfen on adult malaria vectors in susceptibility bioassays and for evaluating the efficacy of pyriproxyfen-treated nets.

Nicotinamide Mononucleotide and Nicotinamide Riboside Reverse Ovarian Aging in Rats Via Rebalancing Mitochondrial Fission and Fusion Mechanisms.

PURPOSE: This study examined the effects of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) on folliculogenesis and mitochondrial dynamics (fission and fusion mechanisms) in ovaries of middle-aged female rats. METHODS: Experimental groups were young, middle-aged (control), middle-aged + NMN and middle-aged + NR. NMN was administered at a concentration of 500 mg/kg intraperitoneally but NR at a concentration of 200 mg/kg by gavage. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels were analyzed by ELISA. Hematoxylin-eosin staining sections were used for histopathological examination and follicles-counting. Expression levels of mitochondrial fission (Drp1, Mff and Fis1) and fusion (Mfn1, Mfn2, Opa1, Fam73a and Fam73b) genes as well as Sirt1 gene were analyzed by RT-PCR. Expression levels of fission-related proteins (DRP1, MFF, FIS1 and SIRT1) were analyzed by Western Blot. RESULTS: Higher ovarian index, more corpus luteum and antral follicles were detected in NMN and NR groups compared to the control. NMN or NR could rebalance LH/FSH ratio. The control group was determined to possess higher expression levels of fission genes and lower expression levels of fusion genes when compared the young group. In comparison with the control group, both NMN and NR group were found to exhibit less mitochondrial fission but more mitochondrial fussion. Higher gene and protein levels for Sirt1 were measured in NMN and NR groups compared to the control group. CONCLUSION: This study reveals that NMN alone or NR alone can rebalance mitochondrial dynamics by decreasing excessive fission in middle-aged rat ovaries, thus alleviating mitochondrial stress and correcting aging-induced folliculogenesis abnormalities.

Systemic immunological responses are dependent on sex and ovarian hormone presence following acute inhaled woodsmoke exposure.

BACKGROUND: Rural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility. METHODS: This exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode. RESULTS: Metals analysis of WS particulate matter (PM) revealed significantly increased levels of 63Cu, 182W, 208Pb, and 238U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1ß, TNF-α, CXCL-1, CCL-5, TGF-ß, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1ß, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1ß, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche. CONCLUSIONS: In summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.

Ti3C2 nanosheet-induced autophagy derails ovarian functions.

BACKGROUND: Two-dimensional ultrathin Ti3C2 (MXene) nanosheets have gained significant attention in various biomedical applications. Although previous studies have described the accumulation and associated damage of Ti3C2 nanosheets in the testes and placenta. However, it is currently unclear whether Ti3C2 nanosheets can be translocated to the ovaries and cause ovarian damage, thereby impairing ovarian functions. RESULTS: We established a mouse model with different doses (1.25, 2.5, and 5 mg/kg bw/d) of Ti3C2 nanosheets injected intravenously for three days. We demonstrated that Ti3C2 nanosheets can enter the ovaries and were internalized by granulosa cells, leading to a decrease in the number of primary, secondary and antral follicles. Furthermore, the decrease in follicles is closely associated with higher levels of FSH and LH, as well as increased level of E2 and P4, and decreased level of T in mouse ovary. In further studies, we found that exposure toTi3C2 nanosheets increased the levels of Beclin1, ATG5, and the ratio of LC3II/Ι, leading to autophagy activation. Additionally, the level of P62 increased, resulting in autophagic flux blockade. Ti3C2 nanosheets can activate autophagy through the PI3K/AKT/mTOR signaling pathway, with oxidative stress playing an important role in this process. Therefore, we chose the ovarian granulosa cell line (KGN cells) for in vitro validation of the impact of autophagy on the hormone secretion capability. The inhibition of autophagy initiation by 3-Methyladenine (3-MA) promoted smooth autophagic flow, thereby partially reduced the secretion of estradiol and progesterone by KGN cells; Whereas blocking autophagic flux by Rapamycin (RAPA) further exacerbated the secretion of estradiol and progesterone in cells. CONCLUSION: Ti3C2 nanosheet-induced increased secretion of hormones in the ovary is mediated through the activation of autophagy and impairment of autophagic flux, which disrupts normal follicular development. These results imply that autophagy dysfunction may be one of the underlying mechanisms of Ti3C2-induced damage to ovarian granulosa cells. Our findings further reveal the mechanism of female reproductive toxicity induced by Ti3C2 nanosheets.

