Rat Estrous Cycle Research Articles

Staging of Estrous Cycle in Rats using Vaginal Cytology and Visual Assessment

The determination of different phases of estrous cycle in lab animals is important for reproductive interventions and other experiments related to animal reproduction where rats are used as experimental models. A total 21 Wistar albino (which had completed their pubertal stage ( greater than 4 weeks) weighing approximately 100- 200 g were used in this experiment. Visual assessment was done to observe vaginal opening during different phases of the estrous cycle. This was followed by collection of vaginal discharge samples by flushing it with 0.1 ml of distilled water using micropipette. Vaginal cytology was performed in 21 rats thrice daily, along with visual assessment. The average length of estrous cycle in rats was recorded to be 4-5 days with average duration of proestrus, estrus, metestrus and diestrus of 24, 24-36, 12 and 48 hours, respectively. The average length of estrous cycle in rats was 4-5 days with recorded average duration of proestrus, estrus, metestrus and diestrus of 24, 24-36, 12 and 48 hours, respectively. Vaginal cytology can be considered as a better option compared to visual assessment for assessment of reproductive status in rats.

Neuroactive steroids fluctuate with regional specificity in the central and peripheral nervous system across the rat estrous cycle

Neuroactive steroids (i.e., sex steroid hormones and neurosteroids) are important physiological regulators of nervous function and potential neuroprotective agents for neurodegenerative and psychiatric disorders. Sex is an important component of such effects. However, even if fluctuations in sex steroid hormone level during the menstrual cycle are associated with neuropathological events in some women, the neuroactive steroid pattern in the brain across the ovarian cycle has been poorly explored. Therefore, we assessed the levels of pregnenolone, progesterone, and its metabolites (i.e., dihydroprogesterone, allopregnanolone and isoallopregnanolone), dehydroepiandrosterone, testosterone and its metabolites (i.e., dihydrotestosterone, 3α-diol and 17β-estradiol) across the rat ovarian cycle to determine whether their plasma fluctuations are similar to those occurring in the central (i.e., hippocampus and cerebral cortex) and peripheral (i.e., sciatic nerve) nervous system. Data obtained indicate that the plasma pattern of these molecules generally does not fully reflect the events occurring in the nervous system. In addition, for some neuroactive steroid levels, the pattern is not identical between the two brain regions and between the brain and peripheral nerves. Indeed, with the exception of progesterone, all other neuroactive steroids assessed here showed peculiar regional differences in their pattern of fluctuation in the nervous system during the estrous cycle. These observations may have important diagnostic and therapeutic consequences for neuropathological events influenced by the menstrual cycle.

Determination of the rat estrous cycle vased on EfficientNet.

In the field of biomedical research, rats are widely used as experimental animals due to their short gestation period and strong reproductive ability. Accurate monitoring of the estrous cycle is crucial for the success of experiments. Traditional methods are time-consuming and rely on the subjective judgment of professionals, which limits the efficiency and accuracy of experiments. This study proposes an EfficientNet model to automate the recognition of the estrous cycle of female rats using deep learning techniques. The model optimizes performance through systematic scaling of the network depth, width, and image resolution. A large dataset of physiological data from female rats was used for training and validation. The improved EfficientNet model effectively recognized different stages of the estrous cycle. The model demonstrated high-precision feature capture and significantly improved recognition accuracy compared to conventional methods. The proposed technique enhances experimental efficiency and reduces human error in recognizing the estrous cycle. This study highlights the potential of deep learning to optimize data processing and achieve high-precision recognition in biomedical research. Future work should focus on further validation with larger datasets and integration into experimental workflows.

Yu Linzhu alleviates primary ovarian insufficiency in a rat model by improving proliferation and energy metabolism of granulosa cells through hif1α/cx43 pathway

