Progesterone Pellets Research Articles

Human vaginal microbiota colonization is regulated by female sex hormones in a mouse model.

Clinically, a Lactobacillus rich vaginal microbiota (VMB) is considered optimal for reproductive outcomes, while a VMB populated by anaerobes is associated with dysbiosis and the clinical condition bacterial vaginosis (BV), which is linked to increased susceptibility to sexually transmitted infections and adverse reproductive outcomes. Mouse models that mimic eubiotic and dysbiotic VMB are currently lacking but could play a critical role in improving protective interventions. In this study, probiotic, eubiotic, and dysbiotic models were developed in C57BL/6 mice, using probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, eubiotic Lactobacillus crispatus, or dysbiotic Gardnerella vaginalis strains. Endogenous sex hormones were manipulated by either ovariectomizing (OVX) mice or administering 17β-estradiol or progesterone pellets in OVX mice. Hormone-altered mice were inoculated with probiotic Lactobacillus species, L. crispatus, or G. vaginalis, and colonization was tracked using quantitative plating assays. Glycogen and MUC-1 levels in hormone-treated mice were determined with ELISA and MUC-1 staining. Following a single administration, L. rhamnosus and L. reuteri persisted in the mouse vaginal tract for up to eight days, L. crispatus persisted for up to three days, and G. vaginalis persisted for up to two days, as measured by quantitative plating assays and qPCR. Colonization of G. vaginalis was facilitated by the presence of mucin. The lack of endogenous hormones in OVX mice dramatically decreased VMB bacterial load compared to normal mice. None of the exogenous bacteria including Lactobacilli could colonize OVX mice for more than 24 hours. Treatment with 17β-estradiol but not progesterone restored the endogenous VMB and colonization with Lactobacilli and G. vaginalis. Interestingly, 17β-estradiol treated mice had significantly increased levels of glycogen compared to OVX and progesterone-treated mice. Based on the results, we have shown that estrogen played a significant role in the ability for human VMB species to colonize in our mouse models, potentially through a glycogen mediated mechanism. These results suggest there is a dynamic interaction between sex hormones and the VMB, which can affect bacterial diversity and the ability for a VMB to colonize.

Abstract 4840: Radiation therapy as a tool to sensitize HR+ breast cancer and mantle cell lymphoma to CDK4/6 inhibitors

Abstract Loss of cell cycle control is a common feature of most (if not all) human cancers. Thus, cell cycle genes that are frequently dysregulated in malignant cells, including CDK4 and CDK6, have attracted considerable attention as targets for the development of novel anticancer agents. This approach has culminated with the FDA approval of selective CDK4/6 inhibitors (e.g. palbociclib) for the treatment of hormone receptor (HR)+ breast cancer (BC). The same inhibitors are currently being tested in patients with a variety of solid and hematological tumors, including mantle cell lymphoma (MCL). However, not all BC and MCL patients achieve durable complete responses on CDK4/6 inhibitors, calling for the development of mechanism-based combination therapies. In this context, focal radiation therapy (RT) stands out as a promising therapeutic partner because both RT and CDK4/6 inhibitors have cytostatic/cytotoxic as well as immunostimulatory effects, but they operate with different mechanisms. We therefore decided to investigate the potential synergy/cooperation between RT and CDK4/6 inhibitors, with a particular focus on administration schedule, to inform the design of clinical trials testing the combination of these therapeutic agents in patients with HR+HER2-BC and MCL. We harnessed palbociclib-sensitive and -resistant MCL cells (MAVER-1 and MAVER-1R) and BC cells (MCF7 and MBA-MB-231) to investigate the ability of RT to synergize with palbociclib in vitro. In addition, we tested these combinations in vivo, in a unique immunocompetent mouse model of mammary carcinogenesis that recapitulates multiple immunobiological features of human HR+HER2-BC. In this model endogenous mammary carcinomas are driven by a slow-release progesterone pellet plus DMBA as an oral carcinogen. We first determined the lowest doses of RT and palbociclib that mediate robust short-term cytostatic/cytotoxic effects in BC and MCL cells. Optimized dose combinations and sequencing experiments pointed to RT followed by palbociclib as the approach with superior therapeutic potential, in vitro. This largely reflects the ability of palbociclib to induce G1 arrest of cells that escaped RT killing during G2/M arrest. In addition, low doses of RT were able to enhance palbociclib sensitivity in MAVER-1R cells. Both RT and palbociclib were active in the endogenous mammary carcinomas model, but failed to mediate disease eradication. In vivo experiments showed that RT preceding CDK4/6 inhibition achieved superior disease control, compared to other sequencing regimens. In conclusion, our results identified an optimal therapeutic sequence to combine RT and CDK4/6 inhibition in preclinical models of BC and MCL, providing a rational for combining stereotactic body RT and palbociclib in a clinical trial for patients with newly diagnosed HR+ oligo-metastatic BC (up to five metastatic lesions). Future directions include the evaluation of the immunostimulatory effects of RT plus palbociclib in the same models of BC and MCL. Supported by SU2C-Ziskin's Award # ZP-6177. Citation Format: Giulia Petroni, Maurizio Di Liberto, Aitziber Buqué Martinez, Selina Chen-Kiang, Lorenzo Galluzzi, Silvia C. Formenti. Radiation therapy as a tool to sensitize HR+ breast cancer and mantle cell lymphoma to CDK4/6 inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4840.

