Progesterone increases metabolism via the pentose phosphate pathway in bovine uterine epithelial cells.

BACKGROUND: During early pregnancy, glucose is essential for the uterine epithelium and the developing embryo. In cows, progesterone increases the secretion of glucose into the uterine lumen. The uterine epithelium can convert glucose to fructose, but other fates of glucose in the uterine epithelium have been sparsely investigated. Therefore, our objective was to investigate how progesterone influences glucose metabolism in immortalized bovine uterine epithelial (BUTE) cells. METHODS: BUTE cells were grown to 80% confluence and treated with vehicle (DMSO) or 10 µM progesterone for 24 h. Cells were collected and analyzed. Immunohistochemistry was performed on endometrial samples collected from the bovine endometrium on days 1 and 11 of the reproductive cycle. RESULTS: Progesterone treatment increased glucose consumption of BUTE cells. RNAseq identified 3,072 genes regulated by progesterone. KEGG analysis indicated that progesterone altered genes associated with metabolic pathways and glutathione metabolism. Manually examining genes unique to specific glucose metabolic pathways identified an increase in the rate-limiting enzyme in the pentose phosphate pathway-glucose-6-phosphate dehydrogenase. Functionally, a major product of the pentose phosphate pathway is NADPH, and progesterone treatment increased NADPH levels in BUTE cells. In cows, immunohistochemistry confirmed that glucose-6-phosphate dehydrogenase levels were higher in the uterine epithelium in the luteal phase when progesterone concentrations are high. CONCLUSIONS: Progesterone increased glucose-6-phosphate dehydrogenase expression and metabolism via the pentose phosphate pathway in the bovine uterine epithelium. This metabolism could provide substrates for cell proliferation, molecules to be secreted into the uterine lumen, or maintain reduction/oxidation balance in the uterine epithelium.

Effect of the Application of PGF2α Associated With Ovulation Induction in a Fixed-Time Superovulation Programme for Precocious Nellore Heifers.

This study evaluated the effect of prostaglandin F2α (PGF2α) associated with gonadotropin-releasing hormone (GnRH) for ovulation induction in precocious indicus heifers submitted to a fixed-time superovulation (SOV) programme. Precocious Nellore heifers (n = 35), aged 13 months, were subjected to the SOV protocol. On day 0 (D0), all animals received intravaginal insertion of a progesterone (P4) device along with intramuscular administration of 2 mg of oestradiol benzoate, plus 200 IU of follicle-stimulating hormone in decreasing doses, with 12-h intervals between D4 and D7, in addition to 150 µg of D-cloprostenol on D6 and device removal on D7. On D8, the donors received 10.5 µg of buserelin acetate and the treatment group received 300 µg of D-cloprostenol/PGF2α. Artificial insemination was performed 12 h and 24 h after GnRH administration using frozen semen. On D15 of the protocol (i.e., D7 after insemination), the embryos were collected and evaluated. All animals passed through the control and treatment groups. Results were evaluated by analysis of variance using an adjusted mixed-effects model (p < 0.05). There was no difference in the total number of embryos between the control and treatment groups (10.40 ± 1.52 vs. 9.60 ± 1.36; p = 0.63) or viable embryos (6.30 ± 1.22 vs. 4.30 ± 0.71). For precocious indicus heifers, treatment with PGF2α in association with GnRH did not affect embryo production in the fixed-time SOV protocol.

A new hormonal protocol supports early development of in vitro-produced embryos after transfer to anoestrus mares.

The present study aimed to evaluate whether primed anoestrus mares are suitable recipients for embryos produced by intracytoplasmic sperm injection (ICSI). Anoestrus was confirmed in four mares and daily doses of oestradiol benzoate (6 mg in total) over 5 days were administered; after 3 days of rest, oral altrenogest was administered at 0.088 mg/kg and embryos (1 to 5 embryos per mare; 15 in total) were transferred 3.5 days after progesterone onset. Uterine lavage was conducted 48 h after transfer. The results revealed an 80% embryo recovery rate, and among the retrieved embryos, 67% showed evident intrauterine development. Hence, ICSI-derived embryos can be successfully transferred to primed anoestrus mares, but more studies are required to ensure further embryo development and foaling.

Escherichia coli induced matrix metalloproteinase-9 activity and type IV collagen degradation is regulated by progesterone in human maternal decidual.