Erythropoietin effects on cryopreserved/transplanted cat ovarian tissue: A comparison of two incubation methods.

Many feline species are currently threatened with extinction. Therefore, germplasm bank establishment has become imperative. However, cryoinjury and ischemia-reperfusion injury pose significant obstacles to both cryopreservation and xenotransplantation. In this regard, erythropoietin (Epo) represents a potential alternative strategy due to its properties. This study aimed to assess the incubation of domestic cat ovarian tissue in Epo, both before and after cryopreservation, and investigate its effectiveness in promoting revascularization following xenotransplantation. Sixteen ovaries from 8 healthy cats were sliced following elective bilateral ovariohysterectomy (OHE). Subsequently, 8 fragments measuring 3 mm³ each were obtained from the cortical region of each ovary. The fragments were allocated into 3 treatment groups: Cryo group, fragments were cryopreserved, thawed and immediately transplanted; Cryo + Epo group, fragments were first cryopreserved in nitrogen, thawed, incubated in Epo (100 IU) for 2h and transplanted; and the Epo + Cryo group, in which fragments were first incubated in Epo (100 IU) for 2h, cryopreserved, thawed and immediately transplanted. The fragments were then xenotransplanted into the dorsal subcutaneous region of ovariectomized female nude mice and retrieved at 7, 14, 21, and 28 days post-transplantation. The results indicated that Epo effectively enhanced follicular survival, preservation of viability, and tissue revascularization. The Epo + Cryo group displayed better revascularization rates on D14 and D21 post-transplantation and an increase in primordial and growing follicles on D28, the Cryo + Epo group exhibited significantly more follicles on D14 and D21, with fewer degenerated follicles.

Effect of globin peptide on female fertility in aging granulosa cell-specific Nrg1 knockout mice.

Ovarian fibrosis contributes to age-related ovarian dysfunction. In our previous study, we observed ovarian fibrosis in both obese and aging mice with intracellular lipid droplets in the fibrotic ovaries. Although the importance of mitochondria in ovarian fibrosis has been recognized in pharmacological studies, their role in lipid metabolism remains unclear. Globin peptide (GP), derived from hemoglobin, enhances lipid metabolism in obese mice. This study aimed to elucidate the importance of lipid metabolism in ovarian fibrosis by using GP. Treatment of ovarian stromal cells with GP increased mitochondrial oxygen consumption during ß-oxidation. Lipid accumulation was also observed in the ovaries of granulosa cell-specific Nrg1 knockout mice (gcNrg1KO), and the administration of GP to gcNrg1KO mice for two months reduced ovarian lipid accumulation and fibrosis in addition to restoring the estrous cycle. GP holds promise for mitigating lipid-related ovarian issues and provides a novel approach to safeguarding ovarian health by regulating fibrosis via lipid pathways.

Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries.

Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.

Androgens regulate ovarian gene expression by balancing Ezh2-Jmjd3 mediated H3K27me3 dynamics.

