A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior.

Sex hormones exert a profound influence on gendered behaviors. How individual sex hormone-responsive neuronal populations regulate diverse sex-typical behaviors is unclear. We performed orthogonal, genetically targeted sequencing of four estrogen receptor 1-expressing (Esr1+) populations and identified 1,415 genes expressed differentially between sexes or estrous states. Unique subsets of these genes were distributed across all 137 transcriptomically defined Esr1+ cell types, including estrous stage-specific ones, that comprise the four populations. We used differentially expressed genes labeling single Esr1+ cell types as entry points to functionally characterize two such cell types, BNSTprTac1/Esr1 and VMHvlCckar/Esr1. We observed that these two cell types, but not the other Esr1+ cell types in these populations, are essential for sex recognition in males and mating in females, respectively. Furthermore, VMHvlCckar/Esr1 cell type projections are distinct from those of other VMHvlEsr1 cell types. Together, projection and functional specialization of dimorphic cell types enables sex hormone-responsive populations to regulate diverse social behaviors.

Limbic Neurons Shape Sex Recognition and Social Behavior in Sexually Naive Males.

Sexually naive animals have to distinguish between the sexes because they show species-typical interactions with males and females without meaningful prior experience. However, central neural pathways in naive mammals that recognize sex of other individuals remain poorly characterized. We examined the role of the principal component of the bed nucleus of stria terminalis (BNSTpr), a limbic center, in social interactions in mice. We find that activity of aromatase-expressing BNSTpr (AB) neurons appears to encode sex of other animals and subsequent displays of mating in sexually naive males. Silencing these neurons in males eliminates preference for female pheromones and abrogates mating success, whereas activating them even transiently promotes male-male mating. Surprisingly, female AB neurons do not appear to control sex recognition, mating, or maternal aggression. In summary, AB neurons represent sex of other animals and govern ensuing social behaviors in sexually naive males.

An unusual aromatase/cyclase programs the formation of the phenyldimethylanthrone framework in anthrabenzoxocinones and fasamycin.

Aromatic polyketides are renowned for their wide-ranging pharmaceutical activities. Their structural diversity is mainly produced via modification of limited types of basic frameworks. In this study, we characterized the biosynthesis of a unique basic aromatic framework, phenyldimethylanthrone (PDA) found in (+)/(-)-anthrabenzoxocinones (ABXs) and fasamycin (FAS). Its biosynthesis employs a methyltransferase (Abx(+)M/Abx(-)M/FasT) and an unusual TcmI-like aromatase/cyclase (ARO/CYC, Abx(+)D/Abx(-)D/FasL) as well as a nonessential helper ARO/CYC (Abx(+)C/Abx(-)C/FasD) to catalyze the aromatization/cyclization of polyketide chain, leading to the formation of all four aromatic rings of the PDA framework, including the C9 to C14 ring and a rare angular benzene ring. Biochemical and structural analysis of Abx(+)D reveals a unique loop region, giving rise to its distinct acyl carrier protein-dependent specificity compared to other conventional TcmI-type ARO/CYCs, all of which impose on free molecules. Mutagenic analysis discloses critical residues of Abx(+)D for its catalytic activity and indicates that the size and shape of its interior pocket determine the orientation of aromatization/cyclization. This study unveils the tetracyclic and non-TcmN type C9 to C14 ARO/CYC, significantly expanding our cognition of ARO/CYCs and the biosynthesis of aromatic polyketide framework.

Casein kinase 1α mediates estradiol secretion via CYP19A1 expression in mouse ovarian granulosa cells.