BACKGROUND: Escherichia coli (E. coli) is one of the main bacteria associated with preterm premature rupture of membranes by increasing pro-matrix metalloproteinase 9 (proMMP-9) and degradation of type IV collagen in human feto-maternal interface (HFMi). proMMP-9 is regulated by progesterone (P4) but it is unclear whether P4 inhibits proMMP in human maternal decidual (MDec). This study aimed to determine a role of P4 on proMMP-2 and - 9 and type IV collagen induced by E. coli infection in MDec. METHODS: Nine HFMi were mounted in a Transwell system. MDec was stimulated with P4 or E. coli for 3-, 6-, or 24-hours. proMMP-2, -9 and type IV collagen were assessed. RESULTS: Gelatin zymography revealed an increase in proMMP-9 after 3, 6, and 24 h of stimulating MDec with E. coli. Using immunofluorescence, it was confirmed the increase in the HFMi tissue and a reduction on the amount of type IV collagen leading to the separation of fetal amniochorion and MDEc. The degradative activity of proMMP-9 was reduced by 20% by coincubation with P4. CONCLUSIONS: P4 modulates the activity of proMMP-9 induced by E. coli stimulation but it was unable to completely reverse the degradation of type IV collagen in human MDec tissue.

Role of Membrane Estrogen Receptor Alpha on the Positive Feedback of Estrogens on Kisspeptin and GnRH Neurons.

Estrogens act through nuclear and membrane-initiated signaling. Estrogen receptor alpha (ERα) is critical for reproduction, but the relative contribution of its nuclear and membrane signaling to the central regulation of reproduction is unclear. To address this question, two complementary approaches were used: estetrol (E4) a natural estrogen acting as an agonist of nuclear ERs, but as an antagonist of their membrane fraction, and the C451A-ERα mouse lacking mERα. E4 dose- dependently blocks ovulation in female rats, but the central mechanism underlying this effect is unknown. To determine whether E4 acts centrally to control ovulation, its effect was tested on the positive feedback of estradiol (E2) on neural circuits underlying luteinizing hormone (LH) secretion. In ovariectomized females chronically exposed to a low dose of E2, estradiol benzoate (EB) alone or combined with progesterone (P) induced an increase in the number of kisspeptin (Kp) and gonadotropin-releasing hormone (GnRH) neurons coexpressing Fos, a marker of neuronal activation. E4 blocked these effects of EB, but not when combined to P. These results indicate that E4 blocked the central induction of the positive feedback in the absence of P, suggesting an antagonistic effect of E4 on mERα in the brain as shown in peripheral tissues. In parallel, as opposed to wild-type females, C451A-ERα females did not show the activation of Kp and GnRH neurons in response to EB unless they are treated with P. Together these effects support a role for membrane-initiated estrogen signaling in the activation of the circuit mediating the LH surge.

Chlorophyll Derivatives Exert Greater Potency Over Progesterone in the Prevention of Infection-Induced Preterm Birth in Murine Models.

PROBLEM: Preterm birth (PTB) is a significant cause of maternal and neonatal morbidity and mortality worldwide. However, the effectiveness of progesterone (P4) which is clinically used for PTB management remains controversial and necessitates research into new therapeutic options METHOD OF STUDY: In the current study, we investigated the effectiveness of two chlorophyll derivatives, pheophorbide a (PBa) and pheophytin a (PTa), in counteracting PTB. Timed-pregnant mice (gestation day 17 ± 0.5) received lipopolysaccharide (LPS) (25 µg/mouse) or phosphate-buffered saline (PBS) intraperitoneally, with PBa, PTa, progesterone (P4), and co-administration of P4 and ibuprofen (IBP), administered orally 2 h prior. RESULTS: The LPS group experienced PTB and 100% fetal mortality, whereas the PBa and PTa groups showed a delayed onset of LPS-induced PTB, with significantly decreased PTB rate and fetal mortality. In addition, PBa and PTa suppressed LPS-induced pro-inflammatory cytokines and NF-κB transcription factor while increasing anti-inflammatory cytokines in the placenta and uterus. CONCLUSIONS: Our findings indicate that the chlorophyll derivatives, PBa and PTa increase fetal survival in infection-induced PTB and demonstrate greater efficacy than P4 in preventing PTB.

