Progesterone Pretreatment Research Articles

THU639 17α-Estradiol Signals Primarily Through Nongenomic Mechanisms: Implications For Its Beneficial Longevity Effects

Abstract Disclosure: P.S. Cooke: None. V.K. Sirohi: None. H.D. Zomer: None. J.B. Graceli: None. A. Mesa: None. T. Medrano: None. Recent work indicates that exogenous 17α-estradiol (17α-E2), a stereoisomer of 17β-E2, increases longevity 20% in male mice, despite previous literature that 17α-E2 is a weak estrogen with low affinity for nuclear (n)ESR1. This study aimed to obtain mechanistic insights into uterine 17α-E2 actions in vivo. We hypothesized that 17α-E2 signals preferentially through membrane (m)ESR1 to affect target tissues and promote longevity. To test this, we treated pre-pubertal (postnatal day 20) female CD-1 mice with 3 daily ip injections of vehicle or 10 mg/kg of 17α-E2 or 17β-E2. 17α-E2 treatment did not significantly increase uterine weight - a marker of nuclear (n)ESR1 signaling (20.3 ± 2.1 vs. vehicle 15.7 ± 2.0, p>0.05, n=4), while 17β-E2 strongly increased it to 41.4 + 1.1 mg; p<0.05). Accordingly, histological analysis revealed reduced cell proliferation in 17α-E2- vs. 17β-E2-treated uteri. Both 17α-E2 and 17β-E2 produced robust and similar epithelial p-ERK and p-AKT expression, the active phosphorylated forms of these protein kinases. Western blotting and immunofluorescence showed that 17α-E2 and 17β-E2 stimulated a strong and comparable stimulation of EZH2, a critical mediator of estrogen’s epigenetic effects linked to mESR1 signaling, as well as increases in NRF2 and CBP signaling. Progesterone pre-treatment blocks both the 17α-E2- and 17β-E2-mediated increases in p-ERK. In conclusion, despite limited actions through nESR1, 17α-E2 robustly signals through mESR1 to activate uterine protein kinase cascades and epigenetic effects. Thus, 17α-E2 acts preferentially through mESR1, which may explain its powerful longevity effects despite limited effects on classical markers of estrogen action such as uterine weight. Presentation: Thursday, June 15, 2023

Evaluation of hirsutism and hormonal parameters in acne vulgaris patients treated with isotretinoin.

Although the effect of isotretinoin use on hormonal changes in acne pathogenesis is not fully known, there are limited studies on its effects on the development of hirsutism. In this study, it was aimed to evaluate the effect of isotretinoin use on hirsutism and hormonal parameters in patients with acne vulgaris. In this study, 30 female acne patients and 30 healthy females were evaluated prospectively. Menstrual irregularity, LH, FSH, prolactin, progesterone, 17-OH progesterone, oestradiol, total testosterone, DHEA-S, insulin, glucose, TSH levels, Ferriman-Gallwey (FG) score and ultrasonography (USG) findings of control group and patient group were recorded. Pre-treatment progesterone (P=.007) and oestradiol (P=.001) levels of the patients were statistically lower than the control group. In the patient group, menstrual irregularity (P<001) and FG hirsutism score at the third month of treatment were significantly higher than before treatment. In 10% of the patients, there were abnormal findings on pelvic USG in the third month of treatment. In our study, it could not be revealed that isotretinoin has a significant effect on pituitary, adrenal hormones and insulin resistance. We found that 3months of isotretinoin treatment caused an increase in menstrual irregularity and FG hirsutism score.

Progesterone pretreatment reduces the incidence of drug-induced torsades de pointes in atrioventricular node-ablated isolated perfused rabbit hearts.

