Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0 = day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17β (E₂) were detected in day 25 conceptuses. Concentrations of E₂ were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E₂ and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1α and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E₂ and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.     

Sexual maturity,seasonal estrus,and gestation in female Indo-Pacific bottlenose dolphins Tursiops aduncus inferred from serum reproductive hormones

Reproductive hormones in serum concentrations of progesterone, estradiol, and testosterone in female Indo-Pacific bottlenose dolphins (Tursiops aduncus, n = 12) housed in Ocean Park Hong Kong were investigated in the present study. Results showed that, onset of puberty of captive Indo-Pacific bottlenose dolphins was at 5 years while sexual maturity was at 6. Average serum progesterone concentrations in non-pregnant sexually mature individuals was 0.33 (0.25–0.97) ng/mL (interquartile), significantly higher than in immature ones 0.26 (0.25–0.38) ng/mL. This study found significant difference in serum estradiol concentrations between individuals at the onset of puberty (9.5 ± 1.7 pg/mL, ±SD) and not (below detection limit 9 pg/mL). A slightly seasonal breeding pattern, with progesterone values tend to be higher from February to October (0.38 [0.25–1.07] ng/mL) was inferred. During pregnancy, serum progesterone concentrations range from 10.54 ± 8.74 ng/mL (indexed month post-conception [IMPC] 0) to 25.49 ± 12.06 ng/mL (IMPC 2), and display a bimodal pattern with 2 peaks in early- (25.49 ± 12.06 ng/mL, IMPC 2) and late-pregnancy (21.71 ± 10.25 ng/mL, IMPC 12), respectively. Serum estradiol concentrations can seldom be detected in early-pregnancy and increase constantly in mid- (9.45 ± 1.83 pg/mL) and late-pregnancy (11.88 ± 3.81 pg/mL), with a spike (15.45 ± 6.78 pg/mL) 1 month prior to delivery. Serum testosterone concentrations elevate significantly in IMPC 7 (0.36 ± 0.10 ng/mL) compared to other months (0.16 ± 0.10 ng/mL) of the year. The present study provides normal concentration profiles for some reproductive hormones in female Indo-Pacific bottlenose dolphins and can contribute to the breeding monitoring of this species. Also, our study would shed further light on the reproductive physiology of small cetaceans.     

Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P₄) (5 ng/ml or 1 μg/ml) or estradiol 17β (E₂β) (50 pg/ml or 1 μg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E₂β (1 μg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.     

Elevated Serum Anti-Müllerian Hormone in an Arabian Mare with Granulosa Cell Tumor

Anti-Müllerian hormone (AMH), produced by granulosa cells, has been reported to be elevated in mares with granulosa cell tumor. A 13-year-old Arabian mare was referred after 3 years of infertility. Rectal and ultrasonographic examination revealed enlargement and multicystic appearance of the left ovary. The mare had substantially higher concentration of AMH (21.6 ng/mL) compared with normal cyclic mares (n = 5; 0.36 ± 0.02 ng/mL). Testosterone (50 pg/mL) concentration was also elevated, but progesterone concentration (0.4 ng/mL) was lowered. Accordingly, the mare was presumptively diagnosed with granulosa-theca cell tumor and a left ovariectomy implemented using ventral midline approach under general anesthesia. Histopathological examination confirmed the diagnosis of granulosa-theca cell tumor. Serum concentrations of AMH decreased to 2 ng/mL and 0.5 ng/mL 2 and 4 weeks after tumor removal, respectively. In conclusion, the current report presents confirmatory evidence that evaluation of serum AMH could be useful for diagnosis of granulosa cell tumor in mare.     

Identification of elevated concentrations of estradiol in bovine uterine endometrium

We have investigated the estradiol content of bovine endometrium and related this to circulating plasma estradiol content. In 9 heifers, mean ± S.E.M. plasma estradiol concentration was 0.64 ± 0.25 pg/ml while the mean ± S.E.M. endometrial estradiol content was 43.0 ± 14.7 pg/g tissue; there was a close relationship between plasma and tissue estradiol levels (R² = 0.81; P < 0.001). During culture of endometrial tissue there was a progressive transfer of estradiol from tissue to culture media but no change in total estradiol. Culture of endometrium from 4 heifers with 5 ng/ml testosterone for 72 h resulted in no increase in estradiol. Furthermore, immunohistochemistry revealed no aromatase protein in uterine endometrium. These results confirm high stored tissue concentrations of estradiol in bovine endometrium while providing no evidence for estradiol synthesis by this tissue. The mechanism(s) through which this sequestration of estradiol into uterine tissue occurs remains to be determined.     

