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Frontiers in Endocrinology 2023Insulin resistance (IR) is found in patients with polycystic ovary syndrome (PCOS), but the effects and mechanisms of IR on diminished ovarian reserve (DOR) remain...
INTRODUCTION
Insulin resistance (IR) is found in patients with polycystic ovary syndrome (PCOS), but the effects and mechanisms of IR on diminished ovarian reserve (DOR) remain unclear. This study set out to investigate the effects of IR on ovarian reserve; to explore the effects of high concentrations of insulin on the function of ovarian cells ; and to validate the hypothesis that the Gengnianchun recipe (GNC) helps to attenuate DOR caused by IR through reducing the senescence of granulosa cells.
METHODS
Estrus cycle, follicle count, and sex hormone levels were detected to evaluate ovarian function in mice with IR caused by feeding a high-fat diet (HFD). In addition, KGN cells (human granulosa cell line) were treated with high concentrations of insulin. The staining for senescence-associatedβ-galactosidase (SA-β-gal), cell cycle, and expression levels of mRNA and gene proteins related to cell aging were detected in KGN cells treated with high concentrations of insulin. Mice treated with an HFD were fed metformin, GNC, or saline solution for 6 weeks by oral gavage. HOMA-IR, the area under the curve (AUC) of the oral glucose tolerance test (OGTT), levels of fasting blood glucose (FBG), and fasting serum insulin (FINS) were examined to confirm the IR status. Then estrus cycle, follicle count, and sex hormone levels were detected to evaluate ovarian function. Expression levels of mRNA and gene proteins related to cell aging were detected in the ovarian tissue of mice in each group.
RESULTS
The results demonstrated that IR reduced murine ovarian reserves, and high doses of insulin caused granulosa cells to senesce. There was a considerable improvement in HFD-induced IR status in the metformin (Met) and GNC treatment groups. In addition, the expression levels of aging-associated biomarkers were much lower in GNC mice than Met mice; and both the latter groups had considerably lower levels than the HFD group. Moreover, higher follicle counts in different stages and shorter diestrus in the Met or GNC groups compared to the HFD group indicated that ovarian aging could be largely reversed.
DISCUSSION
This work showed that: IR impaired ovarian reserve; high concentrations of insulin induced granulosa cell aging; and GNC attenuated ovarian function through inhibiting IR-induced cell aging.
Topics: Female; Humans; Mice; Animals; Insulin Resistance; Ovarian Reserve; Granulosa Cells; Insulin; Gonadal Steroid Hormones; Metformin; RNA, Messenger
PubMed: 36936173
DOI: 10.3389/fendo.2023.1133280 -
Endocrinology Apr 2023Despite the importance of the mouse in biomedical research, the levels of circulating gonadal steroids across the estrous cycle are not established with any temporal...
Despite the importance of the mouse in biomedical research, the levels of circulating gonadal steroids across the estrous cycle are not established with any temporal precision. Using liquid chromatography-mass spectrometry, now considered the gold standard for steroid hormone analysis, we aimed to generate a detailed profile of gonadal steroid levels across the estrous cycle of C57BL/6J mice. For reference, luteinizing hormone (LH) and prolactin concentrations were measured in the same samples by sandwich enzyme-linked immunosorbent assay. Terminal blood samples were collected at 8-hour intervals (10 Am, 6 Pm, 2 Am) throughout the 4 stages of the estrous cycle. As expected, the LH surge was detected at 6 Pm on proestrus with a mean (±SEM) concentration of 11 ± 3 ng/mL and occurred coincident with the peak in progesterone levels (22 ± 4 ng/mL). Surprisingly, estradiol concentrations peaked at 10 Am on diestrus (51 ± 8 pg/mL), with levels on proestrus 6 Pm reaching only two-thirds of this value (31 ± 5 pg/mL). We also observed a proestrus peak in prolactin concentrations (132.5 ± 17 ng/mL) that occurred earlier than expected at 2 Am. Estrone and androstenedione levels were often close to the limit of detection (LOD) and showed no consistent changes across the estrous cycle. Testosterone levels were rarely above the LOD (0.01 ng/mL). These observations provide the first detailed assessment of fluctuating gonadal steroid and reproductive hormone levels across the mouse estrous cycle and indicate that species differences exist between mice and other spontaneously ovulating species.
Topics: Female; Mice; Animals; Prolactin; Estrus; Mice, Inbred C57BL; Luteinizing Hormone; Estrous Cycle; Estradiol; Progesterone
PubMed: 37165692
DOI: 10.1210/endocr/bqad070 -
Reproductive Sciences (Thousand Oaks,... Oct 2022Oviductal extracellular vesicles (OEVs) play an important role in fertilization and embryo development. However, it remains largely unknown whether the size and protein...
