Progesterone affects the transcription of genes in the circadian rhythm signaling and hypothalamic-pituitary-gonadal axes and changes the sex ratio in crucian carp (Carassius auratus).

Progesterone (P4) is an extensively applied progestin in human and veterinary medicine that has been widely detected in ambient aquatic environments, which can be detrimental to the health of aquatic organisms. Here we investigate the long-term effects of P4 on the transcription of genes related to the circadian rhythm signaling pathway and hypothalamic-pituitary-gonadal (HPG) axes in the crucian carp, which may have a potentially negative on endocrine-disrupting and sex differentiation impacts. Our results suggest that the expression of genes associated with the circadian rhythm signaling pathway are altered following exposure for 10, 20, 30, 40, 50 and 60 d, leading to disorders in the endocrine system disorders and the regulation of HPG axes-related gene expression. These maladies may affect gonadal development and the reproductive systems of crucian carp and provide a plausible mechanism for the observed change in sex ratio toward females after 180 d.

The effects of binary mixtures of estradiol and progesterone on transcriptional expression profiles of genes involved in hypothalamic-pituitary-gonadal axis and circadian rhythm signaling in embryonic zebrafish (Danio rerio).

Natural and synthetic estrogens and progestins are present in the various aquatic environments, leading to potential exposure of aquatic organisms to their mixtures. However, very little is known about their combined effects in aquatic organisms. The aim of this study was to analyze the effects of binary mixtures of estradiol (E2) and progesterone (P4) by measuring transcriptional changes of up to 42 selected target genes related to hypothalamic-pituitary-gonadal axis and circadian rhythm signaling in zebrafish (Danio rerio) eleuthero-embryos. Zebrafish embryos were exposed to E2 and P4 alone or in combination at concentrations between 45 and 5217 ng L-1 for 96 h post fertilization (hpf). The results showed that P4 led to slight up-regulation of the cyp11a1, hsd17b3 and fshb transcripts, while a strong induction of cyp19a1b and lhb mRNA by E2 was observed. Also, cyp19a1b and lhb mRNAs expression were strongly up-regulated in the mixtures, which were the same to E2 alone. This finding suggests the mixture activity of E2 and P4 followed the independent action in zebrafish eleuthero-embryos. These transcriptional alterations may translate to adverse effects on sex differentiation and reproduction in fish.

Molecular cloning, characterization of dax1 gene and its response to progesterone in Misgurnus anguillicaudatus.

Progesterone (P4) are aquatic contaminants that can impair fish reproduction even in low concentrations. The aim of this study was to investigate the effects of P4 on the sex differentiation, by quantitative determination of transcriptional changes of a candidate target gene (dax1, has a function in the sex determination and gonadal differentiation of several vertebrate species) in Misgurnus anguillicaudatus. We first cloned and characterized the full-length cDNAs for the dax1 in M. anguillicaudatus (designated as Ma-dax1). Sequence analysis reveals that Ma-dax1 shares high homology with dax1 in other species. Quantitative real-time PCR (qRT-PCR) and in situ hybridization showed that Ma-dax1 gene was highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, central nervous system development and gonad development. For the P4 administration assay, groups of mature fish were exposed for 1, 7, 14, 21 and 28 days to nominal concentrations of 10, 100, and 1000 ng/L P4 in a flow-through system. Quantification of Ma-dax1 transcripts revealed the expression of Ma-dax1 mRNA is altered after P4 treatment in mature gonads. Those showed that P4 could influence the sexual development and sex differentiation in M. anguillicaudatus by disturbing sex differentiation-associated gene expression, and dax1 can be used as a sensitive molecular biomarker for early warning to monitor the environmental progestins chemicals in fresh water environment.

Transcriptional effects of androstenedione and 17α-hydroxyprogesterone in zebrafish embryos.