Optimization of Endometrial Decidualization in the Menstruating Mouse Model for Preclinical Endometriosis Research

To induce endometrial decidualization in rodents, an intrauterine oil stimulus can be delivered via the nontraumatic vagina or via the traumatic laparotomy. However, there is considerable variation in amount of decidualization using these inducing methods. Therefore, we studied which oil delivery route could achieve the highest rate of endometrial decidualization along the full length of both uterine horns. To induce decidualization, ovariectomized C57Bl/6J mice were injected with estrogen (100 ng/day; 3 days). A progesterone pellet (5 mg) was implanted subcutaneously, followed by estrogen injections (5 ng/day; 3 days). Oil (20 µL/horn) was injected in the uterus via laparotomy, laparoscopy, or vagina. Four days later, the pellet was removed, followed by hysterectomy after 4 to 6 hours. Endometrial decidualization was evaluated macroscopically and microscopically using hematoxylin and eosin and desmin staining. Furthermore, uterine weight and hormone levels were measured. The proportion of animals with macroscopic bicornuate decidualization was higher after laparoscopic (83%) and laparotomic (89%) injection than after sham injection (11%). Furthermore, macroscopic bicornuate decidualization was significantly higher after laparotomic injection (89%) compared to the vaginal injection (38%). Uterine weight and endometrial surface area were significantly higher in both laparotomy and laparoscopy groups compared to the sham group, while the relative desmin-positive endometrial surface area was only significantly different between the laparotomy and the sham animals. Methods using laparoscopic and laparotomic intrauterine oil injection resulted in a higher amount of decidualized endometrium compared to sham injection, although further optimization is needed to reach full bicornuate decidualization.

Of Mice and Women: A Laparoscopic Mouse Model for Endometriosis

Study ObjectiveTo demonstrate how a novel laparoscopic approach allows the development of a mouse model for endometriosis after seeding menstrual endometrium from donor mice into the abdominal cavity of syngeneic recipient mice. DesignA step-by-step video description of the techniques used to adapt the estrous cycle of mice towards a menstrual cycle and to subsequently induce endometriosis via laparoscopic seeding of menstrual endometrium. SettingUniversity research institute. EthicsAll animal experiments were ethically approved by KU Leuven, Belgium (ethical approval number: P031/2013). Interventions, Measurements, and Main ResultsOophorectomized female C57BL/6JRj mice received a series of estrogen injections. Next, a progesterone pellet was administered, together with a second series of estrogen injections. In addition, decidualization of the endometrium was induced with an intrauterine sesame oil stimulus. Four days later the progesterone pellet was removed and menstruation started [1]. Five hours after the progesterone pellet was removal the uterus was harvested, and the menstrual endometrium was dissected and seeded into the abdominal cavity of syngeneic recipient mice to induce endometriosis [2] using a laparoscopic approach [3]. Uterus and lesions were removed from the recipient mice 1 week after induction, and tissues were immunohistochemically stained for H&E, vimentin, and cytokeratin. ConclusionIn this video we show a novel methodology to induce endometriosis in mice using laparoscopic inoculation of syngeneic menstrual endometrium, mimicking Sampson's theory of retrograde menstruation [4]. Compared with currently available rodent models, our model offers a less invasive and more physiologic way for fundamental and preclinical endometriosis research, with a high endometriosis incidence and lesion take rate.