BackgroundYu Linzhu (YLZ) is a classical Chinese traditional formula, which has been used for more than 600 years to regulate menstruation to help pregnancy. However, the mechanism of modern scientific action of YLZ needs to be further studied.MethodsThirty SD female rats were divided into three groups to prepare the blank serum and drug-containing serum, and then using UHPLC-QE-MS to identify the ingredients of YLZ and its drug-containing serum. Twenty-four SD female rats were divided into four groups, except the control group, 4-vinylcyclohexene dicycloxide (VCD) was intraperitoneally injected to establish a primary ovarian insufficiency (POI) model of all groups. Using vaginal smear to show that the estrous cycle of rats was disturbed after modeling, indicates that the POI model was successfully established. The ELISA test was used to measure the follicle-stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH) levels in the serum of rats. HE stain was used to assess the morphology of ovarian tissue. The localization and relative expression levels of CX43 protein were detected by tissue immunofluorescence. Primary ovarian granulosa cells (GCs) were identified by cellular immunofluorescence. CCK8 was used to screen time and concentration of drug-containing serum and evaluate the proliferation effect of YLZ on VCD-induced GCs. ATP kit and Seahorse XFe24 were used to detect energy production and real-time glycolytic metabolism rate of GCs. mRNA and protein expression levels of HIF1α, CX43, PEK, LDH, HK1 were detected by RT-PCR and WB.ResultsUHPLC-QE-MS found 1702 ingredients of YLZ and 80 constituents migrating to blood. YLZ reduced the FSH while increasing the AMH and E2 levels. In ovarian tissues, YLZ improved ovarian morphology, follicle development, and the relative expression of CX43. In vitro studies, we found that YLZ increased the proliferative activity of GCs, ATP levels, glycolytic metabolic rate, HIF1α, CX43, PEK, HK1, LDH mRNA, and protein levels.ConclusionsThe study indicated that YLZ increased the proliferation and glycolytic energy metabolism of GCs to improve follicular development further alleviating ovarian function.

Generalization of a positive-feature interoceptive morphine occasion setter across the rat estrous cycle

IntroductionInteroceptive stimuli elicited by drug administration acquire conditioned modulatory properties of the induction of conditioned appetitive behaviours by exteroceptive cues. This effect may be modeled using a drug discrimination task in which the drug stimulus is trained as a positive-feature (FP) occasion setter (OS) that disambiguates the relation between an exteroceptive light conditioned stimulus (CS) and a sucrose unconditioned stimulus (US). We previously reported that females are less sensitive to generalization of a FP morphine OS than males, so we investigated the role of endogenous ovarian hormones in this difference. MethodsMale and female rats received intermixed injections of 3.2 mg/kg morphine or saline before each daily training session. Training consisted of 8 presentations of the CS, each followed by access to sucrose on morphine, but not saline sessions. Following acquisiton, rats were tested for generalization of the morphine stimulus to 0, 1.0, 3.2, and 5.4 mg/kg morphine. Female rats were monitored for estrous cyclicity using vaginal cytology throughout the study. ResultsBoth sexes acquired stable drug discrimination. A gradient of generalization was measured across morphine doses and this behaviour did not differ by sex, nor did it differ across the estrous cycle in females. ConclusionsMorphine generalization is independent of fluctuations in levels of sex and endogenous gonadal hormones in females under these experimental conditions.

Track-by-Day: A standardized approach to estrous cycle monitoring in biobehavioral research

Despite known sex differences in brain function, female subjects are underrepresented in preclinical neuroscience research. This is driven in part by concerns about variability arising from estrous cycle-related hormone fluctuations, especially in fear- and anxiety-related research where there are conflicting reports as to whether and how the cycle influences behavior. The inconsistency may arise from a lack of common standards for tracking and reporting the cycle as opposed to inherent unpredictability in the cycle itself. The rat estrous cycle is conventionally tracked by assigning vaginal cytology smears to one of four qualitatively-defined stages. Although the cytology stages are of unequal length, the stage names are often, but not always, used to refer to the four cycle days. Subjective staging criteria and inconsistent use of terminology are not necessarily a problem in research on the cycle itself, but can lead to irreproducibility in neuroscience studies that treat the stages as independent grouping factors. We propose the explicit use of cycle days as independent variables, which we term Track-by-Day to differentiate it from traditional stage-based tracking, and that days be indexed to the only cytology feature that is a direct and rapid consequence of a hormonal event: a cornified cell layer formed in response to the pre-ovulatory 17β-estradiol peak. Here we demonstrate that cycle length is robustly regular with this method, and that the method outperforms traditional staging in detecting estrous cycle effects on Pavlovian fear conditioning and on a separate proxy for hormonal changes, uterine histology.