Steroid hormones in the aquatic environment may pose a risk to fish health. Here we evaluated effects of two different class steroids that frequently occur in the aquatic environment, the androgen androstenedione (A4) and the progestin 17α-hydroxyprogesterone (17-OHP4). Zebrafish embryos were exposed to four concentrations of A4 and the positive control testosterone and to 17-OHP4, and transcriptional changes were determined at 96 h post fertilization (hpf) and 120 hpf. Transcriptional changes of 18 selected genes were assessed upon exposure to measured concentrations of 0.004, 0.046, 0.62 and 6.56 µg/L A4. Significant induction of the genes encoding sulfotransferase (sult2st3) and aromatase (cyp19b) occurred in 120 hpf embryos at 6.56 µg/L A4 and 1 µg/L testosterone. Additionally, cyp2k7 was significantly induced in two of three independent experiments. 17-OHP4 did not induce physiological effects (muscle contraction, heart rate, hatching success, swimming activity) at concentrations between 0.01 and 10 µg/L. Of the analyzed 15 genes, slight transcriptional alterations occurred for the genes encoding progesterone receptor, aromatases (cyp19a) and (cyp19b) and cyp2k7 at 10 µg/L. Our study highlights sult2st3, cyp19b and cyp2k7 as potential markers of androgen exposure in fish and indicates that 17-OHP4 is not likely to pose a risk for fish at environmental concentrations.

BRCA1-associated mammary tumorigenesis is dependent on estrogen rather than progesterone signaling.

Hereditary breast cancers in BRCA1 mutation carriers are mostly estrogen receptor α (ERα)-negative and progesterone receptor (PR)-negative; however, hormone depletion via bilateral oophorectomy does result in a marked reduction in breast cancer risk, suggesting that BRCA1-associated breast tumorigenesis is dependent on hormone signaling. We used geneticaly engineered mouse models to determine the individual influences of ERα and PR signaling on the development of BRCA1-deficient breast cancer. In line with the human data, BRCA1-deficient mouse mammary tumors are ERα-negative, and bilateral ovariectomy leads to abrogation of mammary tumor development. Hormonal replacement experiments in ovariectomized mice showed that BRCA1-deficient mammary tumor formation is promoted by estrogen but not by progesterone. In line with these data, mammary tumorigenesis was significantly delayed by the selective ERα downregulator fulvestrant, but not by the selective PR antagonist Org33628. Together, our results illustrate that BRCA1-associated tumorigenesis is dependent on estrogen signaling rather than on progesterone signaling, and call into question the utility of PR antagonists as a tumor prevention strategy for BRCA1 mutation carriers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Non-clinical studies of progesterone.

Progesterone is a steroid hormone that is essential for the regulation of reproductive function. Progesterone has been approved for several indications including the treatment of anovulatory menstrual cycles, assisted reproductive technology, contraception during lactation and, when combined with estrogen, for the prevention of endometrial hyperplasia in postmenopausal hormonal therapy. In addition to its role in reproduction, progesterone regulates a number of biologically distinct processes in other tissues, particularly in the nervous system. This physiological hormone is poorly absorbed when administered in a crystalline form and is not active when given orally, unless in micronized form, or from different non-oral delivery systems that allow a more constant delivery rate. A limited number of preclinical studies have been conducted to document the toxicity, carcinogenicity and overall animal safety of progesterone delivered from different formulations, and these rather old studies showed no safety concern. More recently, it has been shown in animal experiments that progesterone, its metabolite allopregnanolone and structurally related progestins have positive effects on neuroregeneration and repair of brain damage, as well as myelin repair. These recent preclinical findings have the potential to accelerate therapeutic translation for multiple unmet neurological needs.

Two synthetic progestins and natural progesterone are responsible for most of the progestagenic activities in municipal wastewater treatment plant effluents in the Czech and Slovak republics.

Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and progesterone, which contributed 65-96% of such activity in samples where no antagonistic activity was found. The progestins medroxyprogesterone acetate, megestrol acetate and progesterone contributed most to the progestagenic activity detected in municipal effluents. Anti-progestagenic activities were found in some municipal effluents, but no causative agents were revealed because two analysed selective progesterone receptor modulators (SPRMs) with anti-progestagenic activities, mifepristone and ulipristal acetate, were not present in the effluents.

Preparation and Evaluation of Progesterone Nanocrystals to Decrease Muscle Irritation and Improve Bioavailability.