Protective effects of the neurosteroid allopregnanolone in a mouse model of spontaneous motoneuron degeneration

Amyotrophic lateral sclerosis (ALS) is a devastating disorder characterized by progressive death of motoneurons. The Wobbler (WR) mouse is a preclinical model sharing neuropathological similarities with human ALS. We have shown that progesterone (PROG) prevents the progression of motoneuron degeneration. We now studied if allopregnanolone (ALLO), a reduced metabolite of PROG endowed with gabaergic activity, also prevents WR neuropathology. Sixty-day old WRs remained untreated or received two steroid treatment regimens in order to evaluate the response of several parameters during early or prolonged steroid administration. ALLO was administered s.c. daily for 5days (4mg/kg) or every other day for 32days (3, 3mg/kg), while another group of WRs received a 20mg PROG pellet s.c. for 18 or 60days. ALLO administration to WRs increased ALLO serum levels without changing PROG and 5 alpha dihydroprogesterone (5α-DHP), whereas PROG treatment increased PROG, 5α-DHP and ALLO. Untreated WRs showed higher basal levels of serum 5α-DHP than controls. In the cervical spinal cord we studied markers of oxidative stress or associated to trophic responses. These included nitric oxide synthase (NOS) activity, motoneuron vacuolation, MnSOD immunoreactivity (IR), brain derived neurotrophic factor (BDNF) and TrkB mRNAs, p75 neurotrophin receptor (p75NTR) and, cell survival or death signals such as pAKT and the stress activated kinase JNK. Untreated WRs showed a reduction of MnSOD-IR and BDNF/TrkB mRNAs, associated to high p75NTR in motoneurons, neuronal and glial NOS hyperactivity and neuronal vacuolation. Also, low pAKT, mainly in young WRs, and a high pJNK in the old stage characterized WŔs spinal cord. Except for MnSOD and BDNF, these alterations were prevented by an acute ALLO treatment, while short-term PROG elevated MnSOD. Moreover, after chronic administration both steroids enhanced MnSOD-IR and BDNF mRNA, while attenuated pJNK and NOS in glial cells. Long-term PROG also increased pAKT and reduced neuronal NOS, parameters not modulated by chronic ALLO. Clinically, both steroids improved muscle performance. Thus, ALLO was able to reduce neuropathology in this model. Since high oxidative stress activates p75NTR and pJNK in neurodegeneration, steroid reduction of these molecules may provide adequate neuroprotection. These data yield the first evidence that ALLO, a gabaergic neuroactive steroid, brings neuroprotection in a model of motoneuron degeneration.

Physical activity induced protection against breast cancer risk associated with delayed parity

Epidemiological evidence indicates that physical activity between menarche and first pregnancy is associated with a lower risk of breast cancer among women with at least 20years between these reproductive events. The mechanism by which physical activity during this interval confers protection is unknown. This study used a novel animal model to assess potentially protective effects of physical activity on tumor development in delayed parity. Thirty-six female Sprague Dawley rats received an i.p. injection of 50mg/kg N-methyl-N-nitrosourea (MNU) at 5weeks of age. Estrogen and progesterone pellets were implanted subcutaneously 1week (early parity, EP, n=8) or 4weeks (delayed parity, DP, n=11) following MNU injection. An additional group of DP rats were progressively exercise trained (Ex+DP, n=9) on a treadmill following MNU injection for 7weeks (up to 20m/min at 15% incline for 30min). We observed the greatest tumor latency and smallest tumor burden in Ex+DP animals. Ductal hyperplasia and inflammation of non-tumor bearing mammary glands were only found in DP, and we detected a significant increase in collagen for DP and Ex+DP compared to EP. Exercise induced differential gene expression of cyclin-dependent kinase-inhibitor 1C (Cdkn1c) and urokinase-plasminogen activator (Plau) in mammary tissue of Ex+DP animals compared to DP alone. While there are delayed parity-induced changes in mammary gland collagen and gene expression levels, Ex+DP animals had longer tumor latency, smaller tumor burden, and glandular tissue resistant to ductal hyperplasia. Exercise may induce protection through beneficial regulation of gene expression profiles.