Higher progesterone concentrations are protective against drug-induced prolongation of ventricular repolarization. We tested the hypothesis that pretreatment with progesterone reduces the incidence of drug-induced torsades de pointes (TdP). Female New Zealand white rabbits (2.5-3.2 kg) underwent ovariectomy and were randomized to undergo implantation with subcutaneous 21-day sustained release pellets containing progesterone 50 mg (n = 22) or placebo (n = 23). After 20 days, hearts were excised, mounted, and perfused with modified Krebs-Henseleit solution. The atrioventricular (AV) node was destroyed manually. Following a 15-minute equilibration period, hearts were perfused with dofetilide 100 nM for 30 minutes, during which the electrocardiogram was recorded continuously. Incidences of spontaneous TdP, other ventricular arrhythmias and mean QTc intervals were compared. Median serum progesterone concentrations were higher in progesterone vs placebo-treated rabbits (3.8 [range, 2.8-5.1] vs 0.7 [0.4-1.7] ng/mL, P < 0.0001). Median serum estradiol concentrations were similar (58 [22-72] vs 53 [34-62] pg/mL), P = 0.79). The incidence of TdP was lower in hearts from progesterone-treated rabbits (27% vs 61%, P = 0.049). The incidences of bigeminy (36% vs 74%, P = 0.03) and trigeminy (18% vs 57%, P = 0.01) were also lower in hearts from progesterone-treated rabbits. There was no significant difference between groups in incidence of couplets (59% vs 74%, P = 0.54) or monomorphic ventricular tachycardia (14% vs 30%, P = 0.28). Maximum QT c interval and short-term beat-to-beat QT interval variability during dofetilide perfusion were significantly shorter in hearts from progesterone-treated rabbits. Pretreatment with progesterone reduces the incidence of drug-induced TdP, bigeminy, and trigeminy in isolated perfused AV node-ablated rabbit hearts.

SAT-196 Maternal immunity: Preimplantation Preparation

The maternal uterus in mammals undergoes extensive remodeling in preparation for implantation of the semi-allogeneic embryo. Activation of the T cell homing receptor, CCR7, regulates multiple aspects of adaptive immunity. Genetic deletion of CCR7 reduces T regulatory (Treg) cell migration into mouse uteri and decreases embryo implantation. CCL19 and CCL21 are the sole ligands for CCR7. We hypothesized that CCL19 and/or CCL21 expression could attract Treg cells into the pre-implantation uterus and provide local immune suppression prior to implantation. Sprague Dawley rat uteri were removed from pregnant rats at Days 3-6. RNA was isolated and real time PCR was used to measure CCL19 and CCL21 expression. To determine the spatial distribution of these ligands, rat uteri were fixed, embedded in paraffin and uterine sections were analyzed by immunocytochemistry. The influence of sex steroids on the spatial distribution of CCL19 was evaluated in uterine sections from steroid treated rats. Hormonal control of CCL19 and CCL21 expression was investigated in a rat uterine stromal cell line treated with female sex steroids. Expression of CCL19 and CCL21 peaked at Day 4 of pregnancy compared with Days 3, 5 or 6 (Mann Whitney, p <0.05). Interestingly, expression of both ligands was significantly higher (p < 0.05) in the inter-site regions compared with the implantation sites at Day 6 of pregnancy. At Day 3 of pregnancy, CCL21 expression was limited to the glandular epithelium. Expression appeared in the antimesometrial uterine stroma at Day 4 of pregnancy. The distribution of CCL21 was similar between Days 4 and 5 of pregnancy but expression was less robust at Day 5. CCL21 expression was lost from the glands at Day 6 but was maintained in the antimesometrial stroma of the inter-site regions. CCL19 was expressed in the luminal and glandular epithelia of ovariectomized rat uteri. Progesterone pretreatment (2 mg daily, 3 days) stimulated expression of CCL19 in the periluminal stroma, which extended into the antimesometrial regions. Estradiol (0.2 µg) administration to progesterone pretreated rats, increased the expression of CCL19 but did not change its spatial distribution. Both ligands were detected in a rat uterine stromal cell line. CCL19 expression increased in response to estradiol (10 nM) at 6 h after hormone addition. Taken together, these results suggest that the maternal preparation for implantation includes a peak in the expression of CCL19 and CCL21 at Day 4 of pregnancy, one day prior to implantation in the rat. Unlike the constitutive expression of these ligands in primary and secondary lymphoid tissue, the spatial distribution and expression of CCL19 and CCL21 is regulated by female sex steroids in the uterus. The data support the concept that immune-mediated events, necessary for successful implantation and pregnancy, are induced in females prior to implantation. Funded by NIH.