Progesterone (PG) is a crucial immunomodulatory agent during early pregnancy, and nowadays PG oil-based injection (PG/OI) has a huge market all over the world. However, PG/OI may accumulate the local muscle and further cause irritations after long-term administration. In this study, PG nanocrystals (PG/NCs) injection was developed to decrease muscle toxicity. PG/NCs injection containing 10% (w/v) PG was first prepared using a wet grinding method. Then, particle size, zeta potential, morphology powder, X-ray diffraction (PXRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) studies were carried out to evaluate the characteristics of dosage form. The rabbit muscle irritation, hemolysis, and rat pharmacokinetics tests were used to estimate the in vivo characteristics of PG/NCs. The results showed that the mean particle size and the zeta potentials of NCs were 299.5 ± 9.0 nm and - 36.8 ± 1.5 mV, respectively. The crystalline state of PG/NCs was not altered during particle size reduction according to PXRD, DSC, and FTIR results. Muscle irritation presented that PG/NCs had lower irritation than that of PG/OI. Hemolysis test suggested that PG/NCs injection was functioned without hemolysis and red cell agglutination. The pharmacokinetics study showed that the AUC0-t and Cmax of PG/NCs was 3.2-fold (p < 0.05) and 3.1-fold higher than PG/OI, which demonstrated that PG/NCs injection had greater bioavailability than PG/OI. Therefore, it was obvious that PG/NCs injection exhibited a lower muscle irritation, hemolysis rate, and higher bioavailability, which was a better dosage form than OI.

Regulation of zebrafish (Danio rerio) locomotor behavior and circadian rhythm network by environmental steroid hormones.

Environmental exposure of fish to steroid hormones through wastewater and agricultural runoff may pose a health risk. Thus far, ecotoxicological studies have largely been focused on the disruption of the sex hormone system, but additional effects have been poorly investigated. Here we report on the effects of a series of different natural and synthetic steroid hormones on the locomotor behavior and the transcriptional levels of core clock genes in zebrafish eleuthero-embryos (Danio rerio). Of the 20 steroids analyzed, progestins and corticosteroids, including progesterone and cortisol, significantly decreased the locomotor activities of eleuthero-embryos at concentrations as low as 16 ng/L, while estrogens such as 17ß-estradiol led to an increase. Consistently, progestins and corticosteroids displayed similar transcriptional effects on core clock genes, which were remarkably different from those of estrogens. Of these genes, per1a and nr1d2a displayed the most pronounced alterations. They were induced upon exposure to various progestins and corticosteroids and could be recovered using the progesterone receptor/glucocorticoid receptor antagonist mifepristone; this, however, was not the case for estrogens and the estrogen receptor antagonist 4-hydroxy-tamoxifen. Our results suggest that steroid hormones can modulate the circadian molecular network in zebrafish and provide novel insights into their mode of actions and potential environmental risks.

Physiological responses and gene expression changes in the western mosquitofish (Gambusia affinis) exposed to progesterone at environmentally relevant concentrations.

Progesterone (P4) is a natural and synthetic steroid, widely distributed in the aquatic environments. It can lead to adverse effects on the endocrine system in aquatic organisms. This study investigated the toxicological effects of exposure to environmentally relevant concentrations (4, 44, and 410ng/L) of progesterone for 42 d on adult female mosquitofish, Gambusia affinis. We performed morphological and histological analyses on gonads, anal fins, liver, and gills after the exposure of mosquito fish to P4. The expression levels of genes (vtg, er, and ar isoforms) related to fish reproduction and detoxification (cyp1a) in the liver were quantified by quantitative real-time polymerase chain reaction. The results showed that the progesterone exposure induced slight masculinization in female mosquitofish, influenced the oocyte maturation as revealed by histology of the ovaries, and caused severe damages to the liver and gills of adult female mosquitofish. It also suppressed the mRNAs expression of vtg, er, cyp1a, and significantly enhanced the expression of ar mRNA in the liver. This study reveals the molecular and physiological effects of progesterone at environmentally relevant concentrations, which might further be translated to alterations in the reproduction of mosquitofish.

Effect of progesterone and its synthetic analogs on reproduction and embryonic development of a freshwater invertebrate model.