BACKGROUND: Casein kinase 1α (CK1α), expressed in both ovarian germ and somatic cells, is involved in the initial meiosis and primordial follicle formation of mouse oocytes. Using in vitro and in vivo experiments in this study, we explored the function and mechanism of CK1α in estrogen synthesis in mice ovarian granulosa cells. METHODS: A CK1α knockout (cKO) mouse model, targeted specifically to ovarian granulosa cells (GCs), was employed to establish the influence of CK1α on in vivo estrogen synthesis. The influence of CK1α deficiency on GCs was determined in vivo and in vitro by immunofluorescence analysis and Western blot assay. Transcriptome profiling, differentially expressed genes and gene functional enrichment analyses, and computation protein-protein docking, were further employed to assess the CK1α pathway. Furthermore, wild-type female mice were treated with the CK1α antagonist D4476 to elucidate the CK1α's role in estrogen regulation. RESULTS: Ovarian GCs CK1α deficiency impaired fertility and superovulation of female mice; also, the average litter size and the estradiol (E2) level in the serum of cKO female mice were decreased by 57.3% and 87.4% vs. control mice, respectively. This deficiency disrupted the estrous cycle and enhanced the apoptosis in the GCs. We observed that CK1α mediated the secretion of estradiol in mouse ovarian GCs via the cytochrome P450 subfamily 19 member 1 (CYP19A1). CONCLUSIONS: These findings improve the existing understanding of the regulation mechanism of female reproduction and estrogen synthesis. TRIAL REGISTRATION: Not applicable.

Estrogens in Male Physiology.

Estrogens have historically been associated with female reproduction, but work over the last two decades established that estrogens and their main nuclear receptors (ESR1 and ESR2) and G protein-coupled estrogen receptor (GPER) also regulate male reproductive and nonreproductive organs. 17ß-Estradiol (E2) is measureable in blood of men and males of other species, but in rete testis fluids, E2 reaches concentrations normally found only in females and in some species nanomolar concentrations of estrone sulfate are found in semen. Aromatase, which converts androgens to estrogens, is expressed in Leydig cells, seminiferous epithelium, and other male organs. Early studies showed E2 binding in numerous male tissues, and ESR1 and ESR2 each show unique distributions and actions in males. Exogenous estrogen treatment produced male reproductive pathologies in laboratory animals and men, especially during development, and studies with transgenic mice with compromised estrogen signaling demonstrated an E2 role in normal male physiology. Efferent ductules and epididymal functions are dependent on estrogen signaling through ESR1, whose loss impaired ion transport and water reabsorption, resulting in abnormal sperm. Loss of ESR1 or aromatase also produces effects on nonreproductive targets such as brain, adipose, skeletal muscle, bone, cardiovascular, and immune tissues. Expression of GPER is extensive in male tracts, suggesting a possible role for E2 signaling through this receptor in male reproduction. Recent evidence also indicates that membrane ESR1 has critical roles in male reproduction. Thus estrogens are important physiological regulators in males, and future studies may reveal additional roles for estrogen signaling in various target tissues.

Sex-specific vulnerabilities in human astrocytes underpin the differential impact of palmitic acid.

Obesity and neurometabolic diseases have been linked to neurodegenerative diseases. Our hypothesis is that the endogenous estrogenic component of human astrocytes plays a critical role in cell response during lipotoxic damage, given that obesity can disrupt hormonal homeostasis and cause brain inflammation. Our findings showed that high concentrations of palmitic acid (PA) significantly reduced cell viability more in male astrocytes, indicating sex-specific vulnerabilities. PA induced a greater increase in cytosolic reactive oxygen species (ROS) production in males, while female astrocytes exhibited higher superoxide ion levels in mitochondria. In addition, female astrocytes treated with PA showed increased expression of antioxidant proteins, including catalase, Gpx-1 and Nrf2 suggesting a stronger cellular defence mechanism. Interestingly, there was a difference in the expression of estrogenic components, such as estrogen, androgens, and progesterone receptors, as well as aromatase and 5α-reductase enzymes, between males and females. PA induced their expression mainly in females, indicating a potential protective mechanism mediated by endogenous hormones. In summary, our findings highlight the impact of sex on the response of human astrocytes to lipotoxicity. Male astrocytes appear to be more susceptible to cellular damage when exposed to high concentrations of fatty acids.