1019: Progesterone attenuates brain inflammatory response and protects the brain from inflammation-induced immature myeloid cells increase

Pregnancy is a challenge for the maternal immune system since it has to defend against pathogens while tolerating alloantigenic fetal tissue. Little is known about maternal brain immune system during pregnancy. During pregnancy there is a marked increase in endogenous progesterone (PRO) and PRO supplementation is used for prevention of preterm birth. We sought to determine the maternal brain immune response to inflammation in pregnant (P) and non-pregnant (NP) mice and whether PRO supplementation attenuates this response. NP & P mice were pretreated with vaginal PRO or carrier from E13-E16. Lipopolysaccharide (LPS 30μg) or vehicle were administered intraperitoneally at E15 & E16. Serum was analyzed for IL-6 levels by ELISA. Brain was analyzed for nNOS, NFkB, IL-6 & TNF-a levels by western blot. Brain immature myeloid cells (IMC; CD45high, CD11b+, Gr1+) as % of CD45high hematopoietic brain cells and microglial (CD45low cells) activity as medial CD45 intensity was analyzed by FACS. LPS significantly increased brain nNOS, NFkB, IL-6 & TNF-a protein brain levels in NP & P mice. PRO pretreat ment significantly reduced nNOS and NFkB brain levels following LPS treatment in both groups. In NP mice, LPS treatment significant increased brain IMC population, and PRO pretreatment reduced the increase. In P mice, neither LPS nor LPS & PRO altered the brain IMC population. LPS led to a significant increase in microglial activity both groups. PRO pretreatment led to significant decrease in microglial activity in P mice. In NP mice, there was a trend but it did not reach statistical significance. PRO had no effect on serum IL-6 levels in both groups. PRO attenuates the inflammatory response in the brain in NP & P mice but had no effect on systemic (IL-6) inflammation. LPS increased brain IMC population in NP mice but not in P mice and PRO pretreatment reduced the IMC increase. These results suggest that endogenous PRO during pregnancy may protect the brain from LPS-induced central inflammation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Progesterone improves perinatal neuromotor outcomes in a mouse model of intrauterine inflammation via immunomodulation of the placenta

To assess the fetal neuroprotective potential of progesterone using a well-validated mouse model of lipopolysaccharide (LPS)-induced intrauterine inflammation (IUI). Embryonic day 17 pregnant mouse dams (n=69) were randomly allocated to receive 17-hydroxyprogesterone caproate (17-OHPC), micronized progesterone (MP), or vehicle 1hour prior to intrauterine injection of phosphate-buffered saline (PBS) or LPS. After 6hours, mice were killed for the collection of placentas and fetal brains, or pregnancy continued for the evaluation of preterm birth (PTB) and offspring neuromotor function. Placentas and fetal brains were analyzed by mini-mRNA array for 96 immune markers with individual confirmatory qPCR. Progesterone pre-treatment before LPS-induced IUI improved neuromotor tests in offspring at PND5 compared to no pre-treatment (P<.05). In placentas, 17-OHPC, but not MP, significantly reduced CXCL9 (P<.05) with a trend toward a lower level of CXCL10. In fetal brains, 17-OHPC significantly reduced CXCL9 compared to no pre-treatment (P<.05) and IL-1β compared to pre-treatment with MP (P<.01). Progesterone pre-treatment prior to LPS-induced IUI improved offspring neuromotor outcomes. 17-OHPC, but not MP, resulted in greater immunomodulation of T cell-mediated immunity in placenta and fetal brain, suggesting a possible mechanism for the observed neuroprotective effects.

Neurosteroidogenesis and progesterone anti-inflammatory/neuroprotective effects.

Progesterone shows anti-inflammatory and promyelinating effects in mice with experimental autoimmune encephalomyelitis (EAE), a commonly used model for multiple sclerosis (MS). Because neurosteroids have been implicated as protective factors for MS and EAE, we analysed the expression of neurosteroidogenic enzymes in the compromised spinal cord of EAE mice. EAE was induced in female C57Bl6 mice, which were then killed on day 16 after induction. Progesterone was given by pellet implantation 1week before EAE induction. Untreated EAE mice showed decreased mRNAs for the steroidogenic acute regulatory protein (Star), voltage-dependent anion channel (VDAC), cholesterol side-chain cleavage (P450scc), 5α-reductase, 3α-hydroxysteroid dehydrogenase (3α-HSOR) and aromatase, whereas changes of 3β-hydroxysteroid dehydrogenase (3β-HSD) were not significant. mRNA translocator protein (18kDa) (TSPO) was elevated, concomitantly with a reactive microgliosis. EAE mice also showed abnormal mitochondrial ultrastructure in axons and neuronal bodies, as well as reduced expression of fission and fusion protein mRNAs. Progesterone pretreatment before EAE induction increased Star, VDAC, P450scc, 5α-reductase type I, 3α-HSOR and aromatase mRNAs and did not modify 3β-HSD. TSPO mRNA was decreased, possibly as a result of reversal of microgliosis. Progesterone pretreatment also improved mitochondrial ultrastructure and increased fission/fusion protein mRNAs. These mitochondrial effects may be part of the progesterone recovery of neurosteroidogenesis. The enzymes 3β-HSD, 3α-HSOR and 5α-reductase are also responsible for the formation of androgens. Because MS patients and EAE rodents show changes of central androgen levels, it is likely that, together with progestins and oestrogens, neuroandrogens afford neuroprotection for EAE and MS. The data reviewed suggest that enhanced synthesis of neurosteroids contributes in an auto/paracrine manner to reinforce the neuroprotective and anti-inflammatory effects of exogenous progesterone given to EAE mice.