The presence of a mixture of progestogens at ng/L concentration levels in surface waters is a worldwide problem. Only a few studies explore the effect of progestogen treatment in a mixture as opposed to individual chemicals to shed light on how non-target species respond to these contaminants. In the present study, we used an invertebrate model species, Lymnaea stagnalis, exposed to a mixture of four progestogens (progesterone, levonorgestrel, drospirenone, and gestodene) in 10ng/L concentration for 3 weeks. Data at both physiological and cellular/molecular level were analyzed using the ELISA technique, stereomicroscopy combined with time lapse software, and capillary microsampling combined with mass spectrometry. The treatment of adult Lymnaeas caused reduced egg production, and low quality egg mass on the first week, compared to the control. Starting from the second week, the egg production, and the quality of egg mass were similar in both groups. At the end of the third week, the egg production and the vitellogenin-like protein content of the hepatopancreas were significantly elevated in the treated group. At the cellular level, accelerated cell proliferation was observed during early embryogenesis in the treated group. The investigation of metabolomic changes resulted significantly elevated hexose utilization in the single-cell zygote cytoplasm, and elevated adenylate energy charge in the egg albumen. These changes suggested that treated snails provided more hexose in the eggs in order to improve offspring viability. Our study contributes to the knowledge of physiological effect of equi-concentration progestogen mixture at environmentally relevant dose on non-target aquatic species.

Inhibitory effect of progesterone during early embryonic development: Suppression of myocardial differentiation and calcium-related transcriptome by progesterone in mESCs: Progesterone disturb cardiac differentiation of mESCs through lower cytosolic Ca(2.).

Progesterone (PG) and its derivates are used in prevention of spontaneous miscarriage. However, some studies have reported that exposure to PG and its derivates during pregnancy can cause malformations and affect both blood pressure and the cardiovascular system. The effect of PG on cardiomyogenesis of mouse embryonic stem cells (mESCs) is not well known. Expression of Pgr mRNA showed an opposite pattern of beating-ratio during differentiation. PG treatment resulted in reduction of the beating ratio to 60.45±1.54% from 92.17±2.98% in normal differentiation, reduced transcripts of heart morphogenesis and Ca(2+) binding-related genes in the next generation sequencing data and significantly decreased expression levels of Ca(2+)/contraction-related genes including Ryr2, Calm2, Trpv2, and Mylk3, the intracellular Ca(2+) level, and the beating frequency. These results suggest that PG exerts inhibitory effects on differentiation of mESCs into functional cardiomyocytes.

Developmental exposure to progestins causes male bias and precocious puberty in zebrafish (Danio rerio).

Progestins are aquatic contaminants that in low concentrations can impair fish reproduction. The mechanisms are likely multiple since different progestins interact with other steroid receptors in addition to progesterone receptors. Puberty is the process when animals first acquire the capability to reproduce and it comprises maturation of sperm and eggs. In zebrafish, puberty is initiated around 45days post fertilization (dpf) in females and around 53-55 dpf in males, and is marked by increased production of pituitary gonadotropins. We exposed juvenile zebrafish from 20 to 80 dpf to the androgenic progestin levonorgestrel at concentrations of 5.5, 79 and 834ngL(-1) and to the non-androgenic progestin progesterone at concentrations of 3.7, 77 and 1122ngL(-1), during sexual differentiation and puberty. Levonorgestrel exposure caused 100% males even at the lowest concentration tested whereas progesterone did not affect the sex ratio. Transcript levels of the gonadal genes amh, CYP11B and CYP19a1a indicated that the masculinizing effect of levonorgestrel occurred very rapidly. Transcript concentrations of gonadotropins in pituitaries were low in control fish at 44 dpf, but high at 55 dpf and onward. In fish exposed to levonorgestrel or progesterone gonadotropin transcript concentrations were high already at 44 dpf, indicating that both progestins caused precocious puberty. Gonad histology at 50 dpf confirmed a well advanced sexual maturation, but only in males. Our results show that progestins can affect sexual development in fish and that the androgenic progestin levonorgestrel induces a male phenotype at concentrations similar to those detected in aquatic environments.

Androgens regulate scarless repair of the endometrial "wound" in a mouse model of menstruation.