Cardiovascular impact of near complete estrogen deprivation in premenopausal women with breast cancer: The CROWN study.

Survival with operable breast cancer has improved markedly in recent decades, however, treatment-related cardiovascular toxicities threaten to offset these gains. Ovarian function suppression paired with aromatase inhibition, for premenopausal women with hormone receptor (HR)-positive breast cancer, is a newer widely adopted therapy with the potential for significant long-term cardiovascular toxicity. Abrupt estrogen deprivation for non-cancer reasons is associated with accelerated coronary artery disease. Women with breast cancer treated with aromatase inhibition in addition to ovarian function suppression experience a dual hit with regards to estrogen exposure. The CaRdiac Outcomes With Near-complete estrogen deprivation (CROWN) study seeks to understand the early, subclinical natural history of cardiovascular compromise in young women undergoing near-complete estrogen deprivation (NCED) therapy. It is critical to understand the early subclinical development of cardiovascular disease to identify a window for therapeutic intervention before overt cardiovascular events occur. This three-site regional study (Atrium Health Wake Forest, Duke, and Virginia Commonwealth University) uses serial stress cardiac magnetic resonance (CMR) imaging and cardiac computed tomography angiography (CCTA) obtained during the initial two years of NCED therapy to study myocardial prefusion reserve (MPR), large cardiovascular vessel changes, left ventricular function, and other cardiovascular parameters. The CROWN cohort will consist of 90 premenopausal women with breast cancer, 67 with HR-positive disease receiving NCED and 23 comparators with HR-negative disease. Participants will undergo three annual CMR scans and 2 CCTA scans during the 2-year study period. After initial activation hurdles, accrual has been brisk, and the study is expected to complete accrual in December 2024. Efforts are in place to encourage participant retention with the study primary outcome, change in MPR between the two groups, to be reported in 2026 to 2027. The results of this study will enable premenopausal women with breast cancer to balance the health burdens of cancer at a young age and treatment-related cardiovascular morbidity. Finally, the tools developed here can be utilized to study cardiovascular risk across a range of cancer types and cancer therapies with the ultimate goals of both developing generalizable risk stratification tools as well as validating interventions which prevent overt cardiovascular compromise.

Conceptus estrogen and prostaglandins provide the maternal recognition of pregnancy signal to prevent luteolysis during early pregnancy in the pig†.

Conceptus estrogens and prostaglandins have long been considered the primary signals for maternal recognition of pregnancy (MRP) in the pig. However, loss-of-function studies targeting conceptus aromatase genes (CYP19A1 and CYP19A2) and prostaglandin-endoperoxide synthase 2 (PTGS2) indicated that conceptuses can not only signal MRP without estrogens or prostaglandins but can maintain early pregnancy. However, complete loss of estrogen production leads to abortion after day 25 of gestation. Although neither conceptus estrogens nor prostaglandins had a significant effect on early maintenance of corpora lutea (CL) function alone, the two conceptus factors have a biological relationship. To investigate the role that both conceptus estrogens and prostaglandins have on MRP and maintenance of pregnancy, a triple loss-of function model (TKO) was generated for conceptus CYP19A1, CYP19A2, and PTGS2. In addition, a conceptus CYP19A2-/- model (A2KO) was established to determine the role of placental estrogen during later pregnancy. Estrogen and prostaglandin synthesis were greatly reduced in TKO concept uses which resulted in a failure to inhibit luteolysis after day 15 of pregnancy despite the presence of conceptuses in the uterine lumen. However, A2KO placentae not only maintained functional CL but were able to maintain pregnancy to day 32 of gestation. Despite the loss of placental CYP19A2 expression, the allantois fluid content of estrogen was not affected as the placenta compensated by expressing CYP19A1 and CYP19A3, which are normally absent in controls. Results suggest conceptuses can signal MRP through production of conceptus PGE or stimulating PGE synthesis from the endometrium through conceptus estrogen. Failure of conceptuses to produce both factors results in failure of MRP and loss of pregnancy.