Direct infiltration effects of dimethyl sulfoxide into ovarian bursas on spontaneous ovulation and the estrous cycle of the rat: antagonism on the type 2 dopaminergic receptor

We studied effects of microinjection (MI) into ovarian burses (OB) of infiltering agent dimethyl sulfoxide (DMSO) on number of ova shed (NOS) and estrous cycle (EC) of adult female rats with fourth day regular estrous cycles (REC), and we compared these effects with a MI of 100µg sulpiride solution in DMSO. Between 13:00- 14:00h of different days of EC, CER groups received 20µL of pure DMSO into both OB; the sham groups received MI with 20µL distillated water (H2O) into each OB. All rats with MI performed and an intact cyclic rat group were autopsied in the morning of next estrous vaginal shown (EVS). Both DMSO and H2O MI does not modify the EC duration and NOS. Other groups of cyclic rats received a MI with 20µL sulpiride solution into each OB and were autopsied at next EVS. Just the sulpiride MI performed on diestrous-1 day delayed 24h the next EVS, but that don’t occur in other sulpiride groups. The NOS were not modified in all experimental group. The DMSO direct administration in ovarian tissue does not affect gonadal primordial functions and its use is recommended like an infiltrating agent of non-polar drugs.

Investigation of the Presence of Stem Cells in Rat Uterus Tissue in Postnatal Development Periods

Uterine tissue is an organ with a high proliferation capacity where regeneration, differentiation and shedding are seen. Although this tissue is regenerated every month, the source of it hasn’t been determined yet. Studies are carried out on stem cells being a source that can provide regeneration of the endometrium and the markers expressed by these cells. The purpose of this study was to examine the immunreactivity of CD9 and CD13 expressed by essential population of uterus and CD34, marker of hematopoietic stem cells, at various stages of the postnatal developing pro- cess. In this study, was used 42 female Wistar-albino rats split into six groups; Group I; newborn (2-days), Group II; pubertal (38-days), and Group III: fertile group (12 weeks), by identifying the stages of the estrous cycle in rats a) pro- estrus, b) estrus, c) metestrus, d) diestrus. The expression of CD9, CD13, and CD34 in uterine tissues excised from rats was investigated. In the groups of 38-days and 12-weeks, CD34-expressing cells were present in the stroma of the endometrium next to the myometrium, but such cells were absent in the 2-days group. All groups' uterine epithelium displayed CD9 expression, except for group 2-days. Endometrial stromal cells that expressed CD13 showed only little immureactivity in groups 2 and 38-days, while CD13 expression is noticeable in group 12-weeks. It was thought that the uterine endometrium could be regenerated in puberty and adulthood with the contribution of bone marrow-derived stem cells and uterine-derived epithelial and stromal cells.

FRI279 Submaximal Loss Of KNDy Neurons Accelerates And Amplifies Pulsatile LH Secretion In Female Rats

Abstract Disclosure: A.C. Campideli-Santana: None. K.S. da Costa Silva: None. R. Araújo-Lopes: None. L.M. Antunes: None. R.E. Szawka: None. Kisspeptin is a powerful stimulator neuropeptide in the hypothalamus-pituitary-gonadal axis and is essential for fertility. Neurons in the arcuate nucleus (ARC) of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (KNDy) play a crucial role in the control of luteinizing hormone (LH) secretion, but the neuroendocrine mechanisms involved in this regulation are not fully understood. Our previous study demonstrated that the moderate loss of KNDy neurons amplifies the LH surge induced by estradiol in ovariectomized rats (1). The current study aimed to perform a longitudinal evaluation of female rats in which KNDy neurons were ablated to determine the integrated role of this neuronal population in the control of pulsatile LH secretion. Adult female rats underwent a neurochemical ablation of KNDy neurons via intra-ARC stereotaxic injections of neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP), while control animals received blank-saporin (Control). Estrous cyclicity was monitored over 21 days before and after the stereotaxic surgery. Three weeks after surgery, blood samples (10 µL) were withdrawn from the tail tip at 6-minute intervals for 180 minutes on the day of diestrus. Rats were then ovariectomized and the ovaries were processed for histological analysis. Whole blood LH levels were measured by ELISA and brains were immunohistochemically labeled for neurokinin B in the ARC. NK3-SAP rats bearing a submaximal loss of KNDy neurons (between 55-95%; n = 8) displayed irregular estrous cycles. The ovarian weight and the number of corpora lutea and atretic follicles were unaltered, whereas the number of healthy early antral follicles decreased in NK3-SAP rats. The frequency of LH pulses was increased and both pulse amplitude and mean LH levels were higher in NK3-SAP compared with Control rats. Thus, the submaximal loss of KNDy neurons affects the regularity of estrous cyclicity but does not preclude ovulation, while seems to stimulate the maturation of early antral follicles. Notably, KNDy neuron loss accelerates and amplifies LH pulsatile secretion during the rat estrous cycle. These results suggest a new role for KNDy neurons in restraining the frequency and amplitude of LH pulses in ovary-intact animals, with possible implications in the rate of recruitment of early antral follicles. Reference: Campideli-Santana et al., J Neuroendocrinol. 2022 Nov;34(11):e13204. Support: FAPEMIG, CNPq, CAPES, and Pro-reitoria de Pesquisa-UFMG. Presentation: Friday, June 16, 2023