The human endometrium undergoes regular cycles of synchronous tissue shedding (wounding) and repair that occur during menstruation before estrogen-dependent regeneration. Endometrial repair is normally both rapid and scarless. Androgens regulate cutaneous wound healing, but their role in endometrial repair is unknown. We used a murine model of simulated menses; mice were treated with a single dose of the nonaromatizable androgen dihydrotestosterone (DHT; 200 µg/mouse) to coincide with initiation of tissue breakdown. DHT altered the duration of vaginal bleeding and delayed restoration of the luminal epithelium. Analysis of uterine mRNAs 24 h after administration of DHT identified significant changes in metalloproteinases (Mmp3 and -9; P < 0.01), a snail family member (Snai3; P < 0.001), and osteopontin (Spp1; P < 0.001). Chromatin immunoprecipitation analysis identified putative androgen receptor (AR) binding sites in the proximal promoters of Mmp9, Snai3, and Spp1. Striking spatial and temporal changes in immunoexpression of matrix metalloproteinase (MMP) 3/9 and caspase 3 were detected after DHT treatment. These data represent a paradigm shift in our understanding of the role of androgens in endometrial repair and suggest that androgens may have direct impacts on endometrial tissue integrity. These studies provide evidence that the AR is a potential target for drug therapy to treat conditions associated with aberrant endometrial repair processes.-Cousins, F. L., Kirkwood, P. M., Murray, A. A., Collins, F., Gibson, D. A., Saunders, P. T. K. Androgens regulate scarless repair of the endometrial "wound" in a mouse model of menstruation.

Activity of binary mixtures of drospirenone with progesterone and 17α-ethinylestradiol in vitro and in vivo.

Despite potential exposure of aquatic organisms to mixtures of steroid hormones, very little is known on their joint activity in fish. Drospirenone (DRS) is a new synthetic progestin used in contraceptive pills in combination with 17α-ethinylestradiol (EE2). Here we systematically analyzed effects of DRS in binary mixtures with progesterone (P4) and EE2. First, we determined the in vitro activity of single compounds in recombinant yeast assays that express the human progesterone, androgen, or estrogen receptor, followed by determination of mixture activities of DRS and P4, DRS and EE2, as well as medroxyprogesterone acetate (MPA) and dydrogesterone (DDG). Mixtures of DRS and P4, as well as of DRS and EE2 showed additive progestogenic and androgenic activities. However, DDG and MPA showed non-additive progestogenic and androgenic activities. We then analyzed the in vivo activity of single compounds and mixtures of DRS and P4, as well as DRS and EE2, by assessing transcriptional changes of up to 14 selected target genes in zebrafish embryos at 48h post fertilization (hpf), and in eleuthero-embryos at 96hpf and 144hpf. DRS, P4, and EE2 led to significant transcriptional alteration of genes, including those encoding hormone receptors (pgr, esr1), a steroidogenic enzyme (hsd17b3), and estrogenic markers (vtg1, cyp19b), in particular at 144 hpf. In general, DRS showed stronger transcriptional changes than P4. In mixtures of DRS and P4, they were mainly non-additive (antagonistic interaction). In mixtures of DRS and EE2, transcriptional responses of esr1, vtg1 and cyp19b were dominated by EE2, suggesting an antagonistic interaction or independent action. Equi-effective mixtures of DRS and EE2, based on progesterone receptor transcripts, showed antagonistic interactions. Our data suggest that interactions in mixtures assessed in vitro in recombinant yeast cannot be translated to the in vivo situation. The receptor-based responses did not correspond well to the transcriptional responses in embryos which are much more complex due to the interplay between hormonal pathways, receptor crosstalk, and hormonal feedback loops.

Avaliação de danos genéticos e apoptose em mulheres menopausadas que fazem uso da terapia de reposição hormonal / Assessment of genetic damage and apoptosis in postmenopausal women under hormone replacement therapy