Esr1 but Not CYP19A1 Overexpression in Mammary Epithelial Cells during Reproductive Senescence Induces Pregnancy-Like Proliferative Mammary Disease Responsive to Anti-Hormonals.

Molecular-level analyses of breast carcinogenesis benefit from vivo disease models. Estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) overexpression targeted to mammary epithelial cells in genetically engineered mouse models induces largely similar rates of proliferative mammary disease in prereproductive senescent mice. Herein, with natural reproductive senescence, Esr1 overexpression compared with CYP19A1 overexpression resulted in significantly higher rates of preneoplasia and cancer. Before reproductive senescence, Esr1, but not CYP19A1, overexpressing mice are tamoxifen resistant. However, during reproductive senescence, Esr1 mice exhibited responsiveness. Both Esr1 and CYP19A1 are responsive to letrozole before and after reproductive senescence. Gene Set Enrichment Analyses of RNA-sequencing data sets showed that higher disease rates in Esr1 mice were accompanied by significantly higher expression of cell proliferation genes, including members of prognostic platforms for women with early-stage hormone receptor-positive disease. Tamoxifen and letrozole exposure induced down-regulation of these genes and resolved differences between the two models. Both Esr1 and CYP19A1 overexpression induced abnormal developmental patterns of pregnancy-like gene expression. This resolved with progression through reproductive senescence in CYP19A1 mice, but was more persistent in Esr1 mice, resolving only with tamoxifen and letrozole exposure. In summary, genetically engineered mouse models of Esr1 and CYP19A1 overexpression revealed a diversion of disease processes resulting from the two distinct molecular pathophysiological mammary gland-targeted intrusions into estrogen signaling during reproductive senescence.

Overexpression of Estrogen Receptor α in Mammary Glands of Aging Mice Is Associated with a Proliferative Risk Signature and Generation of Estrogen Receptor α-Positive Mammary Adenocarcinomas.

Age is a risk factor for human estrogen receptor-positive breast cancer, with highest prevalence following menopause. While transcriptome risk profiling is available for human breast cancers, it is not yet developed for prognostication for primary or secondary breast cancer development utilizing at-risk breast tissue. Both estrogen receptor α (ER) and aromatase overexpression have been linked to human breast cancer. Herein, conditional genetically engineered mouse models of estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) were used to show that induction of Esr1 overexpression just before or with reproductive senescence and maintained through age 30 months resulted in significantly higher prevalence of estrogen receptor-positive adenocarcinomas than CYP19A1 overexpression. All adenocarcinomas tested showed high percentages of ER+ cells. Mammary cancer development was preceded by a persistent proliferative transcriptome risk signature initiated within 1 week of transgene induction that showed parallels to the Prosigna/Prediction Analysis of Microarray 50 human prognostic signature for early-stage human ER+ breast cancer. CYP19A1 mice also developed ER+ mammary cancers, but histology was more divided between adenocarcinoma and adenosquamous, with one ER- adenocarcinoma. Results demonstrate that, like humans, generation of ER+ adenocarcinoma in mice was facilitated by aging mice past the age of reproductive senescence. Esr1 overexpression was associated with a proliferative estrogen pathway-linked signature that preceded appearance of ER+ mammary adenocarcinomas.

Novel mitochondrial and DNA damaging fluorescent Calix[4]arenes bearing isatin groups as aromatase inhibitors: Design, synthesis and anticancer activity.