The mixed legacy of the rat estrous cycle

BackgroundThe rat estrous cycle first characterized by Long and Evans in 1922 profoundly affected the course of endocrine research. Investigators took advantage of sex steroid hormone fluctuations associated with the cycle to assess hormonal influences on anxiety, depression, food intake, stress, brain structure and other traits. Similarities of the rat estrous and human menstrual cycles facilitated understanding of human reproductive physiology. I assessed the impact of awareness of the estrous cycle on the emergence of a sex bias that excluded female rats from biomedical research.MethodsBeginning with the 1918 volume of the American Journal of Physiology and ending in 1976 when the journal subdivided into several separate disciplinary journals, all studies conducted on rats were downloaded; the use of females, males, both sexes and sex left unspecified was tabulated for 485 articles. A second analysis tracked the number of rat estrous cycle studies across all disciplines listed in PubMed from 1950 to 2021.ResultsThe description and awareness of variability associated with the rat estrous cycle was correlated with a precipitous decline in investigations that incorporated both sexes, a marked increase in male-only studies and a striking sex bias that excluded female rats. The number of rat estrous cycles studies increased markedly from earlier decades to a peak in 2021.ConclusionsThe initial description the rat estrous cycle was correlated with a substantial decline in investigations that incorporated both sexes; one result was a marked increase in male-only studies and a striking sex bias that excluded female rats from biomedical research. Recognition of the advantages of studies that incorporate the rat estrous cycle has resulted in recent years in an increase of such investigations. Female rats and females of several other species are not more variable than their male counterparts across traits, arguing for female inclusion without requiring cycle monitoring. There, remain, however, many advantages of incorporating the estrous cycle in contemporary research.

Irpex lacteus polysaccharide exhibits therapeutic potential for ovarian fibrosis in PCOS rats via the TGF-β1/smad pathway

Polycystic ovarian syndrome (PCOS) is one of the commonest endocrinopathies in childbearing women. The research was conducted to assess the impact of Irpex lacteus polysaccharide (ILP, 1000 mg/kg) on the letrozole (1 mg/kg)-induced PCOS model in female rats. Metformin (Met, 265 mg/kg) as the positive control. The study suggested that ILP restored the estrous cycle in rats with PCOS as well as lowered relative ovarian weight and body weight, in comparison to normal. Rats with PCOS showed improvement in ovarian structure and fibrosis when given ILP. ILP decreased the testosterone (T), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), total cholesterol (TC), luteinizing hormone (LH), homeostasis model assessment-insulin resistance (HOMA-IR), fasting blood glucose (FBG), and insulin (INS) levels and elevated the follicle-stimulating hormone (FSH) and estrogen (E2) levels in PCOS rats. In addition, ILP increased the content of superoxide dismutase (SOD) in serum and the antioxidant enzymes (Prdx3, Sod1, Gsr, Gsta4, Mgst1, Gpx3, Sod2 and Cat) expression levels in the ovaries and decreased the serum expression of malondialdehyde (MDA). In addition, ILP treatment slowed down the process of the fibrosis-associated TGF-β1/Smad pathway and downregulated α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) levels in PCOS rats ovaries. According to these findings, ILP may be able to treat letrozole-induced PCOS in rats by ameliorating metabolic disturbances, sex hormone levels, oxidative stress, and ovarian fibrosis.

Effects of Intracerebroventricular Injection of the Steroidal and Non-Steroidal Anti-Inflammatory Drugs on the Seizures during the Estrous Cycle in Rat.