Relationship between the effect of human chorionic gonadotropin treatment on the fifth day after artificial insemination and ovarian ultrasonographic findings and blood nutritional metabolic factors in dairy cows.

The objective of the present study was to determine the ovarian ultrasonographic findings and metabolic factors that influence the effect of human chorionic gonadotropin (hCG) treatment on the fifth day after artificial insemination (AI) in dairy cows. Thirty-seven lactating Holstein cows were assigned to two groups: the hCG group (n = 25), which received 3000 IU of hCG intramuscularly on Day 5 after AI (day of AI = Day 0), and the control group (n = 12), which received no treatment. Ovarian ultrasonography measured luteal tissue area (LTA), luteal blood flow area (LBF), relative LBF (= LBF/LTA), and dominant follicle area on Day 5. Blood tests measured plasma insulin-like growth factor-I, insulin, and metabolite concentrations on Day 5 and plasma progesterone concentrations on Days 5 and 7. LBF was greater in pregnant cows than in non-pregnant cows, and plasma Glu concentration was lesser in pregnant cows than in non-pregnant cows, but in both cases there was no interaction between group and pregnancy outcome. For plasma insulin concentration, there was an interaction between group and pregnancy outcome, with pregnant cows in the hCG group having lesser concentrations than the other groups. Logistic regression analysis showed that group and the interaction between group and plasma insulin concentration were associated with pregnancy outcome. These results suggest that the effect of hCG treatment on Day 5 after AI is related to plasma insulin concentration and is more effective in cows with lesser plasma insulin concentrations.

Myo-Inositol and D-Chiro-Inositol Reduce DHT-Stimulated Changes in the Steroidogenic Activity of Adult Granulosa Cell Tumors.

Considering the properties of myo-inositol (MI) and D-chiro-inositol (DCI), which are well known in polycystic ovary syndrome therapy, and the limitations of adult granulosa cell tumor (AGCT) treatment, especially for androgen-secreting tumors, we studied the role of MI and DCI in the androgen-rich environment of AGCTs. For this purpose, we analyzed the mRNA expression of steroidogenic genes and the secretion of progesterone (P4) and 17ß-estradiol (E2) in an unstimulated and/or dihydrotestosterone (DHT)-stimulated environment under MI and DCI influence. Thus, we used the HGrC1 and KGN cell lines as in vitro models of healthy and cancerous granulosa cells. We found that DHT, the most potent androgen, increased E2 secretion and steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage gene (CYP11A1) mRNA expression without affecting 450 aromatase (CYP19A1) in AGCTs. However, after the MI and DCI treatment of KGN cells, both compounds strongly reduced StAR and CYP11A1 expression. Interestingly, in DHT-stimulated KGN cells, only DCI alone and its cotreatment with MI reduced both CYP11A1 mRNA and E2 secretion. These findings suggest that CYP11A1 is responsible for the antiestrogenic effect of DCI in the androgen-rich environment of AGCTs. Therefore, MI and DCI could be used as effective agents in the adjuvant treatment of AGCT, but further detailed studies are needed.

Effects of separate and combined estradiol and progesterone administration on fear extinction in healthy pre-menopausal women.

Altered fear conditioning and extinction learning are discussed as key etiological features in anxiety disorders. Women have an increased risk for anxiety disorders and fear conditioning has been shown to be influenced by the menstrual cycle phase and circulating gonadal hormones. The objective of our study was to investigate the effects of separate and combined estradiol and progesterone administration on fear extinction in healthy women. We conducted a placebo-controlled, randomized study in healthy women, who completed a fear conditioning paradigm on three consecutive days: fear acquisition training on day 1, fear extinction training on day 2, and return of fear test on day 3. Skin conductance responses (SCRs) served as main outcome variable. Two hours before testing on day 2, participants received pills containing either placebo, estradiol (2 mg), progesterone (400 mg) or the combination of both. We examined 116 women (mean age 25.7 ± 6.0 years), who showed significantly stronger conditioned SCRs to the CS+ than CS- during fear acquisition training indicating successful fear learning. At the beginning of the fear extinction training, estradiol administration reduced the differentiation between the conditioned stimuli. In the return of fear test, the estradiol groups showed heightened SCR responses to the previously extinguished stimulus, i.e., impaired extinction recall. Administration of progesterone did not have any significant influence on SCRs. There were also no effects on fear potentiated startle response. In our interpretation, exogenous estradiol administration affected the extinction of the conditioned fear response which led subsequently to a stronger return of fear. From a clinical perspective our findings suggest that estradiol levels may have an influence on the success of exposure therapy and could be taken into consideration when planning exposure sessions.