Objetivo: Avaliar por meio do teste de micronúcleo em células esfoliadas da mucosa oral a genotoxicidade de hormônios empregados na terapia de reposição hormonal (TRH). Material e métodos: Quarenta mulheres menopausadas foram selecionadas e distribuídas em quatro grupos de dez: G0 (não submetidas à TRH), G1 (TRH com estrógeno), G2 (TRH com estrógeno e progestágeno) e G3 (TRH com tibolona). Células esfoliadas da mucosa oral foram coletadas e processadas para análise, sob microscopia óptica, de danos cromossômicos (micronúcleos) e de apoptose, inferida pelo somatório de alterações nucleares degenerativas (picnose, cariorréxis e cromatina condensada). Duas mil células foram analisadas por participante. A análise estatística foi feita com o uso do teste condicional para comparação de proporções em situação de eventos raros. Resultados: A frequência de micronúcleo não diferiu entre os grupos (p > 0,05). As alterações nucleares indicativas de apoptose foram significativamente mais altas entre as mulheres do G0 em relação às dos demais grupos (p < 0,01). Os grupos submetidos à TRH não diferiram quanto à ocorrência de apoptose (p > 0,05). Conclusões: Esses resultados indicam que, tal como avaliado neste estudo, a TRH não induz danos cromossômicos. Entretanto, devido ao efeito antiapoptótico, permite a proliferação de células geneticamente danificadas, está assim potencialmente associada ao processo de transformação maligna.(AU)

Objective: To evaluate through the micronucleus test in exfoliated cells of oral mucosa the genotoxicity of hormones used in hormone replacement therapy (HRT). Material and methods: Forty postmenopausal women were selected and divided into four groups of ten: G0 (not submitted to HRT), G1 (HRT with estrogen), G2 (HRT with estrogen and progestogen) and G3 (HRT with tibolone). Exfoliated cells of oral mucosa were collected and processed for analysis of chromosomal damage (micronuclei) and apoptosis, inferred by the sum of nuclear degenerative alterations (picnosis, cariorréxis and condensed chromatin). Two thousand cells were analyzed by participant. Statistical analysis was performed using the conditional test to compare proportions in a situation of rare events. Results: The frequency of micronuclei did not differ between groups (p > 0.05). Apoptotic cells were significantly higher among women from G0 when compared to the other groups (p < 0.01). The groups submitted to HRT did not differ in apoptosis occurrence (p > 0.05). Conclusions: These results indicate that, as assessed in this study, TRH does not induce chromosomal damage, however, due to the anti-apoptotic effect, cells genetically damaged can proliferate, favoring the malignant transformation process.(AU)

Environmental Progestins Progesterone and Drospirenone Alter the Circadian Rhythm Network in Zebrafish (Danio rerio).

Progestins alter hormone homeostasis and may result in reproductive effects in humans and animals. Thus far, studies in fish have focused on the hypothalamic-pituitary-gonadal (HPG)-axis and reproduction, but other effects have little been investigated. Here we report that progesterone (P4) and drospirenone (DRS) interfere with regulation of the circadian rhythm in fish. Breeding pairs of adult zebrafish were exposed to P4 and DRS at concentrations between 7 and 13 650 ng/L for 21 days. Transcriptional analysis revealed significant and dose-dependent alterations of the circadian rhythm network in the brain with little effects in the gonads. Significant alterations of many target transcripts occurred even at environmental relevant concentrations of 7 ng/L P4 and at 99 ng/L DRS. They were fully consistent with the well-described circadian rhythm negative/positive feedback loops. Transcriptional alterations of the circadian rhythm network were correlated with those in the HPG-Liver-axis. Fecundity was decreased at 742 (P4) and 2763 (DRS) ng/L. Dose-dependent alterations in the circadian rhythm network were also observed in F1 eleuthero-embryos. Our results suggest a potential target of environmental progestins, the circadian rhythm network, in addition to the adverse reproductive effects. Forthcoming studies should show whether the transcriptional alterations in circadian rhythm translate into physiological effects.

Neonatal Progesterone Programs Adult Uterine Responses to Progesterone and Susceptibility to Uterine Dysfunction.

In this report, we investigated the consequences of neonatal progesterone exposure on adult rat uterine function. Female pups were subcutaneously injected with vehicle or progesterone from postnatal days 3 to 9. Early progesterone exposure affected endometrial gland biogenesis, puberty, decidualization, and fertility. Because decidualization and pregnancy success are directly linked to progesterone action on the uterus, we investigated the responsiveness of the adult uterus to progesterone. We first identified progesterone-dependent uterine gene expression using RNA sequencing and quantitative RT-PCR in Holtzman Sprague-Dawley rats and progesterone-resistant Brown Norway rats. The impact of neonatal progesterone treatment on adult uterine progesterone responsiveness was next investigated using quantitative RT-PCR. Progesterone resistance affected the spectrum and total number of progesterone-responsive genes and the magnitude of uterine responses for a subset of progesterone targets. Several progesterone-responsive genes in adult uterus exhibited significantly dampened responses in neonatally progesterone-treated females compared with those of vehicle-treated controls, whereas other progesterone-responsive transcripts did not differ between female rats exposed to vehicle or progesterone as neonates. The organizational actions of progesterone on the uterus were dependent on signaling through the progesterone receptor but not estrogen receptor 1. To summarize, neonatal progesterone exposure leads to disturbances in endometrial gland biogenesis, progesterone resistance, and uterine dysfunction. Neonatal progesterone effectively programs adult uterine responsiveness to progesterone.