Because of the mutual relationship between neural inflammation and seizure, this study aimed to determine the effects of intracerebroventricular (ICV) injection of the steroidal and non-steroidal anti-inflammatory drugs on pentylenetetrazol (PTZ)-induced seizures during the estrous cycle in rats. A total of 105 adult female Wistar rats were selected and divided into seven groups, including the control (saline), ketorolac tris salt (7.5, 15, and 30 µg), and methylprednisolone acetate (0.15, 0.3, and 0.6 µg), each with four subgroups (proestrus, estrus, metestrus, and diestrus) and three replicates (n=5). After a week of acclimatization, the estrous phase determination and synchronization were performed. Acute epilepsy was inspired by the intraperitoneal injection of 80 mg/kg of PTZ 30 min after the ICV injection of ketorolac and methylprednisolone acetate. The initiation time of myoclonic seizures (ITMS), the initiation time of tonic-clonic seizures (ITTS), seizure duration (SD), and mortality rate (MR) were measured for 30 min. Data were shown as mean±SD and analyzed using One-way ANOVA followed by Tukey-Kramer multiple comparison post hoc test (P<0.05). According to the results, ketorolac (15 and 30 µg) and methylprednisolone acetate (0.3 and 0.6 µg) significantly increased the ITTS and ITMS but decreased SD during the estrous cycle, compared to the control (P<0.05). Moreover, MR and SD were significantly decreased by ketorolac (7.5, 15, and 30 µg) and methylprednisolone (0.3 and 0.6 µg), compared to the control during the estrous cycle (P<0.05). Therefore, it seems that both ketorolac and methylprednisolone possess dose-dependent anticonvulsant effects that may decrease neural inflammation.

Electroacupuncture promotes expression of glutathione related regulatory enzymes in ovary tissue of rats with diminished ovarian reserve

To observe the effect of electroacupuncture (EA) on ovarian function and expression of glutathione (GSH) related regulatory enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione reductase (GR) protein and gene in rats with diminished ovarian reserve (DOR), so as to explore its mechanisms underlying up-regulation of antioxidant stress ability. A total of 30 female SD rats with normal estrous cycle were randomly divided into blank control, model and EA groups, with 10 rats in each group. The DOR model was established by gavage of tripterygium wilfordii polyglycoside suspension (50 mg·kg-1·d-1) for 14 consecutive days, while the rats in the blank group were given equal volume of 0.9% sodium chloride solution. One hour after daily gavage, EA (1.0 mA, 100 Hz) was applied alternately to bilateral "Shenshu"(BL23), and "Zhongwan"(CV12)+"Guanyuan"(CV4) for 10 min, for 14 consecutive days. Estrous cycles of rats in each group were observed and recorded daily during intervention.After the intervention, H.E.staining was used to observe histopathological changes of the ovarian tissue. The contents of serum sex hormones ［follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), estradiol (E2)］ and oxidative damage markers ［8-hydroxydeoxyguanosine (8-OHDG) and nitrotyrosine (NTY)］ were determined by ELISA. The contents of GSH and oxidized glutathione (GSSG) in the liver tissue were determined by colorimetry, and their ratios were calculated. Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the immunoactivity and gene expression levels of γ-GCS and GR in the ovarian tissues, respectively. Compared with the blank group, the model group had a marked increase in the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents (P<0.01) and a considerable decrease in the levels of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number as well as the expression of γ-GCS and GR mRNAs (P<0.05, P<0.01). After EA intervention, the increase of the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents and the decrease of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number of γ-GCS and GR as well as the expression of γ-GCS genes were all reversed (P<0.01, P<0.05). H.E. staining showed degenerative changes of the ovarian tissue, fewer follicles at every level and increase of atretic follicles, disarrangement and layer number decrease of granulosa cells, and atrophy of corpus luteum in the model group, which were relatively milder in the EA group. EA can improve ovarian function, and reduce oxidative stress damage in DOR rats, which may be associated with its functions in up-regulating the expression of γ-GCS and GR protein and gene in the ovarian tissue.

Redundant roles of AKT1 and AKT2 in fertility, estrous cyclicity and endometrial gland development.