Neuregulin 1 Signaling Attenuates Tumor Necrosis Factor α-Induced Female Rat Luteal Cell Death.

The corpus luteum (CL) is a transient ovarian endocrine structure that maintains pregnancy in primates during the first trimester and in rodents during the entire pregnancy by producing steroid hormone progesterone (P4). CL lifespan, growth, and differentiation are tightly regulated by survival and cell death signals through luteotrophic and luteolytic factors, including the epidermal growth factor (EGF)-like factor family. Neuregulin 1 (NRG1), a member of the EGF family, mediates its effect through ErbB2/3 receptors. However, the functional role of NRG1 in luteal cells (LCs) is unknown. Thus, this study investigated the role of NRG1 and its molecular mechanism of action in rat LC. Our experimental results suggest a strong positive correlation between steroidogenic acute regulatory protein (StAR) and NRG1 expression in mid-CL and serum P4 and estrogen (E2) production. In contrast, there was a decrease in StAR and NRG1 expression and P4 and E2 production with an increase in tumor necrosis factor α (TNFα) expression in regressing CL. Further in vitro studies in LCs showed that the knockdown of endogenous Nrg1 promoted the expression of proinflammatory and proapoptotic factors and decreased prosurvival factor expression. Subsequently, treatment with exogenous TNFα under these experimental conditions profoundly elevated proinflammatory and proapoptotic factors. Further analysis demonstrated that the phosphorylation status of ErbB2/3, PI3K, Ak strain transforming or protein kinase B (Akt), and ErK1/2 was significantly inhibited under these experimental conditions, whereas the treatment of TNFα further inhibited the phosphorylation of ErbB2/3, PI3K, Akt, and ErK1/2. Collectively, these studies provide new insights into the NRG1-mediated immunomodulatory and prosurvival role in LCs, which may maintain the function of CL.

Effect of intrafollicular administration of PGE2 or PGF2α in early estrus on ovulation, hemorrhagic anovulatory follicles formation, progesterone secretion and pregnancy outcome in the mare.

This experiment was performed to evaluate whether intrafollicular treatment of PGE2 or PGF2α administered in early estrus would induce normal ovulation, progesterone production (Experiment 1) and pregnancy (Experiment 2). In Experiment 1, mares in estrus after 2 days of endometrial edema were injected in all largest dominant follicles (28-35 mm in diameter) with 0.5 mL of sterile water containing 500 µg PGE2 (n = 6), 125 µg PGF2α (n = 6) or placebo (n = 7) (Hour 0). Ultrasound examinations were performed daily, until ovulation or anovulation was detected, and daily blood samples were taken for 8 days. In Experiment 2, mares with a dominant follicle ≥35 mm after at least three days of slight-to-moderate endometrial edema, were injected with 500 µg PGE2 diluted in 0.5 mL of sterile water for injection in the follicle (PGE2 group; n = 9 mares and 11 dominant follicles). No puncture was performed in the control group (n = 9 mares and 11 dominant follicles). Mares from both groups were inseminated. In Experiment 1, all mares (6/6) in the PGE2 group ovulated within 24 h of treatment. The mean interval from intrafollicular injection to ovulation was shorter (P < 0.001) in PGE2 mares (24 ± 0 h) than in control mares (77 ± 9 h). Mares from the PGF2α group developed hemorrhagic anovulatory follicles (HAF) more often (7/7) than control mares (2/7); P < 0.05). The progesterone concentration in mares from the PGF2α group was lower (P < 0.004) than control mares in the early post-ovulatory period. The first significant increase in post-ovulatory progesterone concentration occurred earlier (P < 0.05) in mares from the control group than in mares from the PGF2α and PGE2 groups. In Experiment 2, more mares from the control group (7/9, 78 %) became pregnant than from the PGE2 group (2/9, 22 %) (P = 0.015). In conclusion, PGE2 alone induced follicle collapse in all treated mares within 24 h of administrations, while PGF2α blocked ovulation and induced formation of HAFs. However, the post-ovulatory rise in progesterone production was delayed and the fertility reduced in mares with ovulation induced by PGE2 compared to control mares.