Breast cancer causes a high rate of mortality all over the world. Therefore, the present study focuses on the anticancer activity of new lower rim-functionalized calix[4]arenes integrated with isatin and the p-position of calixarenes with 1,4-dimethylpyridinium iodine against various human cancer cells such as MCF-7 and MDA-MB-231 breast cancer cell lines, as well as the PNT1A healthy epithelial cell line. It was observed that compound 6c had the lowest values in MCF-7 (8.83 µM) and MDA-MB-231 (3.32 µM). Cell imaging and apoptotic activity studies were performed using confocal microscopy and flow cytometry, respectively. The confocal imaging studies with 6c showed that the compound easily entered the cell, and it was observed that 6c accumulated in the mitochondria. The Comet assay test was used to detect DNA damage of compounds in cells. It was found that treated cells had abnormal tail nuclei and damaged DNA structures compared with untreated cells. In vitro human aromatase enzyme inhibition profiles showed that compound 6c had a remarkable inhibitory effect on aromatase. Compound 6c displayed a significant inhibition capacity on aromatase enzyme with the IC50 value of 0.104 ± 0.004 µM. Thus, not only the anticancer activity of the new fluorescent derivatives, which are the subject of this study, but the aromatase inhibitory profiles have also been proven.

Estrogen masculinizes neural pathways and sex-specific behaviors.

Sex hormones are essential for neural circuit development and sex-specific behaviors. Male behaviors require both testosterone and estrogen, but it is unclear how the two hormonal pathways intersect. Circulating testosterone activates the androgen receptor (AR) and is also converted into estrogen in the brain via aromatase. We demonstrate extensive sexual dimorphism in the number and projections of aromatase-expressing neurons. The masculinization of these cells is independent of AR but can be induced in females by either testosterone or estrogen, indicating a role for aromatase in sexual differentiation of these neurons. We provide evidence suggesting that aromatase is also important in activating male-specific aggression and urine marking because these behaviors can be elicited by testosterone in males mutant for AR and in females subjected to neonatal estrogen exposure. Our results suggest that aromatization of testosterone into estrogen is important for the development and activation of neural circuits that control male territorial behaviors.

Unraveling Hypothalamus-Pituitary dysregulation: Hypergonadotropism in F1 progeny due to prenatal exposure to hexavalent chromium.

The endocrine disruptor hexavalent chromium [Cr(VI)] is a proven reproductive toxicant. We recently demonstrated that prenatal Cr(VI) exposure causes testicular resistance to gonadotropins, resulting in hypergonadotropic hypoandrogenism in F1 rats. However, the mechanism driving hypergonadotropism in F1 rats exposed to Cr(VI) prenatally remains an enigma. Therefore, we hypothesized that 'Prenatal Cr(VI) exposure may disrupt steroid hormones-mediated negative feedback regulation of the hypothalamic GnRH, and its receptor in the pituitary of F1 rats, leading to hypergonadotropism.' We administered potassium dichromate (50, 100, or 200 mg/L) to pregnant rats through drinking water between days 9 and 14, and their male F1 offspring were euthanized at 60 days of age. Prenatal Cr(VI) exposure in F1 rats resulted in the accumulation of Cr in the hypothalamus and pituitary. Western blot detected decreased hypothalamic GnRH, Kisspeptin1, and its receptor GPR54, along with diminished ERα, AR, aromatase, and 5α reductase, and GnRH regulatory transcription factors Pit-1 and GATA-4 proteins. Immunohistochemical studies revealed increased immunopositivity of GnRH receptor, AR, 5α reductase, ERα, ERß, and aromatase proteins in the pituitary, whereas decreased Kisspeptin1, GPR54, and inhibin ß. Our findings imply that Cr(VI) exposure during the prenatal period disrupts the hypothalamic Kisspeptin-GPR54-Pit-1/GATA4-GnRH network, boosting the pituitary GnRH receptor. We conclude that prenatal exposure to Cr(VI) alters GnRH expression in the hypothalamus and its receptor in the pituitary of F1 progeny through interfering with the negative feedback effect of androgens and estrogens.

Aromatase inhibitors for the treatment of breast cancer: An overview (2019-2023).