Apoptosis and cell survival regulation are crucial processes during the estrous cycle to prepare a receptive uterus during implantation for successful recognition of pregnancy. PI3K/AKT signaling has a crucial role during gestation and AKT isoforms (1, 2 or 3) are regulated differently in the endometrium during the estrous cycle and embryo implantation. However, the specific roles of these isoforms are still unclear. We have previously shown that AKT isoforms expression during the rat estrous cycle and gestation are differently regulated. The present study aimed to establish the specific role of AKT isoforms in the mouse uterus. The hypothesis is that a dysregulation of AKT isoforms expression could cause fertility-related issues in an isoform-specific manner. With four different mouse models and in-house crossbreeding, all isoforms KO combinations (single, double and triple) were obtained in PGR expressing tissues. The results demonstrated that in absence of one or more AKT isoforms, female fertility was decreased. Mainly, we have observed smaller litter size, specifically in Akt1-2 KO mice. Additionally, we have found Akt1-2-3 KO mice to be fully infertile. Estrous cyclicity was also disrupted in Akt1-2 KO mice with longer diestrus stage. Moreover, the number of endometrial glands was decreased throughout the estrous cycle suggesting an important role in gland development for AKT1 and AKT2. Our results suggest specific roles between each isoform, but also a partially redundant function of AKT1 and AKT2 in litter size, estrous cyclicity and endometrial gland development. This highlights the importance of AKT in the physiological regulation of mouse fertility.

Cedrus deodara (Roxb. ex D.Don) G.Don bark fraction ameliorates metabolic, endocrine and ovarian dynamics in rats experiencing polycystic ovarian syndrome.

In the Ayurvedic system of medicine, Cedrus deodara bark has been utilized as a folk medicine to remove ovarian cysts and treat infertility in females. The present study is the first to investigate ameliorating potential of C. deodara bark on testosterone propionate and high-fat diet-induced polycystic ovarian syndrome in experimental rats. LC-MS analysis of the fraction selected through bioassay-guided approach employing uterine relaxant activity was performed to determine the bioactive constituents present in it. Further, the identified compounds were docked on the catalytic site of the androgen receptor and insulin receptor substrate-1. Later, the fraction was investigated against testosterone propionate and high-fat diet-induced PCOS in rats. Chloroform fraction (F1) of the plant bark was found most active in uterine smooth muscle relaxant activity. LC-MS analysis of F1 indicated the presence of key flavonoids namely deodarin, cedrin, deodardione, and cedrusinin. Afterward, a molecular docking study of these compounds revealed impressive binding interactions with androgen receptor and insulin receptor substrate-1. Besides, in vivo studies, treatment with F1 significantly restored the estrous cycle in rats from the diestrus phase in a dose-dependent manner. Also, the disturbed metabolic and endocrine profile was markedly improved in rats. Later, histopathological analysis revealed the presence of a large number of mature follicles and corpora lutea in F1-treated rats. In a nutshell, F1 exhibited promising beneficial effects in PCOS and associated conditions via amelioration of metabolic, endocrine, and ovarian dynamics in experimental rats.

The Track-by-Day Method for Monitoring the Rodent Estrous Cycle.

The exclusion of female subjects from preclinical neuroscience research has traditionally been justified in part by concerns about potential effects of cycling ovarian hormones on brain function. There is evidence that some behavioral and neurobiological measures do change over the estrous cycle and, as the use of female subjects becomes increasingly routine, there is a greater demand for accessible cycle-tracking methods. Conventional estrous cycle staging requires expert training in the qualitative interpretation of vaginal cytology smears, which serves as a barrier for novice researchers. In addition, definitions and reporting practices are not standardized across laboratories, which makes it difficult to compare results across studies and likely contributes to a false perception of the cycle as ephemeral and inconsistent. Here, we describe a streamlined method for monitoring the estrous cycle in rats, which we term Track-by-Day. It is simple to implement and inherently produces consistent reporting. Our protocol should serve to demystify and facilitate adoption of cycle tracking for those new to the practice. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Collection and staining of vaginal smears Basic Protocol 2: Track-by-Day classification of vaginal smears Support Protocol: Preparation of gelatin-subbed slides.

Estrous cycle impacts on dendritic spine plasticity in rat nucleus accumbens core and shell and caudate-putamen.