Reproductive outcomes of prepubertal Bos indicus beef heifers raised in a pasture-based feeding system submitted to ovulation induction strategies prior to a timed-artificial insemination protocol.

Reproductive outcomes were evaluated in Nelore (Bos indicus) heifers submitted to one, two or no ovulation induction protocols based on progesterone (P4) and estradiol (E2) prior to a timed-artificial insemination (TAI) protocol. A total of 1,437 heifers (13.0 ± 0.8 mo old; 3.1 ± 0.1 of body condition score [BCS] and 279.9 ± 25.8 kg of body weight [BW]) were randomly assigned to 1 of 3 treatments: 0IND (n = 486): no ovulation induction protocol; 1IND (n = 481): one ovulation induction protocol; or 2IND (n = 470): two ovulation induction protocols. On Day -47, heifers from 2IND received a disinfected intravaginal P4 device (2 g, previously used for 21 d), kept until Day -40, when 0.5 mg of E2 cypionate (EC) was given. On Day -19, heifers from 2IND and 1IND underwent the same protocol. On Day 0, all heifers were submitted to the same TAI protocol, starting with a P4 device (0.5 g), 0.5 mg of cloprostenol sodium (PGF), and 1.5 mg of E2 benzoate. On Day 7, P4 device was removed, 0.5 mg of PGF, 0.5 mg of EC, and 200 IU of equine chorionic gonadotropin (eCG) were administered. The TAI was performed 2 d later (Day 9). Blood samples were collected on Days -47 and 0, to determine the presence of CL (circulating P4 concentrations ≥ 1.0 ng/mL). Ultrasound was performed on Days 40, 75 and between Day 150 and parturition to assess pregnancy per AI (P/AI) and pregnancy loss (PL). Statistical analyses were performed using SAS 9.4 (a-cP ≤ 0.05; A,B0.05 < P ≤ 0.10). The proportion of heifers with CL on Day -47 was similar among groups (3.4%). A greater proportion of heifers from 1IND had CL on Day 0, followed by 2IND, then 0IND (87.9a; 80.4b; 28.8c%). There was an effect of treatment on expression of estrus (2IND: 66.6a; 1IND: 67.2a; 0IND: 57.4b%), P/AI on Day 40 (2IND: 53.4a; 1IND: 43.9b; 0IND: 46.5b%), P/AI on Day 75 (2IND: 49.8a; 1IND: 40.5b; 0IND: 44.4ab%) and final P/AI (2IND: 45.5a; 1IND: 35.8b; 0IND: 40.5ab%). No differences were observed in PL (40-75 = 6.3%; 75-final = 9.6%; Total = 15.3%). Particularly within lighter heifers, there was an effect of treatment on P/AI on Day 40 (0IND: 39.2b; 1IND: 43.3ab; 2IND: 53.9a%) and on Day 75 (0IND: 36.6B; 1IND: 39.0AB; 2IND: 48.5A%). At the first pregnancy diagnosis, more nonpregnant heifers from 2IND had CL on Day 40 than 0IND, but 1IND did not differ from the other groups (85.4a; 74.8b; 80.8ab%). In conclusion, ovulation induction protocols performed prior to the TAI protocol increased the proportion of heifers with CL on Day 0. The use of two induction protocols resulted in greater fertility, particularly in lighter heifers, and increased cyclicity among nonpregnant heifers. These results indicate that this strategy may be an optimized method for inducing cyclicity and enhancing fertility of prepubertal Nelore heifers raised in pasture-based feeding systems.

Optimisation of Early Pregnancy Diagnosis and Resynchronisation in Beef Cows.