Abstract B29: Effects of prenatal exposure to cancer treatment: The development of a placental murine xenograft model

Abstract The aim of this study was to develop a suitable murine xenograft model to examine the possible effect of drugs or chemotherapeutic agents on the placental tissue. Methods: The test population consisted of 4 examination groups of six immunodefficient nude mice. After the mice were ovariectomised and hormonal stimulated (17b estradiol and progesterone pellets), placental tissue derived from first and third trimester pregnancies was engrafted subcutaneously bilateral in the interscapular fat pad. hCG (human chorionic gonadotrophin) secretion was measured in serum and urine by an enzyme-linked immunosorbent assay (ELISA). To examine the viability and preservation of the placental tissue, factors for proliferation (hCG, Ki67), apoptosis (cleaved caspase-3) and murine angiogenesis (CD31) were evaluated by immunohistochemistry (IHC). The expression level of genes relevant for the angiogenesis (IGF-1, PGF, IGF-2), inflammation (eNOS) and proliferation (Flk-1, Flt-1) was compared by using reverse transcription- quantitative real-time polymerase chain reaction (RT-qPCR). Results: Comparing the placental tissue before and after engraftment, we found preserved structure and histological features. Also, the PCR analysis showed preserved genetic characteristics of the primary engrafted placental tissue. hCG secretion, evaluated by ELISA in the urine and blood, was present for 3 weeks. Conclusion: We created a murine xenograft model with proven stability and preserved structure of the engrafted placental tissue for 3 weeks. This established murine xenograft model will be useful to examine the effect of cancer treatment; moreover, it could serve as a model for other fetal toxicity studies. Citation Format: Magali Verheecke, Wout Devlies, Els Hermans, Frédéric Amant. Effects of prenatal exposure to cancer treatment: The development of a placental murine xenograft model. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B29.

Induction of post-menstrual regeneration by ovarian steroid withdrawal in the functionalis of xenografted human endometrium.

Does the endometrial functionalis have the potential to undergo self-renewal after menstruation and how is this process controlled by ovarian steroids? Endometrial xenografts subjected to withdrawal of estradiol and progesterone shrink but also show signs of proliferation and tissue repair; new estradiol supply prevents atrophy but is not sufficient to increase graft volume. Menstruation, i.e. cyclic proteolysis of the extracellular matrix of endometrial functionalis, is induced by a fall in estrogen and progesterone concentration and is followed by tissue regeneration. However, there is debate about whether regenerating cells must originate from the basalis or from stem cells and whether new estrogen supply is required for the early repair concomitant with menstruation. Fragments from human endometrial functionalis (from 24 hysterectomy specimens) were xenografted in ovariectomized SCID mice and submitted to a 4-day estradiol and progesterone withdrawal (to mimic menstruation) followed by re-exposure to estradiol (to mimic the proliferative phase). We measured signs of proliferation and changes in graft volume. Endometrium was collected from spontaneously cycling women. Cell proliferation was examined by immunolabeling Ki-67, cyclin D1 and phosphorylated-histone H3. Xenograft volume was measured by magnetic resonance imaging. Xenograft histomorphometry was performed to determine how the different tissue compartments contributed to volume change. Hormone withdrawal induced a rapid decrease in graft volume mainly attributable to stroma condensation and breakdown, concomitant with an increase of proliferation markers. Reinsertion of estradiol pellets after induced menstruation blocked volume decrease and stimulated epithelial and stromal growth, but, surprisingly, did not induce graft enlargement. Reinsertion of both estradiol and progesterone pellets blocked apoptosis. Mechanisms of endometrial remodeling are different in women and mice and the contribution of circulating inflammatory cells in both species remains to be clarified. Moreover, during human menstruation, endometrial fragments resulting from tissue proteolysis can be expelled by the menstrual flow, unlike in this model. Menstruation is a multifocal event within the functionalis. This is the first evidence that endometrial fragments that are not shed after menstrual tissue breakdown can support endometrial regeneration. Endometriosis is commonly thought to result from the retrograde migration of menstrual fragments of the degraded functionalis into the peritoneal cavity. Our study supports their potential to regenerate as ectopic endometrium. This work was supported by the Fonds de la Recherche Scientifique Médicale, Concerted Research Actions, Communauté Française de Belgique, Région wallonne, Région bruxelloise and Loterie nationale. P.H. and B.F.J. are research associates of the Belgian Fonds de la Recherche Scientifique (F.R.S.-F.N.R.S.). E.M. is Associate Editor at Human Reproduction. There is no conflict of interest to declare.