An important factor that can modulate neuron properties is sex-specific hormone fluctuations, including the human menstrual cycle and rat estrous cycle in adult females. Considering the striatal brain regions, the nucleus accumbens (NAc) core, NAc shell, and caudate-putamen (CPu), the estrous cycle has previously been shown to impact relevant behaviors and disorders, neuromodulator action, and medium spiny neuron (MSN) electrophysiology. Whether the estrous cycle impacts MSN dendritic spine attributes has not yet been examined, even though MSN spines and glutamatergic synapse properties are sensitive to exogenously applied estradiol. Thus, we hypothesized that MSN dendritic spine attributes would differ by estrous cycle phase. To test this hypothesis, brains from adult male rats and female rats in diestrus, proestrus AM, proestrus PM, and estrus were processed for Rapid Golgi-Cox staining. MSN dendritic spine density, size, and type were analyzed in the NAc core, NAc shell, and CPu. Overall spine size differed across estrous cycle phases in female NAc core and NAc shell, and spine length differed across estrous cycle phase in NAc shell and CPu. Consistent with previous work, dendritic spine density was increased in the NAc core compared to the NAc shell and CPu, independent of sex and estrous cycle. Spine attributes in all striatal regions did not differ by sex when estrous cycle was disregarded. These results indicate, for the first time, that estrous cycle phase impacts dendritic spine plasticity in striatal regions, providing a neuroanatomical avenue by which sex-specific hormone fluctuations can impact striatal function and disorders.

Effects of electroacupuncture on the kisspeptin-gonadotropin-releasing hormone (GnRH) /luteinizing hormone (LH) neural circuit abnormalities and androgen receptor expression of kisspeptin/neurokinin B/dynorphin neurons in PCOS rats

BackgroundPolycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, anovulation, and polycystic ovaries. Electroacupuncture (EA) can effectively improve hyperandrogenism and increase ovulation frequency in patients with PCOS. Pieces of suggest that androgen activity in the brain is associated with impaired steroid negative feedback in such patients. Studies have shown that EA regulated androgen receptor (AR) expression and local factor levels (such as anti-Müllerian hormone and inhibin B) in the ovary of PCOS rats. However, few studies have explored the effect of EA on androgen activity in the brain.ObjectiveThis study investigated the effect of EA on the kisspeptin-gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) neural circuit and sex hormone receptor expression in the hypothalamus of PCOS rats.MethodsPCOS signs were induced by letrozole administration, and the induced rats were treated with low-frequency EA at Guan Yuan acupoint (CV4). The effect of EA on PCOS-like signs was evaluated by observing changes in the body weight, ovarian quality, ovarian morphology, and serum sex hormone levels in rats. To explore the mechanism of the effect of EA on PCOS-like signs, the neuropeptide content of the kisspeptin-GnRH/LH neural circuit was assessed using enzyme-linked immunosorbent assay(ELISA); AR and estrogen receptor α (ERα) coexpression on kisspeptin/neurokinin B/dynorphin (KNDy) neurons was determined via triple-label immunofluorescence; and protein and mRNA expression of Kiss1, Ar, Esr1, and kisspeptin receptor (Kiss1r) was evaluated via western blotting and Reverse Transcription-Polymerase Chain Reaction (RT-PCR).ResultsThe results revealed that the estrous cycle of rats in the EA treatment group recovered, and their body and ovary weight reduced; ovarian morphology improved; serum testosterone and LH levels significantly decreased; and kisspeptin, GnRH, and dynorphin levels in hypothalamic arcuate nucleus significantly decreased. Compared with controls, the number of AR/Kiss1-positive cells increased, number of ERα/Kiss1-positive cells decreased, and protein and mRNA expression of Kiss1, Ar, and Kiss1r significantly increased in PCOS rats. However, EA treatment reversed these changes and reduced the expression of Kiss1, Ar, and Kiss1r significantly.ConclusionImprovement in the reproductive hallmarks of PCOS rats via EA may be achieved by regulating the kisspeptin-GnRH/LH circuit via androgen activity attenuation. Thus, the results provide an experimental basis for acupuncture as an adjuvant medical therapy on PCOS.