The objectives of this study were (1) to investigate the effects of the preovulatory follicle (POF) size on the accuracy of Doppler-based early pregnancy detection, and (2) to determine whether the removal of PGF2α (PGF) treatment during the resynchronisation protocol would affect fertility in beef cows. In Experiment 1, Nelore suckling cows (n = 224) were enrolled in an estradiol-progesterone-based timed artificial insemination (TAI) protocol. At TAI, cows were separated based on the range of POF diameters, as follows: ≤11.0 mm (n = 50), 11.1-12.9 mm (n = 64), 13.0-14.4 mm (n = 62) and ≥14.5 mm (n = 48). On day 22 after TAI, the corpus luteum (CL) blood flow (CLBF) of all cows was examined by colour Doppler ultrasonography to diagnose nonpregnant cows. The cows with the largest POF had the greatest positive predictive value (88.6%; 31 of 35) and diagnostic accuracy (91.7%; 44 of 48). In Experiment 2, Nelore cows (n = 233) were subjected to the same TAI protocol. Fourteen days after TAI, all cows were started on a resynchronisation protocol. Cows diagnosed as nonpregnant based on CLBF, on day 22, received 0.5 mg estradiol cypionate intramuscular (im) and were assigned to receive either 150 µg of PGF (PGF; n = 50) or 2 mL of saline (control; n = 47). Cows treated with PGF had a P/AI of 30.0% compared with a 48.9% P/AI in controls (p = 0.06). Our findings demonstrate that the POF size affects the accuracy of a CLBF-based early pregnancy diagnosis and that the removal of PGF treatment from the resynchronisation protocol tended to increase P/AI of the second TAI.

Mechanism of Zuogui pill enhancing ovarian function and skin elastic repair in premature aging rats based on artificial intelligence medical image analysis.

BACKGROUND: AI medical image analysis shows potential applications in research on premature aging and skin. The purpose of this study was to explore the mechanism of the Zuogui pill based on artificial intelligence medical image analysis on ovarian function enhancement and skin elasticity repair in rats with premature aging. MATERIALS AND METHODS: The premature aging rat model was established by using an experimental animal model. Then Zuogui pills were injected into the rats with premature aging, and the images were detected by an optical microscope. Then, through the analysis of artificial intelligence medical images, the image data is analyzed to evaluate the indicators of ovarian function. RESULTS: Through optical microscope image detection, we observed that the Zuogui pill played an active role in repairing ovarian tissue structure and increasing the number of follicles in mice, and Zuogui pill also significantly increased the level of progesterone in the blood of mice. CONCLUSION: Most of the ZGP-induced outcomes are significantly dose-dependent.

Progesterone promotes CXCl2-dependent vaginal neutrophil killing by activating cervical resident macrophage-neutrophil crosstalk.

Vaginal infections in women of reproductive age represent a clinical dilemma with significant socioeconomic implications. The current understanding of mucosal immunity failure during early pathogenic invasions that allows the pathogen to grow and thrive is far from complete. Neutrophils infiltrate most tissues following circadian patterns as part of normal repair, regulation of microbiota, or immune surveillance and become more numerous after infection. Neutrophils are responsible for maintaining vaginal immunity. Specific to the vagina, neutrophils continuously infiltrate at high levels, although during ovulation, they retreat to avoid sperm damage and permit reproduction. Here we show that, after ovulation, progesterone promotes resident vaginal macrophage-neutrophil crosstalk by upregulating Yolk sac early fetal organs (FOLR2+) macrophage CXCl2 expression, in a TNFA-patrolling monocyte-derived macrophage-mediated (CX3CR1hiMHCIIhi-mediated) manner, to activate the neutrophils' capacity to eliminate sex-transmitted and opportunistic microorganisms. Indeed, progesterone plays an essential role in conciliating the balance between the commensal microbiota, sperm, and the threat of pathogens because progesterone not only promotes a flurry of neutrophils but also increases neutrophilic fury to restore immunity after ovulation to thwart pathogenic invasion after intercourse. Therefore, modest progesterone dysregulations could lead to a suboptimal neutrophilic response, resulting in insufficient mucosal defense and recurrent unresolved infections.

Membrane progesterone receptors mediate progesterone-stimulated glycogenolysis in the bovine uterine epithelium.