Oviductal extracellular vesicles (OEVs) play an important role in fertilization and embryo development. However, it remains largely unknown whether the size and protein cargo of OEVs change during the estrous cycle in mice. This study analyzed the changes in the size distribution and protein cargo of OEVs at four stages of the estrous cycle in mice. The distribution widths of OEVs according to the estrous cycle stage were as follows: proestrus, 20-690 nm in diameter, with two peaks at 50 and 250 nm; estrus, 22-420 nm in diameter, with two peaks at 40 and 200 nm; metestrus, 30-70 nm diameter, with a single peak at 40 nm; and diestrus, 10-26 nm diameter, with a single peak at 20 nm. The estrogen receptor (ER) level in OEVs at the proestrus stage differed significantly from that at estrus (P = 0.013) and diestrus (P = 0.005). The levels of CD9 and Hsc70 fluctuated across the four stages, although with no significant differences. Furthermore, OEVs were observed among the cilia and microvilli of epithelial cells at the proestrus, estrus, and diestrus stages, but not at the metestrus stage. The number of observed OEVs was the highest at the proestrus stage, followed by the estrus, and the diestrus stage. Endosomes were also observed at the estrus and diestrus stages. The change of the OEV size and ER cargo is associated with the estrous cycle in mice. Our findings increase the understanding of the physiological characteristics of OEVs, which may have clinical applications.
Topics: Animals; Estrous Cycle; Extracellular Vesicles; Fallopian Tubes; Female; Humans; Mice; Oviducts; Receptors, Estrogen
PubMed: 35137347
DOI: 10.1007/s43032-022-00862-w -
Animals : An Open Access Journal From... Nov 2022Endometrial immune cells are essential to support uterine functions across the estrous cycle and in preparation for pregnancy. It has been acknowledged that changes in...
Endometrial immune cells are essential to support uterine functions across the estrous cycle and in preparation for pregnancy. It has been acknowledged that changes in phenotype and/or numbers of lymphocytes, such as regulatory T cells (Tregs) and NK cells, might result in lower fertility in women and mice. Little is known about equine endometrial immune cells across the estrous cycle. Here, we compared the populations of endometrial Tregs and NK cells in estrus and diestrus in mares. Endometrial biopsy and blood samples were taken in estrus and diestrus from 11 mares ages 4-12 years. Flow cytometry with anti-CD4, -CD25 and -FOXP3 and anti-NKp46 and -CD3 antibodies was used to determine the populations of Tregs and NK cells, respectively. The concentration of progesterone was measured with chemiluminescence immunoassay. The results were analyzed with paired Student tests. The mean percentage of endometrial CD4FOXP3 Tregs was 13.7 ± 6.2% in diestrus and 14.5 ± 5.9% in estrus, while the mean percentage of endometrial CD4FOXP3CD25 Tregs changed from 3.6 ± 2.1% in diestrus to 2 ± 2% in estrus ( = 0.0947). The mean proportion of CD3NKp46 lymphocytes in the endometrium was not significantly different, with 6 ± 1% in estrus and 6.5 ± 1.4% in diestrus. There was a large variation in the percentage of NK cells between mares of 2.1-12.7%. This study showed, for the first time, the presence of CD4FOXP3CD25 Tregs and CD3NKp46 NK cells in the endometrium of non-pregnant cycling mares. The percentage of Tregs, and to a greater extent NK cells, showed large fluctuations between mares. Both Tregs and NK cells might be important for the preparation of the endometrium for semen deposition and pregnancy; however, further research is required.
PubMed: 36496894
DOI: 10.3390/ani12233373 -
Reproductive Biology and Endocrinology... Mar 2021Long-acting, reversible contraceptives (LARC; progestin only) are an increasingly common hormonal contraceptive choice in reproductive aged women looking to suppress...
BACKGROUND
Long-acting, reversible contraceptives (LARC; progestin only) are an increasingly common hormonal contraceptive choice in reproductive aged women looking to suppress ovarian function and menstrual cyclicity. The overall objective was to develop and validate a rodent model of implanted etonogestrel (ENG) LARC, at body size equivalent doses to the average dose received by women during each of the first 3 years of ENG subdermal rod LARC use.
METHODS
Intact, virgin, female Sprague-Dawley rats (16-wk-old) were randomized to 1 of 4 groups (n = 8/group) of ENG LARC (high-0.30μg/d, medium-0.17μg/d, low-0.09μg/d, placebo-0.00μg/d) via a slow-release pellet implanted subcutaneously. Animals were monitored for 21 days before and 29 days following pellet implantation using vaginal smears, ultrasound biomicroscopy (UBM), saphenous blood draws, food consumption, and body weights. Data were analyzed by chi-square, non-parametric, univariate, and repeated measures 2-way ANOVA.