Aromatase inhibition is considered a legitimate approach for the treatment of ER-positive (ER+) breast cancer as it accounts for more than 70% of breast cancer cases. Aromatase inhibitor therapy has been demonstrated to be highly effective in decreasing tumour size, increasing survival rates, and lowering the chance of cancer recurrence. The present review deliberates the pathophysiology and the role of aromatase in estrogen biosynthesis. Estrogen biosynthesis, various androgens, and their function in the human body have also been discussed. The salient aspects of the aromatase active site, its mode of action, and AIs, along with their intended interactions with presently FDA-approved inhibitors, have been briefly discussed. It has been detailed how different reported AIs were designed, their SAR investigations, in silico analysis, and biological evaluations. Various AIs from multiple origins, such as synthetic and semi-synthetic, have also been discussed.

DMF-DMA catalyzed Synthesis, molecular docking, in-vitro, in-silico design, and binding free energy studies of novel thiohydantoin derivatives as antioxidant and antiproliferative agents targeting EGFR tyrosine kinase and aromatase cytochrome P450 enzyme.

A set of novels 2-thiohydantoin derivatives were synthesized and enaminone function was discussed at position 5 using DMFDMA catalyst which result in formation of pyrazole, isoxazole, benzoxazepine by using reagents such as hydrazine, hydroxylamine and 2-aminothiophenol. These newly synthesized compounds were evaluated for their antioxidant and antiproliferative activity. In vitro studies on the effect of 2-thiohydantoin on scavenging 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) confirmed the free radical scavenging and antioxidant activity of 2-thiohydantoin. The synthesized compounds show significant antioxidant activity. The in vitro antitumor activity of 2-thiohydantoin on MCF7 (breast) and PC3 cells (prostate) was evaluated using MTT assay. Some of the synthesized compounds show significant to moderate antiproliferative properties compared to reference drug erlotinib. Among all, compound 4a exhibit potent antitumor properties against MCF7 and PC3 cancer cell lines with IC50 = 2.53 ± 0.09 /ml & with IC50 = 3.25 ± 0.12 µg/ml respectively and has potent antioxidant activity with IC50 = 10.04 ± 0.49 µg/ml.

The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Evaluation of new alternative methods for the identification of estrogenic, androgenic and steroidogenic effects: a comparative in vitro/in silico study.

A suite of in vitro assays and in silico models were evaluated to identify which best detected the endocrine-disrupting (ED) potential of 10 test chemicals according to their estrogenic, androgenic and steroidogenic (EAS) potential compared to the outcomes from ToxCast. In vitro methods included receptor-binding, CALUX transactivation, H295R steroidogenesis, aromatase activity inhibition and the Yeast oestrogen (YES) and Yeast androgen screen (YAS) assays. The impact of metabolism was also evaluated. The YES/YAS assays exhibited a high sensitivity for ER effects and, despite some challenges in predicting AR effects, is a good initial screening assay. Results from receptor-binding and CALUX assays generally correlated and were in accordance with classifications based on ToxCast assays. ER agonism and AR antagonism of benzyl butyl phthalate were abolished when CALUX assays included liver S9. In silico final calls were mostly in agreement with the in vitro assays, and predicted ER and AR effects well. The efficiency of the in silico models (reflecting applicability domains or inconclusive results) was 43-100%. The percentage of correct calls for ER (50-100%), AR (57-100%) and aromatase (33-100%) effects when compared to the final ToxCast call covered a wide range from highly reliable to less reliable models. In conclusion, Danish (Q)SAR, Opera, ADMET Lab LBD and ProToxII models demonstrated the best overall performance for ER and AR effects. These can be combined with the YES/YAS assays in an initial screen of chemicals in the early tiers of an NGRA to inform on the MoA and the design of mechanistic in vitro assays used later in the assessment. Inhibition of aromatase was best predicted by the Vega, AdmetLab and ProToxII models. Other mechanisms and exposure should be considered when making a conclusion with respect to ED effects.

Lipopolysaccharide-binding protein in follicular fluid is associated with the follicular inflammatory status and granulosa cell steroidogenesis in dairy cows.