In livestock, the amount of glucose needed by the endometrium and embryo increases during early pregnancy. Yet, how glucose concentrations in the endometrium are regulated remains unclear. The bovine uterine epithelium can store glucose as glycogen, and glycogen content decreases in the luteal phase. Our objective was to elucidate the role of progesterone in glycogen breakdown in immortalized bovine uterine epithelial (BUTE) cells. After 48 h of treatment, progesterone decreased glycogen abundance in BUTE cells (P < 0.001) but did not alter glycogen phosphorylase levels. RU486, a nuclear progesterone receptor (nPR; part of the PAQR family) antagonist, did not block progesterone's effect, suggesting that progesterone acted through membrane progesterone receptors (mPRs). RT-PCR confirmed that BUTE cells express all five mPRs, and immunohistochemistry showed that the bovine uterine epithelium expresses mPRs in vivo. An mPRα agonist (Org OD 02-0) reduced glycogen abundance in BUTE cells (P < 0.001). Progesterone nor Org OD 02-0 affected cAMP concentrations. Progesterone increased phosphorylated AMP-activated protein kinase (pAMPK) levels (P < 0.001), indicating that progesterone increases intracellular AMP concentrations. However, AMPK did not mediate the effect of progesterone. AMP allosterically activates glycogen phosphorylase, and D942 (which increases intracellular AMP concentrations) decreased glycogen abundance in BUTE cells. A glycogen phosphorylase inhibitor partially blocked the effect of progesterone (P < 0.05). Progesterone and Org OD 02-0 had similar effects in Ishikawa cells (P < 0.01), a human cell line that lacks nPRs. In conclusion, progesterone stimulates glycogen breakdown in the uterine epithelium via mPR/AMP signaling. Glucose released from glycogen could support embryonic development or be metabolized by the uterine epithelium.

Comparison of two intravaginal progesterone-releasing devices in lactating Holstein cows synchronized with a 5-d GnRH-based timed-AI protocol.

The objective of the study was to compare the effectiveness of CIDR vs. PRID-Delta devices for use in a 5-day Ovsynch protocol for TAI in lactating Holstein cows that were either not in estrus after the end of the voluntary waiting period or non-pregnant and not returning to estrus following the previous AI. Cows fitted with a collar-mounted automated activity monitoring system (Alta Cow Watch) were subjected to a standard 5-d Ovsynch protocol [100 µg of gonadorelin (GnRH) on Day 0 and 500 µg of cloprostenol on Days 5 and 6] and allocated randomly to receive either an intravaginal device containing 1.35 g (CIDR; n = 304) or 1.55 g (PRID ® DELTA; n = 304) of progesterone between Day 0 and 5. All cows received a second administration of GnRH at approximately 56 h and timed-AI (TAI) 72 h after intravaginal device removal. Inseminations were done using conventional frozen-thawed semen. Estrus events prior to TAI were recorded and transrectal ultrasonography was done on Day 0 to determine presence of a corpus luteum (CL) and 33 and 61 d post-TAI, respectively, to diagnose and confirm pregnancy. Cows had an average of 2.2 lactations, 124.3 days in milk, and a milk yield of 43.6 kg/d at enrollment. The overall percentage of cows with a CL at initiation of treatment was 68.8 % and did not differ between treatment groups. Cows with a CL had greater pregnancy per AI (P/AI) at 33 and 61 d post-TAI than cows without a CL (P < 0.01; 46.9 and 42.3 % vs. 32.1 and 27.4 %, respectively). The overall percentage of cows that expressed estrus prior to TAI was 24.8 % and did not differ between treatment groups; however, estrus expression prior to TAI affected P/AI at 33 and 61 d post-TAI (P < 0.01; 53.6 and 49.0 % vs. 38.5 and 33.9 % for those expressing or not expressing estrus, respectively). Pregnancy per AI at 33 d post-TAI tended to differ between treatment groups (P = 0.08; 46.1 vs. 38.5 % for PRID and CIDR groups, respectively) and P/AI at 61 d post-TAI was greater (P < 0.01) for PRID-treated cows (43.8 %) compared to CIDR-treated cows (31.6 %). Thus, PRID-treated cows had lower pregnancy loss than CIDR-treated cows (P < 0.01; 5.0 vs. 17.9 %). Also, treatment with a PRID tended (P = 0.08) to result in fewer twin pregnancies (7.9 vs. 14.5 % for PRID and CIDR treated cows, respectively). In conclusion, lactating dairy cows subjected to a 5-d Ovsynch TAI protocol plus a PRID-Delta had greater P/AI at 61 d post-TAI, lower pregnancy loss between 33 and 61 d post-TAI, and fewer twin pregnancies compared to cows subjected to a 5-d Ovsynch protocol plus a CIDR.

The novel stimulators of feline ovarian granulosa cell functions: Monocyte chemoattractant protein-1 and plasminogen activator inhibitor-1.