Conventionally viewed as male hormone, androgens play a critical role in female fertility. Although androgen receptors (AR) are transcription factors, to date very few direct transcriptional targets of ARs have been identified in the ovary. Using mouse models, this study provides three critical insights about androgen-induced gene regulation in the ovary and its impact on female fertility. First, RNA-sequencing reveals a number of genes and biological processes that were previously not known to be directly regulated by androgens in the ovary. Second, androgens can also influence gene expression by decreasing the tri-methyl mark on lysine 27 of histone3 (H3K27me3), a gene silencing epigenetic mark. ChIP-seq analyses highlight that androgen-induced modulation of H3K27me3 mark within gene bodies, promoters or distal enhancers have a much broader impact on ovarian function than the direct genomic effects of androgens. Third, androgen-induced decrease of H3K27me3 is mediated through (a) inhibiting the expression and activity of Enhancer of Zeste Homologue 2 (EZH2), a histone methyltransferase that promotes tri-methylation of K27 and (b) by inducing the expression of a histone demethylase called Jumonji domain containing protein-3 (JMJD3/KDM6B), responsible for removing the H3K27me3 mark. Androgens through the PI3K/Akt pathway, in a transcription-independent fashion, increase hypoxia-inducible factor 1 alpha (HIF1α) protein levels, which in turn induce JMJD3 expression. Furthermore, proof of concept studies involving in vivo knockdown of Ar in the ovary and ovarian (granulosa) cell-specific Ar knockout mouse model show that ARs regulate the expression of key ovarian genes through modulation of H3K27me3.

Melatonin activates Nrf2/HO-1 signalling pathway to antagonizes oxidative stress-induced injury via melatonin receptor 1 (MT1) in cryopreserved mice ovarian tissue.

Our previous research has shown that melatonin (MLT) can reduce cryopreserved ovarian damage in mice. Yet, the molecular mechanism of MLT protection is still unclear. Some studies have shown that melatonin receptor 1 (MT1) is very important for animal reproductive system. To evaluate whether MLT exerts its protective effect on cryopreserved mice ovarian tissue via MT1, we added antagonist of MT1/MT2 (Luzindor) or antagonist of MT2 (4P-PDOT) to the freezing solution, followed by cryopreservation and thawing of ovarian tissue. The levels of total superoxide dismutase (T-SOD), catalase (CAT), nitric oxide (NO) and malondialdehyde (MDA) were detected. Besides, by using RT-PCR and Western blotting, the expression of Bcl-2, Bax and Nrf2/HO-1 signalling pathway-related proteins was detected. These findings demonstrated that compared with the melatonin group, the addition of Luzindor increased apoptosis, NO and MDA activities, decreased CAT and T-SOD activities and inhibited Nrf2/HO-1 signalling pathway. In conclusion, melatonin can play a protective role in cryopreserved ovarian tissue of mice through MT1 receptor.

The Protective Role of Magnoliae Flos in Preventing Ovotoxicity and Managing Ovarian Function: An In Vitro and In Vivo Study.

Magnoliae Flos (MF) is a medicinal herb widely employed in traditional medicine for relieving sinusitis, allergic rhinitis, headaches, and toothaches. Here, we investigated the potential preventive effects of MF extract (MFE) against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in ovarian cells and a mouse model of premature ovarian insufficiency (POI). The cytoprotective effects of MFE were assessed using CHO-K1 or COV434 cells. In vivo, B6C3F1 female mice were intraperitoneally injected with VCD for two weeks to induce POI, while MFE was orally administered for four weeks, beginning one week before VCD administration. VCD led to a significant decline in the viabilities of CHO-K1 and COV434 cells and triggered excessive reactive oxygen species (ROS) production and apoptosis specifically in CHO-K1 cells. However, pretreatment with MFE effectively prevented VCD-induced cell death and ROS generation, while also activating the Akt signaling pathway. In vivo, MFE increased relative ovary weights, follicle numbers, and serum estradiol and anti-Müllerian hormone levels versus controls under conditions of ovary failure. Collectively, our results demonstrate that MFE has a preventive effect on VCD-induced ovotoxicity through Akt activation. These results suggest that MFE may have the potential to prevent and manage conditions such as POI and diminished ovarian reserve.