Establishment and characterization of a coculture system of equine endometrial epithelial and stromal cells

To investigate the equine endometrium as close to the in vivo situation as possible, we established a coculture system for epithelial and stromal cells (ECs/SCs). ECs and SCs were isolated from nine endometrial tissue specimens. ECs obtained as glandular formations were cultivated on one side of the semipermeable membrane of a Millicell^® insert. After 2 days, SCs (2 × 10⁴ cells/membrane) were seeded onto the other side of the same membrane. During cocultivation, the low serum containing culture medium (Theuß et al., 2010) was supplemented with different concentrations and combinations of 17β‐estradiol (2.0–3.0 pg/ml medium) and progesterone (0.5–15.0 ng/ml medium). Once the cocultures formed continuous cell layers as determined by phase‐contrast microscopy, the membranes were fixed and processed for light microscopical examination. Cytokeratin 19, steroid hormone receptors and the uterine proteins uteroglobin and calbindin_D9k were detected using immunocytochemistry to determine the degree of culture purity and functional cellular differentiation. The culture purity of the EC layer averaged ≥95%. Uteroglobin and calbindin_D9k were consistently expressed in ECs, while hormone receptors were predominantly absent in both cell populations. An explicit cytomorphological epithelial differentiation with formation of round‐oval to polygonal cell forms was encountered in ≤50% of all ECs and independent of supplemented steroids. Based on the findings altogether, and despite the partly absent congruence to the in situ prerequisites, we established a standardized and reproducible coculture system, which offers a basic approach for studies of physiologic and pathophysiologic issues in the mare.     

超数排卵处理沼泽型水牛血清E_2和P_4浓度变化研究

研究利用3种不同的方法超数排卵处理沼泽型水牛,比较研究不同方法处理时水牛血清雌二醇(E2)、孕酮(P4)浓度变化规律。结果表明:进口FolltropinR○-V、国产FSH和PMSG超数排卵处理沼泽型水牛,血清E2浓度峰值分别出现在氯前列烯醇(PGc)处理后的48 h([142.45±94.66)pg/mL]、72 h([87.78±29.62)pg/mL]、48 h([126.38±92.33)pg/mL];血液中P4浓度最低值分别出现在PGc处理后的48 h([0.76±0.21)pg/mL]、24 h([1.18±0.12)pg/mL]和144 h([0.82±0.06)pg/mL]。     

孕酮对奶牛子宫内膜上皮细胞Wnt/β-catenin信号通路关键蛋白表达的影响

为研究孕酮对奶牛子宫内膜上皮细胞Wnt/β-catenin信号通路关键蛋白表达的影响,本试验以原代奶牛子宫内膜上皮细胞为材料,添加不同浓度孕酮(0、1、3、5 ng/mL)对其培养,采用CCK-8法检测孕酮对细胞增殖的影响;免疫印迹法检测孕酮对Wnt/β-catenin信号通路关键蛋白表达的影响。结果表明:与对照组相比,添加1、3 ng/mL孕酮组奶牛子宫内膜上皮细胞增殖率提高(P>0.05),5 ng/mL孕酮组细胞增殖率降低(P>0.05);与对照组相比,1 ng/mL和3 ng/mL孕酮组Wnt/β-catenin信号通路关键蛋白β-catenin、cyclin D1和c-Myc的表达水平均升高(P<0.01),而5 ng/mL孕酮则抑制了β-catenin的表达(P<0.05),且cyclin D1和c-Myc蛋白表达较3ng/mL孕酮组有下降趋势(P>0.05)。综上,高浓度孕酮抑制了体外培养的原代奶牛子宫内膜上皮细胞Wnt/β-catenin信号通路的激活和表达,提示该信号通路参与了奶牛产后子宫复旧延迟进程,为进一步研究奶牛子宫内膜的发病机制提供思路。     

Progesterone and estradiol-17β as a potential method for pregnancy diagnosis in the collared peccary (Pecari tajacu)