RESULTS
Prior to pellet implantation there was no difference in time spent in estrus cycle phases among the treatment groups (p > 0.30). Following pellet implantation there was a dose-dependent impact on the time spent in diestrus and estrus (p < 0.05), with the high dose group spending more days in diestrus and fewer days in estrus. Prior to pellet insertion there was not an association between treatment group and estrus cycle classification (p = 0.57) but following pellet implantation there was a dose-dependent association with cycle classification (p < 0.02). Measurements from the UBM (ovarian volume, follicle count, corpora lutea count) indicate an alteration of ovarian function following pellet implantation.
CONCLUSION
Assessment of estrus cyclicity indicated a dose-response relationship in the shift to a larger number of acyclic rats and longer in duration spent in the diestrus phase. Therefore, each dose in this model mimics some of the changes observed in the ovaries of women using ENG LARC and provides an opportunity for investigating the impacts on non-reproductive tissues in the future.
Topics: Animals; Contraceptive Agents, Female; Desogestrel; Dose-Response Relationship, Drug; Drug Implants; Estrus; Female; Humans; Models, Animal; Progestins; Rats; Rats, Sprague-Dawley; Rodentia
PubMed: 33752672
DOI: 10.1186/s12958-021-00729-w -
Behavioural Brain Research Feb 2018Females are an under-represented research model and the mechanisms through which sleep loss impairs cognition are not clear. Since levels of reproductive hormones and...
Females are an under-represented research model and the mechanisms through which sleep loss impairs cognition are not clear. Since levels of reproductive hormones and the estrous cycle are sensitive to sleep loss and necessary for learning and memory, we hypothesized that sleep deprivation impacts learning and memory in female mice by interfering with the estrous cycle. We used the object recognition task to assess learning and memory in female mice during separate phases of the estrous cycle and after sleep loss. Mice in metestrus/diestrus attended to sample objects less than mice in proestrus/estrus during object acquisition, the first phase of the object recognition task. Subsequently, during the recognition phase of the task, only mice in proestrus/estrus displayed a preference for the novel object. Sleep deprivation for 12h immediately before the object recognition task reduced time attending to sample objects and novel object preference for mice in proestrus/estrus, without changing length of the estrous cycle. These results show that sleep deprived mice in proestrus/estrus had learning deficits and memory impairments, like mice in metestrus/diestrus. Since sleep deprivation did not disrupt the estrous cycle, however, results did not support the hypothesis. Cognitive impairments due to acute sleep loss were not due to alterations to the estrous cycle.
Topics: Animals; Behavior, Animal; Estrous Cycle; Female; Learning; Memory; Metestrus; Mice, Inbred C57BL; Proestrus; Sleep Deprivation
PubMed: 29180134
DOI: 10.1016/j.bbr.2017.11.033 -
Animals : An Open Access Journal From... Nov 2020Lactic acid bacteria (LAB) dominate human vaginal microbiota and inhibit pathogen proliferation. In other mammals, LAB do not dominate vaginal microbiota, however shifts...
Lactic acid bacteria (LAB) dominate human vaginal microbiota and inhibit pathogen proliferation. In other mammals, LAB do not dominate vaginal microbiota, however shifts of dominant microorganisms occur during ovarian cycle. The study objectives were to characterize equine vaginal microbiota in mares by culture-dependent and independent methods and to describe its variation in estrus and diestrus. Vaginal swabs from 8 healthy adult Arabian mares were obtained in estrus and diestrus. For culture-dependent processing, bacteria were isolated on Columbia blood agar (BA) and Man Rogosa Sharpe (MRS) agar. LAB comprised only 2% of total bacterial isolates and were not related to ovarian phases. For culture-independent processing, V3/V4 variable regions of the 16S ribosomal RNA gene were amplified and sequenced using Illumina Miseq. The diversity and composition of the vaginal microbiota did not change during the estrous cycle. Core equine vaginal microbiome consisted of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria at the phylum level. At the genus level it was defined by , uncultured and . comprised only 0.18% of the taxonomic composition in estrus and 0.37% in diestrus. No differences in the relative abundance of the most abundant phylum or genera were observed between estrus and diestrus samples.
PubMed: 33153053
DOI: 10.3390/ani10112020 -
The Journal of Endocrinology Feb 2022Kisspeptin neurons, i.e. KNDy neurons, in the arcuate nucleus (ARC) coexpress neurokinin B and dynorphin and regulate gonadotropin-releasing hormone/luteinizing hormone...