Modulation of HIV replication in monocyte derived macrophages (MDM) by steroid hormones.

Significant sex specific differences in the progression of HIV/AIDS have been reported. Several studies have implicated steroid hormones in regulating host factor expression and modulating HIV transmission and replication. However, the exact mechanism exerted by steroid hormones estrogen and progesterone in the regulation of HIV-1 replication is still unclear. Results from the current study indicated a dose dependent down regulation of HIV-1 replication in monocyte derived macrophages pre-treated with high concentrations of estrogen or progesterone. To elucidate the molecular mechanisms associated with the down regulation of HIV-1 replication by estrogen and progesterone we used PCR arrays to analyze the expression profile of host genes involved in antiviral responses. Several chemokines, cytokines, transcription factors, interferon stimulated genes and genes involved in type-1 interferon signaling were down regulated in cells infected with HIV-1 pre-treated with high concentrations of estrogen or progesterone compared to untreated HIV-1 infected cells or HIV-1 infected cells treated with low concentrations of estrogen or progesterone. The down regulation of CXCL9, CXCL10 and CXCL11 chemokines and IL-1β, IL-6 cytokines in response to high concentrations of estrogen and progesterone pre-treatment in HIV-1 infected cells was confirmed at the protein level by quantitating chemokine and cytokine concentrations in the culture supernatant. These results demonstrate that a potent anti-inflammatory response is mediated by pre-treatment with high concentrations of estrogen and progesterone. Thus, our study suggests a strong correlation between the down-modulation of anti-viral and pro-inflammatory responses mediated by estrogen and progesterone pre-treatment and the down regulation of HIV-1 replication. These findings may be relevant to clinical observations of sex specific differences in patient populations and point to the need for further investigation.

Luteolytic efficiency of reduced doses of cloprostenol in the ewe. Effect of progesterone concentrations at the time of treatment

Seventy six ewes were treated with 7.5, 12.5, 25 or 50μg of cloprostenol on day 6 or 9 post-estrus to compare the luteolytic efficiency of the PGF2α analogue at each stage and to evaluate if progesterone concentrations at the time of treatment affect such efficiency. Blood samples were obtained before cloprostenol administration and 12, 24, 48, and 72h thereafter. There was an effect of dose (p<0.05) but not of day post-estrus on the proportion of animals completing luteolysis. As the dose increased, the proportion of ewes completing luteolysis also increased. Also, as the dose increased from 7.5 to 25μg, more ewes showed a transient progesterone decline instead of an absence of response, indicating that in some ewes reduced doses initiated luteolysis but were not able to finish the process. Since the dose of 25μg resulted in close to 50% luteolytic efficacy, this group was used to study the effects of progesterone concentrations at the time of treatment on the response to cloprostenol. Pre-treatment progesterone concentrations were higher (p<0.01) in ewes experiencing luteolytic failure than in those that completed luteolysis. There was a negative correlation between initial progesterone concentrations and their reduction by 12h post-treatment. It is concluded that high progesterone concentrations are associated with a reduction in sensitivity to small doses of cloprostenol. Possible mechanisms and implications of this luteoprotective effect are discussed.

Oral contraceptive pill, progestogen or oestrogen pretreatment for ovarian stimulation protocols for women undergoing assisted reproductive techniques.

Oral contraceptive pill, progestogen or oestrogen pretreatment for ovarian stimulation protocols for women undergoing assisted reproductive techniques.