In this study, the period of pregnancy of nine collared peccary females has been monitored through the analysis of serum progesterone and estradiol-17β profiles. Serum concentrations of progesterone increased by Day 4 after conception, reaching concentrations of 33.4±5.6ng/mL on Day 10. Between Days 10 and 130 progesterone values were maintained between 20 and 60ng/mL. In the collared peccary, embryonic estradiol synthesis is first observed in the systemic circulation by Day 15 of pregnancy. Between Days 0 and 50 of pregnancy, average estradiol-17β concentrations were between 0 and 30pg/mL. From Day 75 of pregnancy onwards, estradiol concentrations were constantly increasing, reaching maximum concentrations (131.4±40.8pg/mL) on the day of parturition. The combined study of serum progesterone and estradiol-17β concentrations as a potential method for early pregnancy diagnosis presented the best overall accuracy (73%) when the threshold was established at 20ng/mL serum progesterone and 20pg/mL serum estradiol. Nevertheless, the accuracy for diagnosing pregnancy of females at mid and late pregnancy was 78% and 95%, respectively. The analysis of the sexual hormones during pregnancy could be a useful tool as a potential pregnancy diagnosis and an efficient predictor of the day of parturition in the captive collared peccary.     

Production of calcium maintenance factor Stanniocalcin-1 (STC1) by the equine endometrium during the early pregnant period

A factor responsible for progression to pregnancy establishment in the mare has not been definitively characterized. To identify factors possibly involved in the establishment of equine pregnancy, the endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19 and 25 pregnant animals. From initial subtractive hybridization studies, a calcium regulating factor, Stanniocalcin-1 (STC1) mRNA, was found as a candidate molecule expressed uniquely in the pregnant endometrium. Endometrial expression of STC1 mRNA was noted on day 19 and was markedly increased in the day 25 gravid endometrium. STC1 protein was found in the extracts of day 25 gravid endometrium and immunochemically localized in the uterine glands. In addition, STC1 protein was detected in uterine flushing media collected from day 25 pregnant mares. High concentrations of estradiol-17 β (E(2)) were detected in day 25 conceptuses. E(2) levels were much higher in the gravid endometrium than in other regions, whereas progesterone levels did not differ among the samples from different endometrial regions. Expression of STC1 mRNA, however, was not significantly upregulated in cultured endometrial explants treated with various concentrations of E(2) (0.01-100 ng/ml) with or without 10 ng/ml progesterone. These results indicate that an increase in STC1 expression appears to coincide with capsule disappearance in the conceptus, and suggest that STC1 from the uterine glands likely plays a role in conceptus development during the pregnancy establishment period in the mare.     

Equine Endometrial Tissue Concentration of Fluconazole Following Oral Administration

The objective of this study was to determine the plasma and endometrial tissue concentrations of orally administered fluconazole and to determine if these tissue levels surpassed the minimum inhibitory concentration (MIC) for Candida spp. organisms in the reproductive tract of the mare. Mares from study 1 (n = 9) were administered a single oral loading dose of 14 mg/kg fluconazole. Plasma and endometrial tissue samples were collected before fluconazole administration and for 24 hours after the loading dose. Study 2 mares (n = 3), a subset of study 1, were administered the loading dose, followed by maintenance doses of 5 mg/kg every 24 hours for 6 days. Plasma and biopsy samples were collected for 48 hours after the last maintenance dose. High pressure liquid chromatography-tandem mass spectrometry was used to determine the concentration of fluconazole in all samples. The mean plasma and endometrial fluconazole levels 24 hours after the loading dose were 9.53 ± 0.824 μg/mL (mean ± standard deviation) and 11.3 ± 2.38 μg/g, respectively. Fluconazole levels in plasma and endometrial tissue 24 hours after the last maintenance dose were 7.82 ± 1.81 μg/mL and 7.23 ± 3.86 μg/g, respectively. Oral fluconazole administered as a 14-mg/kg loading dose and a 5-mg/kg maintenance dose every 24 hours will result in endometrial tissue levels near the accepted MIC values for most Candida spp. and surpass the MIC for Candida albicans in the reproductive tract of the mare. Consequently, this dosage regimen could be considered for the treatment of infectious endometritis caused by susceptible fungal organisms of Candida spp. in the mare.     

Communications: Serum Progesterone and Estradiol Concentrations in Captive Manatees Trichechus manatus

Abstract

Serum concentrations of progesterone (P) and estradiol (E₂) were measured over a 7-month period in two captive juvenile female manatees Trichechus manatus. The animals, aged 5.17 and 6.58 years, had mean (±95% confidence interval) serum P concentrations of 0.45 ± 0.27 ng/mL and 0.67 ± 0.24 ng/mL, and mean serum E₂ concentrations of 91.1 ± 71.9 pg/ mL and 121.6 ± 50.6 pg/mL, respectively. A mature male was sampled twice: serum P was 0.92 and 0.79 ng/ mL, and serum E₂ was 117.6 and 207.8 pg/mL. A gravid female was sampled once during her first trimester: serum P was 3.42 ng/mL, and E₂ was 27.7 pg/mL.     