Resveratrol and Endometrium: A Closer Look at an Active Ingredient of Red Wine Using In Vivo and In Vitro Models

Resveratrol is a natural phytoestrogen with antiproliferative properties present in red wine, grapes, and berries. Published reports on the effects of resveratrol in human endometrial function are limited. The objective of this study was to investigate the expression of estrogen receptor α (ESR1), Ki-67 (a proliferative marker), aryl hydrocarbon receptor (AhR), and members of the cytochrome P450 superfamily of enzymes (CYP1A1 and CYP1B1) in an in vitro and vivo assay. Alkaline phosphatase assay of estrogenicity was used to compare estrogen activity of different concentrations of resveratrol to estradiol (E2) and diethylstilbestrol (DES), using Ishikawa cell culture. Immunohistochemical expression of ESR1 and Ki67, and reverse transcriptase polymerase chain reaction of AhR, CYP1A1, and CYP1B1 were analyzed from xenograft implants of human endometrial tissue in ovariectomized immunodeficient RAG-2-γ(c) mice, after 30 days of treatment with subcutaneous pellets of E2, E2 plus progesterone (P4), or E2 plus resveratrol (6, 30, or 60 mg) for 30 days. Compared to E2, resveratrol acted as an agonist and antagonist of estrogen in low and high concentrations, respectively, when combined with E2. Xenografts of human endometrial tissues in RAG-2 mice exhibited reduced expression of ESR1 and proliferative activity (Ki67) with 60 mg of resveratrol. This study suggests that resveratrol, at high doses, has the potential benefit to reduce proliferation of human endometrium through ESR1.

Calcineurin activity is required for cardiac remodelling in pregnancy.

Calcium fluctuations and cardiac hypertrophy occur during pregnancy, but the role of the well-studied calcium-activated phosphatase, calcineurin, has not been studied in this setting. The purpose of this study was to determine whether calcineurin signalling is required for cardiac remodelling during pregnancy in mice. We first examined calcineurin expression in the heart of mice during pregnancy. We found both calcineurin levels and activity were significantly increased in early-pregnancy and decreased in late-pregnancy. Since progesterone levels start to rise in early-pregnancy, we investigated whether progesterone alone was sufficient to modulate calcineurin levels in vivo. After implantation of progesterone pellets in non-pregnant female mice, cardiac mass increased, whereas cardiac function was maintained. In addition, calcineurin levels increased, which is also consistent with early-pregnancy. To determine whether these effects were occurring in the cardiac myocytes, we treated neonatal rat ventricular myocytes (NRVMs) with pregnancy-associated sex hormones. We found that progesterone treatment, but not oestradiol, increased calcineurin levels. To obtain a functional read-out of increased calcineurin activity, we measured the activity of the transcription factor NFAT, a downstream target of calcineurin. Progesterone treatment significantly increased NFAT activity in NRVMs, and this was blocked by the calcineurin inhibitor cyclosporine A (CsA), showing that the progesterone-mediated increase in NFAT activity requires calcineurin activity. Importantly, CsA treatment of mice completely blocked pregnancy-induced cardiac hypertrophy. Our results show that calcineurin is required for pregnancy-induced cardiac hypertrophy, and that calcineurin activity in early-pregnancy is due at least in part to increased progesterone.

Progesterone effects on neuronal brain-derived neurotrophic factor and glial cells during progression of Wobbler mouse neurodegeneration

Previous results have shown a depletion of brain-derived neurotrophic factor (BDNF) mRNA in the degenerating motoneurons from clinically afflicted Wobbler mice, whereas progesterone treatment reverts this depletion. We now compared progesterone regulation of BDNF in motoneurons and oligodendrocytes of Wobbler mice at the progressive (EP, 1–3 months), symptomatic (SYM, 5–8 months old), and late stages (LS, 12–13 months). As controls we used NFR/NFR mice. Controls and Wobbler mice of different ages remained untreated or received a 20 mg progesterone pellet during 18 days. BDNF mRNA was determined in the ventral, intermediolateral, and dorsal gray matter by film autoradiography and in motoneurons using in situ hybridization. A depletion of BDNF mRNA already occurred at the EP stage of Wobblers, but progesterone was inactive at this period. In contrast, progesterone upregulated the low levels of BDNF mRNA in SYM Wobblers in the three gray matter regions analyzed. Progesterone also increased BDNF mRNA in LS Wobblers, according to grain counting procedures. BDNF protein analyzed by enzyme-linked immunosorbent assay (ELISA) in ventral horns or immunostaining of motoneurons was normal in steroid-naive SYM Wobblers. BDNF protein was decreased by progesterone, suggesting increased anterograde transport and/or release of neuronal BDNF. Wobbler mice also showed depletion of CC1-immunopositive oligodendrocytes, whereas progesterone treatment enhanced the density of BDNF+ and CC1+ oligodendrocytes in EP, SYM, and LS Wobblers. Our results suggest that BDNF could be involved in progesterone effects on motoneurons at the SYM and LS periods, whereas effects on oligodendrocytes occurred at all stages of the Wobbler disease. These steroid actions may be important to arrest the ongoing neurodegeneration.