Micronized progesterone pretreatment affects the inflammatory response of human gestational tissues and the cervix to lipopolysaccharide stimulation

IntroductionVaginal administration of micronized progesterone (utrogestan capsule, UG) reduces the risk of preterm birth (PTB) in asymptomatic women with a sonographic short cervix at mid-trimester or with a prior history of spontaneous PTB; however, its exact mechanisms remain unclear. We hypothesized that UG limits the inflammatory processes within the gestational tissues and the cervix. MethodsFetal membranes and villous tissues were obtained from normal term placentas from women with cesarean delivery before labor onset. Ectocervical tissues were obtained from premenopausal women undergoing hysterectomies for uterine fibroids. Explant tissue cultures were pretreated with UG for 24 h and then exposed to UG with or without lipopolysaccharide (LPS) for 48 h. Tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1, interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-13, interferon-γ, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 levels in tissue homogenates and culture medium were measured by enzyme-linked immunosorbent assays. Real-time quantitative PCR, Western blot, and gelatine zymography were used to measure matrix metalloproteinase (MMP)-9 and MMP-2 mRNA, protein, and activity levels, respectively. ResultsUG pretreatment did not cause a significant change in basal levels or in LPS-induced production and secretion of cytokines, chemokines, and TIMPs in the three tissues. However, UG pretreatment significantly reduced MMP-9 and MMP-2 expression and activity in fetal membranes stimulated with LPS but not in villous or cervical tissues. DiscussionUG pretreatment significantly reduced MMP-9 and MMP-2 expression and activity in fetal membranes stimulated with LPS, suggesting a possible protective mechanism of micronized progesterone in preventing infection-associated PTB.

The effects of progesterone on the alpha2-adrenergic receptor subtypes in late-pregnant uterine contractions in vitro.

BackgroundThe adrenergic system and progesterone play major roles in the control of the uterine function. Our aims were to clarify the changes in function and expression of the α2-adrenergic receptor (AR) subtypes after progesterone pretreatment in late pregnancy.MethodsSprague Dawley rats from pregnancy day 15 were treated with progesterone for 7 days. The myometrial expressions of the α2-AR subtypes were determined by RT-PCR and Western blot analysis. In vitro contractions were stimulated with (−)-noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C) and spiroxatrine (α2A). The accumulation of myometrial cAMP was also measured. The activated G-protein level was investigated via GTPγS binding assays.ResultsProgesterone pretreatment decreased the contractile effect of (−)-noradrenaline through the α2-ARs. The most significant reduction was found through the α2B-ARs. The mRNA of all of the α2-AR subtypes was increased. Progesterone pretreatment increased the myometrial cAMP level in the presence of BRL 44408 (p < 0.001), spiroxatrine (p < 0.001) or the spiroxatrine + BRL 44408 combination (p < 0.05). Progesterone pretreatment increased the G-protein-activating effect of (−)-noradrenaline in the presence of the spiroxatrine + BRL 44408 combination.ConclusionsThe expression of the α2-AR subtypes is progesterone-sensitive. It decreases the contractile response of (−)-noradrenaline through the α2B-AR subtype, blocks the function of α2A-AR subtype and alters the G protein coupling of these receptors, promoting a Gs-dependent pathway. A combination of α2C-AR agonists and α2B-AR antagonists with progesterone could be considered for the treatment or prevention of preterm birth.

Effects of Lipopolysaccharide and Progesterone Exposures on Embryonic Cerebral Cortex Development in Mice.

Our objective was to determine if progesterone pretreatment could ameliorate the detrimental effects of lipopolysaccharide (LPS)-induced inflammation on cortical neurogenesis. Timed pregnant mouse dams (n = 8) were given intraperitoneal injections of progesterone (42 mg/kg) or vehicle on embryonic day 17.5. Two hours later, mice were given intraperitoneal LPS (140 μg/kg) or vehicle. Mice were sacrificed 16 hours later on embryonic day 18. Two-color immunofluorescence was performed with primary antibodies T-box transcription factor 2 (Tbr2), ionized calcium binding adapter molecule 1 (Iba1), cleaved caspase 3 (CC3), and 5-bromo-2'-deoxyuridine (BrdU). Cells were counted, and statistical analysis was determined using analysis of variance and Tukey-Kramer method. The Tbr2 intermediate neural progenitor cell density decreased after LPS exposure (P = .0022). Pre-exposure to progesterone statistically increased Tbr2 intermediate neural progenitors compared to LPS treatment alone and was similar to controls (P = .0022). After LPS exposure, microglia displayed an activated phenotype, and cell density was increased (P < .001). Cell death rates were low among study groups but was increased in LPS exposure groups compared to progesterone alone (P = .0015). Lipopolysaccharide-induced systemic inflammation reduces prenatal neurogenesis in mice. Pre-exposure with progesterone is associated with increased neurogenesis. Progesterone may protect the preterm brain from defects of neurogenesis induced by inflammation.