Effects of Estrogen and Progesterone on Canine Endometrial Stromal Cells Proliferation and Progesterone Receptor Expression

In this study different digestion and isolation methods were applied to obtain canine endometrial stromal cells (ESCs), different concentration levels were set for estrogen (E₂) and progesterone (P₄), then MTT method was used to measure their effect on cell proliferation in vitro, also cell immunohistochemistry was used for cell identification and measurement of effect on progesterone receptors (PR) expression.The results indicated that E₂ (15, 30 and 100 pg/mL) showed no significant regulations on both cell proliferation and PR expression, P₄ (15 and 30 ng/mL) had significant promoting effect on proliferation of ESCs (P<0.05), P₄ (3, 15 and 30 ng/mL) showed significant inhibitory effect on PR expression (P<0.05), the regulation level was related to concentration and acting time.     

A Retrospective Analysis of 2,253 Cases Submitted for Endocrine Diagnosis of Possible Granulosa Cell Tumors in Mares

Endocrine diagnoses of granulosa cell tumors (GCTs) in the mare are frequently based upon determination of serum concentrations of inhibin, testosterone, and progesterone (GCT panel). In the present study, we examined results from 2,253 samples submitted for determination of GCT panel. In an additional subset of samples (n = 75), diagnosis of GCT was confirmed based upon surgery or necropsy. The objective of the current study was to examine the agreement between serum inhibin and testosterone concentrations in mares with serum progesterone concentrations less than or ≥1 ng/mL. Across all samples, elevations in serum inhibin or testosterone were noted in 15.2% or 17.3% of samples, respectively. For samples with progesterone <1 ng/mL, more (P = .001) samples had elevated inhibin than elevated testosterone, whereas for samples with progesterone ≥1 ng/mL, more (P < .0001) samples had elevated testosterone than elevated inhibin. For samples with progesterone <1 ng/mL, the agreement between elevated inhibin and testosterone was greater than that for samples with progesterone ≥1 ng/mL. In a subset of 53 samples with progesterone ≥1 ng/mL, 28 samples had endocrine evidence of a granulosa cell tumors based upon elevations of serum anti-Müllerian hormone, and 4 of these samples had endocrine evidence of pregnancy based upon estrone sulfate concentrations. For samples from mares with confirmed GCTs, 85% and 55% had elevated inhibin or testosterone, respectively. Based upon endocrine diagnosis, GCTs occur in mares with elevated progesterone and/or estrone sulfate, albeit at a low rate.     

Bilateral ovarian leiomyoma treated by standing laparoscopic ovariectomy

An 11‐year‐old Warmblood mare presented to a referral facility due to behavioural problems. A hormonal profile performed prior to referral included increased systemic concentrations of testosterone and progesterone but a low concentration of inhibin. Transrectal ultrasound revealed ovaries consistent with a normal cycling mare. At the request of the owner, standing bilateral laparoscopic ovariectomy was performed without complication. Both ovaries were submitted for histopathological examination which revealed bilateral ovarian leiomyoma.     

雌激素和孕酮对犬子宫内膜基质细胞增殖和孕酮受体表达的影响

本试验应用不同消化分离途径获取犬子宫内膜基质细胞,调节培养液中雌激素(E₂)和孕酮(P₄)的浓度,采用MTT法测定E₂和P₄浓度水平对犬子宫内膜基质细胞体外增殖的影响,利用细胞免疫组织化学法鉴定细胞并测定细胞孕酮受体(PR)表达与激素浓度水平的相关性。结果表明,E₂浓度变化(15、30、100 pg/mL)对犬子宫内膜基质细胞的增殖和PR的表达均没有显著的调节作用(P>0.05);P₄(15、30 ng/mL)对犬子宫内膜基质细胞的增殖有显著促进作用(P<0.05),P₄(3、15、30 ng/mL)对犬子宫内膜基质细胞PR的表达具有显著的抑制作用(P<0.05),其影响程度与浓度和作用时间关系密切。     

Response of gilts with delayed puberty to pregnant mare serum gonadotropin or estrogen