Long-term exposure to environmentally relevant concentrations of progesterone and norgestrel affects sex differentiation in zebrafish (Danio rerio).

The aim of this study was to investigate the effects of progestins on the sex differentiation of zebrafish by measuring the sex ratio and transcriptions of genes related to sex differentiation (Amh, Dmrt1, Figa, Sox9a and Sox9b genes) as well as sex hormone levels and transcriptional expression profiles along the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes in juvenile zebrafish. Exposure of zebrafish to 4, 33, 63ngL(-1) progesterone (P4) or 4, 34, 77ngL(-1) norgestrel (NGT) started at 20 days post fertilization (dpf) and ended at 60 dpf. The results showed that exposure to P4 caused a significant increase in proportion of females as well as significant down-regulation of Amh gene and up-regulation of Figa at a concentration of 63ngL(-1). However, the shift in the sex ratio toward males was observed following exposure to 34 and 77ngL(-1) NGT, which came along with the significant induction of Dmrt1 gene and inhibition of Figa gene. The sex hormones in exposed fish were measured with estrone being detected only in the fish exposed to the highest P4 concentration; whereas estradiol and androstenedione were detected only in the fish of the control and lowest NGT concentration. Furthermore, the increase in females was associated with the significant up-regulation of several key genes controlling the synthesis of sex hormones (i.e., Cyp17, Cyp19a1a and Hsd3b) following exposure to 63ngL(-1) P4 whereas the significant down-regulation of Cyp11a1, Cyp17, Cyp19a1a and Hsd3b genes was observed in the male-biased populations caused by 34 and 77ngL(-1) NGT. The overall results imply that both P4 and NGT could significantly affect sex differentiation in zebrafish, and that changes may be reflected by altered sex hormone levels and transcriptional expression profiles of genes related to synthesis of sex hormones.

Thermosensitive progesterone hydrogel: a safe and effective new formulation for vaginal application.

PURPOSE: The safe and functional delivery of progesterone through the vaginal route remains an unmet clinical need. The purpose of this work is to prepare a new progesterone (P4) gel for vaginal application using a thermosensitive mucoadhesive polymer, glycol chitin (GC). METHOD: Thermogelling, mucoadhesive, mechanical, and viscoelastic properties of GC and the new formulation were evaluated using rheometry. In vitro release profile and the bioactivity of P4 were determined using vaginal fluid simulant (VFS) pH 4.2, and PR-reporter gene assay, respectively. In vitro safety of the formulations was tested using (VK2/E6E7) vaginal epithelial cell line and Lactobacillus Crispatus. Finally, in vivo safety and the efficacy of this formulation were evaluated using an endometrial hypoplasia mouse model. RESULTS: Results shows the aqueous solution of 5%; (w/v) GC loaded with 0.1%; (w/v) P4 prepared in pH 4.2, (GC-P4), forms a thermosensitive mucoadhesive hydrogel and can maintain stable physical properties at 37 °C. GC-P4 gel release 50% of P4 in 4 h after exposure to VFS, and no significant decrease in % viability of VK2/E6E7 or Lactobacillus was found after exposure to 5% GC or GC-P4. GC-P4 does not exhibit obvious toxicities to vaginal tissue in vivo even after repeated application. Efficacy studies indicated that GC-P4 was capable of preventing the progression of simple endometrial hyperplasia (SEH) to complex atypical endometrial hyperplasia (CAEH) in vivo. CONCLUSIONS: Results indicates that GC-P4 retains many characteristics for an effective vaginal delivery system for P4. Therefore we believe that GC-P4 formulation is a promising alternative to current vaginal P4 formulation.