059 The effect of age, plane of nutrition, and progesterone pretreatment on lambing rate in nulliparous females bred during the breeding season

059 The effect of age, plane of nutrition, and progesterone pretreatment on lambing rate in nulliparous females bred during the breeding season

Progesterone pretreatment increases the stress response to social isolation in ewes

Progesterone pretreatment increases the stress response to social isolation in ewes

Evaluation of the Possible Role of Interlukin-1beta (IL-1β) in Diclofenac-Induced Hepatoxicity and the Exacerbative Effect of Progesterone Hormone in Mice

Diclofenac (DCLF) is one of the most widely prescribed non-steroidal anti-inflammatory drugs (NSAIDs). DCLF is associated with rare but serious idiosyncratic drug-induced liver injury (IDILI) in humans. It ranges from asymptomatic increase of plasma transaminases to life-threatening fulminant hepatitis with the need for liver transplantation. Mechanisms of DCLF-induced IDILI are not yet clarified, but immune responses are suspected to underlie them. In general, it is believed that women exhibit worse outcomes from DILI than men. The aim of this study is to investigate the involvement of immune reaction in mediating DCLF- hepatotoxicity and to evaluate the exacerbative effect of progesterone on DCLF-IDILI. The study was conducted on 100 albino mice divided into 5 main groups; each group was subdivided into a (female subgroup) and b (male subgroup) each of 10 mice; group I (negative control group), group II (normal saline group), group III (progesterone group), group IV (DCLF group), group V (progesterone and DCLF group). Results: DCLF administration, either alone or following progesterone, induced liver toxicity as manifested by abnormalities of hepatic profile (AST, ALT, T.Bil and GGT) together with increased plasma level of immune cytokine interlukin-1beta (IL-1β), with significant higher results encountered in females compared to males. Progesterone pre-treatment led to augmentation of the afro-mentioned indices in female mice only. These changes were substantiated with histopathological observations. From the previous results it can be concluded that an immune factor such as IL-1β is implicated in acute DCLF-induced hepatotoxicity and progesterone inflicts an exacerbative effect on this toxicity.

The effects of female sexual hormones on the expression and function of α1A- and α1D-adrenoceptor subtypes in the late-pregnant rat myometrium

The aim of the study was to investigate the roles of α1-adrenoceptor subtypes in the last-day pregnant rat uterus in vitro by the administration of subtype-specific antagonists (the α1A-adrenoceptor antagonist WB 4101 and the α1D-adrenoceptor antagonist BMY 7378) after 17β-estradiol or progesterone pretreatment.In isolated organ bath studies, contractions were elicited with (-)-noradrenaline (10−8–10−5M) in the presence of propranolol (10−5M) and yohimbine (10−6M) in order to avoid β-, and α2-adrenergic action. The myometrial expressions of the α1-adrenoceptor subtypes were determined by means of the real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques. The activated G protein levels were investigated through radiolabelled GTP binding assays.Both 17β-estradiol and progesterone pretreatment changed the myometrial contracting effect of (-)-noradrenaline. In the presence of WB 4101, progesterone pretreatment decreased the (-)-noradrenaline-induced myometrial contraction. In the presence of BMY 7378, both the 17β-estradiol and the progesterone pretreatment reduced the effect of (-)-noradrenaline. The mRNA and protein expressions of the α1A-adrenoceptors were decreased after 17β-estradiol pretreatment. (-)-Noradrenaline increased the [35S]GTPγS binding of the α1-adrenoceptors, which was most markedly elevated by progesterone. Pertussis toxin inhibited the [35S]GTPγS binding-stimulating effect of (-)-noradrenaline, indicating the role of Gi proteins in the signal mechanisms.17β-estradiol pretreatment blocks the expression of the α1A-adrenoceptors, whereas it does not influence the expression of the α1D-adrenoceptors. Progesterone pretreatment does not have any effect on the myometrial mRNA and protein expressions of the α1-adrenoceptors, but it alters the G protein coupling of these receptors, promoting a Gi-dependent pathway.