The effect of pregnant mare serum gonadotropin (PMSG) or estradiol cyclopentylpropionate (EC) on the induction of estrus, duration of estrus, and serum progesterone concentration after estrus was evaluated in 8 gilts with delayed puberty. Four gilts were given 500 IU of PMSG IM and 4 were given 2 mg of EC, IM. The inactive status of the ovaries at the time of treatment was verified by serum progesterone values of less than 0.5 ng/ml in serial samples collected before treatment. The 4 EC-treated gilts came into estrus at a mean of 3.5 days after treatment, but 1 of the gilts did not form corpora lutea. Three PMSG-treated gilts came into estrus at a mean of 4.0 days after treatment. The remaining PMSG-treated gilt remained anestrus and did not form corpora lutea. The mean duration of estrus in EC-treated gilts was 5.25 days compared with 2.0 days for PMSG-treated gilts (P less than 0.05). Serum progesterone concentrations were higher in PMSG-treated gilts than in EC-treated gilts at 8, 11, and 17 days after treatment (P less than 0.05).     

Effects of Neodymium:Yttnum Aluminum Garnet Laser Irradiation on Endometrium and on Endometrial Cysts in Six Mares

Effects of neodymium:yttrium aluminum garnet (Nd:YAG) laser irradiation on equine endometrium were evaluated In vitro and in six mares with endometrial cysts. The Nd:YAG laser was applied to six endometrial sites, in each of five uterine specimens, with power densities of 5659 to 33,954 J/cm². Depth of tissue ablation was measured and graded on histologic sections of the tissue lesions. Power density had a significant effect on the depth of tissue ablation (p < .001). Grade 3 lesions (full-thickness ablation of the endometrium) were created with energy densities of 16,977 to 33,954 J/cm². Six mares had endometrial cysts treated by photoablation. Two of the four mares that were reproductively sound but barren, despite appropriate breeding, produced foals after treatment. One mare remained reproductively unsound after treatment, and another mare that was treated postpartum was bred successfully.     

水牛发情周期生殖激素变化规律及唾液结晶的分析

试验对水牛发情周期血清和唾液中雌二醇(E_2)和孕酮(P_4)的浓度变化规律、水牛唾液结晶与卵泡发育变化分别进行了分析研究,为进一步探讨水牛发情规律、指导生产提供依据。采用酶联免疫分析法(ELISA)测定发情母水牛血清和唾液中E_2和P_4的浓度变化,并对血清和唾液的激素变化规律进行相关性分析。结果表明,水牛血清和唾液中的E_2和P_4呈波动性变化。发情前期,唾液中P_4浓度一直维持在6.50～7.10 ng/mL,发情第13天达到11.09 ng/mL,随后快速下降。唾液中E_2浓度在发情第3～5天出现一个峰值178.53 pg/mL,在第14～17天唾液中E_2浓度显著升高,出现第二个峰值179.10 pg/mL。母水牛唾液中E_2和P_4浓度的变化趋势与其在血清中的变化趋势基本一致,均呈显著相关(P<0.05);唾液中E_2与P_4浓度呈极显著相关(P<0.01)。水牛发情当天唾液结晶呈现明显的蕨类作物形状且分维值显著低于其他时间点(P<0.05)。水牛发情周期唾液结晶图形的变化与卵巢卵泡发育基本同步,可作为监测水牛发情及预测排卵的可靠指标之一。     

Premature luteal regression in a pregnant mare and subsequent pregnancy maintenance with the use of oral altrenogest

Premature luteal demise or luteal insufficiency is not well characterised as a cause of pregnancy loss in domestic species, including horses. In this report, a mare inseminated with cooled‐transported semen at our facility returned for a routine pregnancy diagnosis at 15 days post ovulation. Ultrasonography per rectum revealed endometrial oedema and the absence of visual indication of a corpus luteum on either ovary. Nonetheless, an embryonic vesicle small for the gestational age was identified. Daily oral altrenogest treatment was implemented immediately. Serum progesterone concentration was 0.67 ng/ml, which is below the threshold considered adequate for pregnancy maintenance in the mare. Examinations were repeated at 17, 25, 30, 39, 49, 72 and 120 days post ovulation. At 25 days post ovulation the embryonic vesicle presented normal development for the gestational age. In addition, sequential blood samples were collected to measure progesterone, equine chorionic gonadotrophin and oestrone sulphate concentrations. Although progesterone concentration did not exceed 2 ng/ml until 72 days post ovulation, all other results were unremarkable and a healthy filly was born uneventfully at 344 days post ovulation.