Environmental Tobacco Smoke and Progesterone Alter Lung Inflammation and Mucous Metaplasia in a Mouse Model of Allergic Airway Disease

The prevalence and severity of asthma is sexually dimorphic. Adult women have a higher incidence of asthma than men. This suggests that this disease may have a hormonal component. Progesterone has been shown to elicit an immune response similar to that seen in allergic asthma and previous studies have shown that progesterone increases total IgE levels in the peripheral blood. In the current study, we examine the effect of environmental tobacco smoke (ETS) and progesterone on hallmarks of asthma pathology in lung tissue with the goal of defining whether progesterone can also exacerbate two key features of airway remodeling: accumulation of eosinophils and increased mucous. We used a mouse model of allergic asthma that includes house dust mite allergen (HDMA). Adult female BALB/c mice were ovariectomized and implanted with time-release progesterone pellets. Mice were housed in filtered air or ETS for 6 weeks (1 mg/m³ total suspended particulate) and exposed to HDMA by inhalation. Progesterone alone did not increase mucous cell mass or the abundance of eosinophils but ETS coupled with progesterone exposure resulted in a significant increase in mucous cell metaplasia and increased accumulation of eosinophils in the asthma model. Levels of cytokines in the bronchoalveolar lavage fluid, measured using a multiplex cytokine assay, revealed elevated levels of both interleukin (IL)-5 and IL-12(p40) in HDMA-exposed animals. The addition of progesterone further exacerbated this response. We conclude that progesterone, in the absence of estrogen, exacerbates airway inflammation and airway remodeling induced by the toxicant ETS.

Stage dependent effects of progesterone on motoneurons and glial cells of wobbler mouse spinal cord degeneration.

In the Wobbler mouse, a mutation in the Vps54 gene is accompanied by motoneuron degeneration and astrogliosis in the cervical spinal cord. Previous work has shown that these abnormalities are greatly attenuated by progesterone treatment of clinically afflicted Wobblers. However, whether progesterone is effective at all disease stages has not yet been tested. The present work used genotyped (wr/wr) Wobbler mice at three periods of the disease: early progressive (1-2 months), established (5-8 months) or late stages (12 months) and age-matched wildtype controls (NFR/NFR), half of which were implanted with a progesterone pellet (20 mg) for 18 days. In untreated Wobblers, degenerating vacuolated motoneurons were initially abundant, experienced a slight reduction at the established stage and dramatically diminished during the late period. In motoneurons, the cholinergic marker choline acetyltransferase (ChAT) was reduced at all stages of the Wobbler disease, whereas hyperexpression of the growth-associated protein (GAP43) mRNA preferentially occurred at the early progressive and established stages. Progesterone therapy significantly reduced motoneuron vacuolation, enhanced ChAT immunoreactive perikarya and reduced the hyperexpression of GAP43 during the early progressive and established stages. At all stage periods, untreated Wobblers showed high density of glial fibrillary acidic protein (GFAP)+ astrocytes and decreased number of glutamine synthase (GS) immunostained cells. Progesterone treatment down-regulated GFAP+ astrocytes and up-regulated GS+ cell number. These data reinforced the usefulness of progesterone to improve motoneuron and glial cell abnormalities of Wobbler mice and further showed that therapeutic benefit seems more effective at the early progressive and established periods, rather than on advance stages of spinal cord neurodegeneration.

Pharmacotherapy with 17β-estradiol and progesterone prevents development of mouse experimental autoimmune encephalomyelitis.

Pregnant women with multiple sclerosis (MS) show disease remission in the third trimester concomitant with high circulating levels of sex steroids. Rodent experimental autoimmune encephalomyelitis (EAE) is an accepted model for MS. Previous studies have shown that monotherapy with estrogens or progesterone exert beneficial effects on EAE. The aim of the present study was to determine if estrogen and progesterone cotherapy of C57BL/6 female mice provided substantial protection from EAE. A group of mice received single pellets of progesterone (100mg) and 17 β-estradiol (2.5mg) subcutaneously 1week before EAE induction, whereas another group were untreated before EAE induction. On day 16 we compared the two EAE groups and control mice in terms of clinical scores, spinal cord demyelination, expression of myelin basic protein and proteolipid protein, macrophage cell infiltration, neuronal expression of brain-derived neurotrophic factor mRNA and protein, and the number of glial fribrillary acidic protein (GFAP)-immunopositive astrocytes. Clinical signs of EAE were substantially attenuated by estrogen and progesterone treatment. Steroid cotherapy prevented spinal cord demyelination, infiltration of inflammatory cells and GFAP+ astrogliocytes to a great extent. In motoneurons, expression of BDNF mRNA and protein was highly stimulated, indicating concomitant beneficial effects of the steroid on neuronal and glial cells. Cotherapy with estrogen and progesterone inhibits the development of major neurochemical abnormalities and clinical signs of EAE. We suggest that a combination of neuroprotective, promyelinating and immuno-suppressive mechanisms are involved in these beneficial effects.