Progesterone pretreatment increases the stress response to social isolation in ewes.

In rodents, progesterone (P4) pretreatment increases anxiety and response to stressors. Social isolation is a stressor that generates physiological and behavioural stress responses in sheep. The aim of the study was to compare the stress response of anoestrous ewes previously treated or not with P4 to the social isolation test. Ten ewes received P4 treatment during 13 d (group P4-W) and another 10 remained untreated as controls (group Con). The ewes were individually isolated in a novel place during 10 min, 24 h after the end of P4 pretreatment and their behaviours were recorded. Cortisol and P4 concentrations as well as body surface temperature were recorded before and after the test. Ewes of the P4-W group presented higher cortisol levels 0, 10, 20 and 30 min after the social isolation and had greater area under the curve of cortisol compared to Con ewes (41,785%±4,156% vs. 25,682%±4,565% during 75 min). Progesterone and body surface temperature increased after social isolation, with no differences between P4-W and Con ewes. There were no differences in behavioural responses to social isolation. P4 pretreatment appears to augment the stress response to social isolation in anoestrous ewes.

Effect of stimulation with a gonadotropin mixture on reproductive outcome in nulliparous ewes bred during seasonal anestrus and early breeding season

To evaluate the effect of gonadotropic stimulation on the reproductive response of nulliparous acyclic female sheep during mid-anestrous and early breeding season, fall-born ewe lambs of mixed breeding (N=191; ∼7 months old; study 1) and yearlings (Dorset X Texel (DT), N=120, study 2) were assigned randomly during May to July to be either pre-treated with progesterone using CIDR-g (0.3g of progesterone) devices for 5 days or to also receive gonadotropins (240IU eCG and 120IU hCG, 3mL i.m. injection of P.G. 600®) at CIDR-g removal. Study 3 was conducted with nulliparous acyclic DT yearling ewes (N=104) in late August and was similar to study 2 except that a group of control ewes that were not treated was included. Gonadotropic stimulation increased estrous response (P=0.0002), pregnancy rate to the first service period (P=0.0007), proportion of ewes lambing (P=0.03) and the lambing rate (P=0.01) in fall-born ewe lambs (study 1), but did not modify reproductive outcome in yearling females (study 2) during mid-anestrus. During the transition into the breeding season (study 3), progesterone pre-treatment increased the pregnancy rate and the proportion of ewes lambing to first service after ram introduction (P=0.003 and P=0.02, respectively). However, gonadotropic stimulation of progesterone pre-treated yearlings did not modify reproductive performance variables. In conclusion, gonadotropic stimulation improved reproductive outcome in fall-born ewe lambs bred during the mid-anestrous period, but did not affect reproductive outcome in yearling females.

Anti-Inflammatory Effects of Progesterone in Lipopolysaccharide-Stimulated BV-2 Microglia

Female sex is associated with improved outcome in experimental brain injury models, such as traumatic brain injury, ischemic stroke, and intracerebral hemorrhage. This implies female gonadal steroids may be neuroprotective. A mechanism for this may involve modulation of post-injury neuroinflammation. As the resident immunomodulatory cells in central nervous system, microglia are activated during acute brain injury and produce inflammatory mediators which contribute to secondary injury including proinflammatory cytokines, and nitric oxide (NO) and prostaglandin E2 (PGE2), mediated by inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. We hypothesized that female gonadal steroids reduce microglia mediated neuroinflammation. In this study, the progesterone’s effects on tumor necrosis factor alpha (TNF-α), iNOS, and COX-2 expression were investigated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Further, investigation included nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways. LPS (30 ng/ml) upregulated TNF-α, iNOS, and COX-2 protein expression in BV-2 cells. Progesterone pretreatment attenuated LPS-stimulated TNF-α, iNOS, and COX-2 expression in a dose-dependent fashion. Progesterone suppressed LPS-induced NF-κB activation by decreasing inhibitory κBα and NF-κB p65 phosphorylation and p65 nuclear translocation. Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs. These progesterone effects were inhibited by its antagonist mifepristone. In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-κB and MAPK activation. This suggests progesterone may be used as a potential neurotherapeutic to treat inflammatory components of acute brain injury.