Kisspeptin neurons, i.e. KNDy neurons, in the arcuate nucleus (ARC) coexpress neurokinin B and dynorphin and regulate gonadotropin-releasing hormone/luteinizing hormone (LH) pulses. Because it remains unclear whether these neurons are associated with reproductive dysfunction in diabetic females, we examined the expression of KNDy neurons detected by histochemistry in streptozotocin (STZ)-induced diabetic female rats 8 weeks after STZ injection. We also evaluated relevant metabolic parameters - glucose, 3-hydroxybutyrate, and non-esterified fatty acids - as indicators of diabetes progression. Severe diabetes with hyperglycemia and severe ketosis suppressed the mRNA expression of KNDy neurons, resulting in low plasma LH levels and persistent diestrus. In moderate diabetes with hyperglycemia and moderate ketosis, kisspeptin-immunoreactive cells and plasma LH levels were decreased, while the mRNA expression of KNDy neurons remained unchanged. Mild diabetes with hyperglycemia and slight ketosis did not affect KNDy neurons and plasma LH levels. The number of KNDy cells was strongly and negatively correlated with plasma 3-hydroxybutyrate levels. The vaginal smear analysis showed unclear proestrus in diabetic rats 3-5 days after STZ injection, and the mRNA expression of kisspeptin in the ARC was decreased 2 weeks after STZ injection in severely diabetic rats. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV), which induce an LH surge, were unaffected at 2 and 8 weeks after STZ injection regardless of the diabetes severity. These results suggest that diabetes mellitus progression in females may negatively affect ARC kisspeptin neurons but not AVPV kisspeptin neurons, implicating a potential role of ARC kisspeptin neurons in menstrual disorder and infertility.
Topics: Animals; Arcuate Nucleus of Hypothalamus; Diabetes Mellitus, Experimental; Female; Gonadotropin-Releasing Hormone; Kisspeptins; Neurokinin B; Neurons; Rats
PubMed: 35084363
DOI: 10.1530/JOE-21-0169 -
Frontiers in Bioscience (Scholar... Jan 2016The term "uterosomes" was first used to classify extracellular membrane vesicles released into the uterine luminal fluid. These extracellular vesicles (EVs), varying in... (Review)
Review
The term "uterosomes" was first used to classify extracellular membrane vesicles released into the uterine luminal fluid. These extracellular vesicles (EVs), varying in sizes, fit the classification of exosomes and microvesicles on the basis of size, the presence of the CD9 biochemical marker, and lateral orientation of the membrane. Uterosomes appear to be formed by the apocrine pathway, similar to other reproductive EVs. In the murine system, the protein cargo carried by uterosomes includes glycosyl phosphatidylinositol (GPI)-linked and transmembrane proteins and these are hormonally regulated, appearing at high levels during proestrus/estrus and only marginally present at diestrus /metestrus. Uterosomes have been shown to deliver proteins in their cargo to sperm, with a functional impact, and are thought to participate in promoting sperm capacitation. Further studies are warranted, particularly those aimed at identifying the contents of their cargo during the estrus and menstrual cycle and the role they play n sperm maturation.
Topics: Animals; Estrous Cycle; Estrus; Exosomes; Female; Fertilization; Humans; Male; Metestrus; Organelle Biogenesis; Proteins; Reproduction; Sperm Maturation; Spermatozoa; Uterus
PubMed: 26709901
DOI: 10.2741/s451 -
Frontiers in Cell and Developmental... 2022Female reproductive cycle, also known as menstrual cycle or estrous cycle in primate or non-primate mammals, respectively, dominates the reproductive processes in...
Female reproductive cycle, also known as menstrual cycle or estrous cycle in primate or non-primate mammals, respectively, dominates the reproductive processes in non-pregnant state. However, in addition to reproductive tissues, reproductive cycle could also perform global regulation because the receptors of two major female hormones fluctuating throughout the cycle, estrogen and progesterone, are widely distributed. Therefore, a multi-tissue gene expression landscape is in continuous demand for better understanding the systemic changes during the reproductive cycle but remains largely undefined. Here we delineated a transcriptomic landscape covering 15 tissues of C57BL/6J female mice in two phases of estrous cycle, estrus and diestrus, by RNA-sequencing. Then, a number of genes, pathways, and transcription factors involved in the estrous cycle were revealed. We found the estrous cycle could widely regulate the neuro-functions, immuno-functions, blood coagulation and so on. And behind the transcriptomic alteration between estrus and diestrus, 13 transcription factors may play important roles. Next, bioinformatics modeling with 1,263 manually curated gene signatures of various physiological and pathophysiological states systematically characterized the beneficial/deleterious effects brought by estrus/diestrus on individual tissues. We revealed that the estrous cycle has a significant effect on cardiovascular system (aorta, heart, vein), in which the anti-hypertensive pattern in aorta induced by estrus is one of the most striking findings. Inspired by this point, we validated that two hypotensive drugs, felodipine and acebutolol, could exhibit significantly enhanced efficacy in estrus than diestrus by mouse and rat experiments. Together, this study provides a valuable data resource for investigating reproductive cycle from a transcriptomic perspective, and presents models and clues for investigating precision medicine associated with reproductive cycle.
PubMed: 36589755
DOI: 10.3389/fcell.2022.983712