Interrelationship between prolactin and progesterone in normal mammary gland growth in SHN virgin mice.

In order to clarify the interrelationship between prolactin and progesterone on normal mammary gland growth, SHN strain of virgin mice were given the pituitary grafting under the kidney capsule, the subcutaneous implantation of progesterone pellet or the bilateral ovariectomy singly or in combination at 40 days of age. All mice were killed 60 days after treatments and mammary end-bud formation, mammary DNA content and plasma prolactin level were examined. The single pituitary grafting elevated plasma prolactin levels in both the intact and the ovariectomized animals, however, mammary gland growth was enhanced only in the former. Progesterone pellet implantation also stimulated mammary gland growth in the intact mice, while prolactin changed little. Progesterone effect was nullified by ovariectomy associated with the decline in plasma prolactin level. The simultaneous single pituitary grafting and progesterone pellet implantation in combination or the grafting with three pituitaries restored mammary gland regression by ovariectomy. All findings have demonstrated that the manifestation of progesterone effect on mammary glands is dependent upon prolactin and that the role of prolactin is both direct on the glands and indirect through its luteotropic effect - through its stimulation of ovarian progesterone secretion.

Progesterone (P4) modulates cytokines expression in temporomandibular joint (TMJ)

OBJECTIVESEvidence suggests that symptoms of TMJD vary over menstrual cycle indicating that changes in steroid sex hormone modulate TMJ pain. The objective of the study is to determine modulatory role of P4 on cytokine/chemokine expression in TMJ.METHODSSprague Dawley ovariectomized female rats were divided in 2 groups with 1 group receiving progesterone pellets & subcutaneous P4 injections to recapitulate physiological plasma levels of progesterone. Groups were subdivided further & were given bilateral TMJ injections of Complete Freund's adjuvant (CFA) or saline within the superior joint space of TMJ during diestrous‐2 and proestrous phase. So 4 groups include P4/diestrous‐2/saline, P4/diestrous‐2/CFA, P4/proestrous/saline & P4/proestrous/CFA.Rats were sacrificed 24 hrs later. The cytokine/chemokine expression was analyzed in retrodiscal tissue, trigeminal ganglia & subnucleus caudalis using arrays.RESULTSThe transcript expression of cytokines IL‐6 and IL‐1<â > decreased by 50 & 82 fold &chemokines CCL3, CCL20, CXCL1, CXCL2 and CXCL3 decreased by 103, 91, 86, 118 and 155 fold, comparing the P4/diestrous‐2/saline group to P4/proestrous/saline group suggesting P4 modulates expression of these molecules in TMJ.CONCLUSIONThe data suggest that cyclical plasma levels of P4 in females can modulate cytokines/chemokines expression in TMJ. Supported by NIDCR DE016059‐01 (LLB) & DE15372 (PRK).

Progesterone and environmental tobacco smoke act synergistically to exacerbate the development of allergic asthma in a mouse model