The aim of the present study was to determine whether adipokines monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) can affect the functions of ovarian cells in cats. The addition of either MCP-1 or PAI-1 increased viability; promoted the accumulation of proliferation markers and progesterone and estradiol release; and decreased the accumulation of apoptosis markers in cultured feline granulosa cells. The present observations suggest that MCP-1 or PAI-1 can be physiological stimulators of ovarian granulosa cell functions.

Safety and efficacy assessment of a synthetic porcine recombinant corticotrophin for the ACTH stimulation test in healthy cats.

Porcine adrenocorticotrophic hormone (ACTH) has been considered valid for the ACTH stimulation test (ACTHST) in humans and dogs; however, its safety and efficacy for use in cats are unknown. Also, the equivalence between 5 µg/kg and 125 µg/cat dose of synthetic corticotropin (1-24 ACTH - cosyntropin/tetracosactide) is assumed for ACTHST in cats. This study evaluated the safety and effectiveness of different porcine recombinant ACTH doses for the ACTHST in healthy cats and its equivalence with tetracosactide. The study was divided into two arms. The first evaluated safety and equivalence of intravenous 1 µg/kg, 5 µg/kg, or 125 µg/cat porcine ACTH in seven healthy cats for the ACTHST evaluating basal and post-ACTH androstenedione, aldosterone, cortisol, and progesterone concentrations. In the second arm, the equivalence of the 125 µg/cat porcine ACTH dose was evaluated compared to results obtained using 125 µg/cat of tetracosactide in ten healthy cats regarding cortisol responses. In all tests, several cat-friendly strategies were adopted, and the ACTHST protocol involved basal and 60-minute post-ACTH blood sampling and intravenous ACTH injection. No adverse reactions were documented, and no tested cat showed any complications during the study. No porcine ACTH tested dose significantly increased androstenedione secretion. In contrast, all tested doses were able to increase progesterone concentration significantly (P < 0.05), and Δ-progesterone in response to 5 µg/kg or 125 µg/cat was considered equivalent (P > 0.99). The 125 µg/cat dose promoted greater responses for both cortisol and aldosterone, characterized by Δ-cortisol (P = 0.009) and Δ-aldosterone (P = 0.004). Despite equivalent Δ-cortisol results in response to 5 µg/kg or 125 µg/cat (P = 0.18); post-ACTH results of cortisol in response to 5 µg/kg only approximate statistical significance when compared with basal (P = 0.07). Porcine ACTH and tetracosactide significantly increased post-ACTH cortisol concentration (P < 0.0001) while the Δ-cortisol was slightly greater in response to the porcine ACTH (P = 0.006). These results suggest porcine ACTH could be an alternative source of corticotropin for the ACTHST in cats; however, maximum corticoadrenal stimulation seemed more reliable in response to a 125 µg/cat regarding cortisol and aldosterone.

Progesterone (P4) ameliorates cigarette smoke-induced chronic obstructive pulmonary disease (COPD).

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease associated with high morbidity and mortality worldwide. Oxidative injury and mitochondrial dysfunction in the airway epithelium are major events in COPD progression. METHODS AND RESULTS: The therapeutic effects of Progesterone (P4) were investigated in vivo and in vitro in this study. In vivo, in a cigarette smoke (CS) exposure-induced COPD mouse model, P4 treatment significantly ameliorated CS exposure-induced physiological and pathological characteristics, including inflammatory cell infiltration and oxidative injury, in a dose-dependent manner. The c-MYC/SIRT1/PGC-1α pathway is involved in the protective function of P4 against CS-induced COPD. In vitro, P4 co-treatment significantly ameliorated H2O2-induced oxidative injury and mitochondrial dysfunctions by promoting cell proliferation, increasing mitochondrial membrane potential, decreasing ROS levels and apoptosis, and increasing ATP content. Moreover, P4 co-treatment partially attenuated H2O2-caused inhibition in Nrf1, Tfam, Mfn1, PGR-B, c-MYC, SIRT1, and PGC-1α levels. In BEAS-2B and ASM cells, the c-MYC/SIRT1 axis regulated P4's protective effects against H2O2-induced oxidative injury and mitochondrial dysfunctions. CONCLUSION: P4 activates the c-MYC/SIRT1 axis, ameliorating CS-induced COPD and protecting both airway epithelial cells and smooth muscle cells against H2O2-induced oxidative damage. PGC-1α and downstream mitochondrial signaling pathways might be involved.