Metabolic stress and subsequent hepatic dysfunction in high-producing dairy cows are associated with inflammatory diseases and declining fertility. Lipopolysaccharide (LPS)-binding protein (LBP) is produced by hepatocytes and controls the immune response, suggesting that it is involved in the pathophysiology of inflammation-related attenuation of reproductive functions during metabolic stress. This study investigated the effect of LBP on the inflammatory status, oocyte quality, and steroidogenesis in the follicular microenvironment of dairy cows. Using bovine ovaries obtained from a slaughterhouse, follicular fluid and granulosa cells were collected from large follicles to evaluate the follicular status of metabolism, inflammation, and steroidogenesis. Cumulus-oocyte complexes were aspirated from small follicles and subjected to in vitro embryo production. The results showed that follicular fluid LBP concentrations were significantly higher in cows with fatty livers and hepatitis than in those with healthy livers. Follicular fluid LBP and LPS concentrations were negatively correlated, whereas LPS concentration showed a positive correlation with the concentrations of non-esterified fatty acids (NEFA) and ß-hydroxybutyric acid in follicular fluid. The blastulation rate of oocytes after in vitro fertilization was impaired in cows in which coexisting large follicles had high NEFA levels. Follicular fluid NEFA concentration was negatively correlated with granulosa cell expression of the estradiol (E2) synthesis-related gene (CYP19A1). Follicular fluid LBP concentration was positively correlated with follicular fluid E2 concentration and granulosa cell CYP19A1 expression. In conclusion, follicular fluid LBP may be associated with favorable conditions in the follicular microenvironment, including low LPS levels and high E2 production by granulosa cells.

Direct evidence that KNDy neurons maintain gonadotropin pulses and folliculogenesis as the GnRH pulse generator.

The gonadotropin-releasing hormone (GnRH) pulse is fundamental for mammalian reproduction: GnRH pulse regimens are needed as therapies for infertile women as continuous GnRH treatment paradoxically inhibits gonadotropin release. Circumstantial evidence suggests that the hypothalamic arcuate KNDy neurons expressing kisspeptin (encoded by Kiss1), neurokinin B (encoded by Tac3), and dynorphin A serve as a GnRH pulse generator; however, no direct evidence is currently available. Here, we show that rescuing >20% KNDy neurons by transfecting Kiss1 inside arcuate Tac3 neurons, but not outside of these neurons, recovered folliculogenesis and luteinizing hormone (LH) pulses, an indicator of GnRH pulses, in female global Kiss1 knockout (KO) rats and that >90% conditional arcuate Kiss1 KO in newly generated Kiss1-floxed rats completely suppressed LH pulses. These results first provide direct evidence that KNDy neurons are the GnRH pulse generator, and at least 20% of KNDy neurons are sufficient to maintain folliculogenesis via generating GnRH/gonadotropin pulses.

d-Chiro-Inositol in Clinical Practice: A Perspective from the Experts Group on Inositol in Basic and Clinical Research (EGOI).

BACKGROUND: d-Chiro-inositol is a natural molecule that, in association with its well-studied isomer myo-inositol, may play a role in treating various metabolic and gynecological disorders. OBJECTIVES: This perspective seeks to explore the mechanisms and functions of d-chiro-inositol, laying the foundations to discuss its use in clinical practice, across dysmetabolism, obesity, and hormonal dysregulation. METHODS: A narrative review of all the relevant papers known to the authors was conducted. OUTCOME: d-Chiro-inositol acts through a variety of mechanisms, acting as an insulin sensitizer, inhibiting the transcription of aromatase, in addition to modulating white adipose tissue/brown adipose tissue transdifferentiation. These different modes of action have potential applications in a variety of therapeutic fields, including PCOS, dysmetabolism, obesity, hypoestrogenic/hyperandrogenic disorders, and bone health. CONCLUSIONS: d-Chiro-inositol mode of action has been studied in detail in recent years, resulting in a clear differentiation between d-chiro-inositol and its isomer myo-inositol. The insulin-sensitizing activities of d-chiro-inositol are well understood; however, its potential applications in other fields, in particular obesity and hyperestrogenic/hypoandrogenic disorders in men and women, represent promising avenues of research that require further clinical study.