Asthma affects males and females differently. Females have a higher incidence than males after the onset of puberty. This suggests a hormonal component to the development of the disease. Progesterone, a female hormone, has previously been shown to illicit a T-helper type 2 (TH2) immune response similar to that seen in allergic asthma. Previous studies performed by our laboratory have shown that exposure to environmental tobacco smoke (ETS) enhances the immune response to allergens. To determine if the combination of exposure to ETS and progesterone would further exacerbate the immune response in a mouse model of allergic asthma. Female mice were ovariectomized and then implanted with time-release progesterone pellets. Mice were housed in either filtered air (FA) or ETS chambers and half were exposed to aerosolized house dust mite allergen (HDMA). Bronchoalveolar lavage was performed for cell differentials; lung and spleen cells were harvested to compare IL-4 and IFN-gamma production by ELISPOT. Progesterone pellet implantation resulted in increased serum progesterone levels (28.3+/-8.43 vs. 13.5+/-7.22 ng/mL in placebo-treated mice, P<0.0001). Serum total IgE levels were significantly greater in progesterone vs. non-progesterone treated animals that were also exposed to HDMA. ETS exposure enhanced total IgE levels as well. Lung homogenate cells from HDMA/progesterone-treated animals stimulated with Concavalin A produced significantly more IL-4 compared with HDMA/placebo-treated animals (200+/-17.6 vs. 146+/-17.5 spots/well, P<0.01 in ETS exposed animals and 221+/-28.9 vs. 167+/-23.4 spots/well, P<0.01 in animals housed in FA). HDMA/ETS-treated animals had higher eosinophilia in lavage than all other groups. Increased serum progesterone levels exacerbate the allergic asthmatic phenotype in a mouse model. These effects are further exacerbated by the addition of environmental tobacco smoke. Progesterone provides a major contribution to the gender differences seen in the development and elicitation of the asthmatic response.

C57BL/6 Mice Infected with Neospora caninum During Administration of Progesterone Show Bias Toward Type 2 Immune Response

To clarify the role of progesterone in the development of immune responses during pregnancy against Neospora caninum infection, C57BL/6 mice were given a progesterone pellet, and measured on Interferon-gamma and interleukin-4 production following the infection. IFN-gamma production in the prescribed group was significantly lower than that in the intact group on day 40 post administration. IL-4 producing cell population in the prescribed group was larger than that in the intact group. These results suggest that progesterone may alter the balance of cytokine production, and that the bias toward type 2 immune response may remain for a certain period after the infection.

Female Sex Hormones and Rat Dural Vasodilatation to CGRP, Periarterial Electrical Stimulation and Capsaicin

The prevalence of migraine is 2 to 3-fold higher in females than in males, and it is intricately related to the levels of female sex hormones. These hormones may regulate the synthesis and receptor expression of calcitonin gene-related peptide (CGRP), which mediates neurogenic dural vasodilatation and is implicated in migraine pathogenesis. To investigate the effects of the female sex steroids, 17beta-estradiol and progesterone, separately and in combination, on dural vasodilatation induced by alphaCGRP, periarterial electrical stimulation and capsaicin in ovariectomized rats, using intravital microscopy. Sprague-Dawley rats were ovariectomized and, 7 days later, subcutaneously implanted with 21-day release pellets of 17beta-estradiol, progesterone, their combination or placebo. On day 19 to 21, the animals were anesthetized, overlying bone thinned to visualize the middle meningeal artery and vasodilator responses to alphaCGRP (10 to 3000 ng kg(-1)), periarterial electrical stimulation (25 to 125 microA) and capsaicin (0.3 to 18 microg kg(-1)) elicited. There were no significant differences in the vasodilator potency or efficacy of alphaCGRP or capsaicin in the different groups studied. In contrast, the vasodilator response to electrical stimulation was significantly higher in rats treated with 17beta-estradiol (Emax:157 +/- 19%) as compared to those observed after placebo treatment (Emax:93 +/- 11%). Our results show that, in contrast to CGRP- or capsaicin-induced dural vasodilatation, 17beta-estradiol enhanced neurogenic vasodilatation, suggesting increased CGRP release from perivascular nerves. This may be one of the mechanisms through which 17beta-estradiol exacerbates migraine in women.

Hormonal stimulation increases the recruitment of bone marrow-derived myoepithelial cells and periductal fibroblasts into the mammary gland

Recent reports have revealed that bone marrow (BM)-derived cells can be constituents in a number of organs, especially in remodeling tissue. Using bone marrow transplantation (BMT) technique, we found that BM can serve as a source of both myoepithelial cells and periductal fibroblasts in the mammary gland. The numbers of BM-derived myoepithelial cell were 4.8-fold, and those of periductal fibroblast were 2.4-fold higher in the mice when BMT which was performed at the pubertal stage, as compared with BMT was performed at the postpubertal stage. Treatment with estrogen + progesterone pellet increased numbers of BM-derived myoepithelial cells and periductal fibroblasts, to levels 4.5- and 2.6-fold higher than in placebo mice, respectively. In situ hybridization revealed BM-derived periductal fibroblasts expressed insulin-like growth factor I mRNAs that are known to regulate mammary gland. These results suggest that drastic structural change that is induced by hormonal stimulation increased the recruitment of BM-derived myoepithelial cells and periductal fibroblasts to the mammary gland context.