Erxian decoction alleviates cisplatin-induced premature ovarian failure in rats by reducing oxidation levels in ovarian granulosa cells

Ethnopharmacological relevantErxian Decoction (EXD) has been used empirically for more than 70 years to treat premature ovarian failure (POF), but more research is needed to understand how it works. Aim of the researchThe study aims to ascertain both in vivo and in vitro rewards of EXD. Materials and methodsEXD is composed of Curculiginis Rhizoma, Epimedii Folium, Morindae Officinalis, Angelicae Sinensis, Anemarrhenae Rhizoma, and Phellodendri Chinensis Cortex. UPLC/MS analysis was used to investigate the components of EXD. Using a POF model created by administering cisplatin to rats intraperitoneally, the pharmacodynamic effects of EXD were investigated. Three dose groups of EXD were garaged into rats: high (15.6 g/kg), medium (7.8 g/kg), and low (3.9 g/kg). By using a vaginal smear, the impact of EXD on the rat estrous cycle was evaluated. An ELISA test was used to measure the anti-Mullerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels in the serum of rats. By using HE stains, pathological alterations in the ovaries may be seen. MDA and SOD levels in ovarian samples were used to measure the degree of ovarian oxidation. TUNEL labeling of ovarian sections was used to find apoptosis levels. By using ATP, energy production was evaluated. The relative expression of proteins connected to aging and the RAGE pathway was assessed using Western blot. Then, using H2O2, a model of senescent human ovarian granulosa cells (KGN) was created in vitro. The impact of EXD and H2O2 on cellular senescence was discovered using-galactosidase staining. Cell apoptosis levels were found using PI/Hoechest33342. By using DCFH-DA, intracellular ROS was examined. MDA and SOD concentrations were used to measure the degree of cellular oxidation. RAGE-related mRNA and protein expression were evaluated using RT-qPCR and western blotting. ResultsUsing UPLC/MS analysis, 39 chemicals in EXD were found. Rats' estrous cycles were enhanced by EXD, which increased ovarian index and follicle count and reduced the proportion of atretic follicles in the rats. EXD reduced LH and FSH output while restoring AMH and E2 secretion. In ovarian tissues, EXD reduced the amount of apoptosis and MDA while raising SOD activity and ATP levels. The protein levels of p16, p21, p53, and Lamin A/C were among the senescence-related proteins that EXD lowered, along with the levels of RAGE, PI3K, BAX, and CASPASE 3. Anti-apoptotic protein BCL-2 was also raised in the RAGE pathway. Senescence, apoptosis, ROS, and MDA levels in the KGN cells were lowered in vitro by EXD. Additionally, EXD increased the anti-apoptotic potential by changing the expression of CAT, SOD2, and SIRT1. RAGE, BAX, BCL-2, CASPASE 3, and p38 expression levels were altered by EXD, enhancing its anti-apoptotic capability. ConclusionEXD boosted the ovary's antioxidant and anti-apoptotic capabilities while enhancing the estrous cycle and hormone output. These findings strongly suggested that EXD may contribute to the alleviation of POF and ovarian granulosa cells senescence.

Resident intruder paradigm-induced PMDD rat model of premenstrual irritability: behavioral phenotypes, drug intervention, and biomarkers.

Premenstrual dysphoric disorder (PMDD) is high in women of childbearing age with obvious premenstrual irritability. However, reliable animal models are still lacking. PMDD rat model of premenstrual irritability was induced by the resident-intruder paradigm (RIP). Behavioral characteristics were determined by the aggressive behavior test, elevated plus maze, open-field test, and breast width measurement. The estrous cycle in rats was artificially manipulated by bilateral ovariectomy and exogenous hormone injection to verify the model phenotype's dependence on the estrous cycle. Fluoxetine and Baixiangdan capsules were administered by gavage to determine the symptom improvement effect of PMDD irritability. Biomarkers in serum and brain were detected using ELISA, and GABRA4 was detected in the brain by RT-PCR and Western blot. Rat models demonstrated similar clinical characteristics as PMDD, such as premenstrual irritability and anxiety, and the above symptoms were estrous cycle-dependent. In addition, the levels of progesterone (P) and ALLO hormones decreased in the serum, hippocampus, amygdala, and frontal lobe in the NR phase. The contents of 5-HT in the brain were significantly increased, while NE and GABA contents were considerably reduced. Moreover, mRNA and protein expression of GABRA4 levels in model rats' amygdala, hippocampus, and frontal lobe were significantly increased, while drug intervention downregulated its expression in these tissues. Premenstrual irritability rat model of PMDD demonstrates a behavioral phenotype consistent with the clinical symptoms of PMDD and micro index. The increased levels of 5-HT, NE, and expression of GABRA4, as well as the decrease of GABA, P, and ALLO levels, may be critical biomarkers of the abnormal changes that occur during the pathogenesis of PMDD.