Intravascular neutrophils partially mediate the endometrial endothelial cell proliferative response to oestrogen in ovariectomised mice

The aim of this study was to investigate the role of intravascular neutrophils in initiating endothelial cell proliferation following oestrogen treatment in ovariectomised mouse endometrium. Uterine tissues were collected from ovariectomised C57/CBA female mice 24 h after oestrogen treatment with or without systemic neutrophil depletion. Neutropenia was achieved with either an in-house anti-neutrophil serum (ANS) or Gr-1 monoclonal antibody. All mice received an i.p. injection of bromodeoxyuridine (BrdU) 4 h prior to dissection to allow visualisation of proliferating cells using immunocytochemistry. Endometrial sections were immunostained for BrdU, vascular endothelial growth factor (VEGF), and neutrophils (using ANS). Oestrogen treatment of ovariectomised mice significantly increased the number of intravascular neutrophils, whereas induction of neutropenia with either ANS or Gr-1 in conjunction with oestrogen treatment prevented this increase. Oestrogen treatment of ovariectomised mice also significantly increased the number of intravascular VEGF-positive cells; however, whereas induction of neutropenia with ANS significantly reduced this increase, Gr-1 did not. In both studies, neutropenia significantly reduced, but did not eliminate, the amount of endometrial endothelial cell proliferation. These results suggest a role for neutrophils in endometrial angiogenesis following acute oestrogen treatment; however, the presence of VEGF-positive cells even after induction of neutropenia suggests that more than one type of leukocyte may be involved.     

Blood and milk progesterone in pregnant and nonpregnant buffalo

Changes in progesterone concentration of blood and milk were measured by radioimmunoassay in 10 Murrah buffalo up to 40 days after insemination. Progesterone concentration in blood plasma at estrus was .1 ng/ml which rose to a peak of 3.6 ng/ml on day 13. It continued to increase in animals that conceived but dropped to .6 ng/ml on 3 days before next estrus in those that failed to conceive. The average concentration of progesterone in milk was .5 ng/ml at estrus; it increased to 18 ng/ml on day 15, and thereafter it declined to 4.4 ng/ml 3 days preceding next estrus in nonpregnant animals. In pregnant animals, it was maintained and elevated further to 24.8 ng/ml on day 37. Progesterone in milk was four to five times higher than in blood plasma.     

Regulation of the strypsin-related proteinase ISP2 by progesterone in endometrial gland epithelium during implantation in mice

Hormones prepare the uterus for the arrival and subsequent invasion of the embryo during pregnancy. Extracellular matrix-degrading proteinases and their inhibitors are involved in this integration process. Recent genetic evidence indicates that there is redundancy within the implantation proteinase cascade, indicating that additional proteinases may be involved. Recently, we described a novel implantation serine proteinase (ISP1) gene that encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching in vitro and the initiation of invasion. The evidence presented in the present study indicates that a second proteinase secreted from the uterus also participates in lysis of the zona pellucida. A second implantation serine proteinase gene (ISP2) was isolated, which encodes a related secreted tryptase expressed specifically within uterine endometrial glands. In pseudopregnancy, ISP2 gene expression is dependent on progesterone priming and is inhibited by the antiprogestin RU486. On the basis of similarities between ISP2 gene expression and that of a progesterone-regulated luminal proteinase associated with lysis of the zona pellucida, it is possible that the strypsin-related protein, ISP2, may encode a zona lysin proteinase.     

Influence of genetic dissimilarity of mother and fetus on progesterone concentrations in pregnant mice and adaptive features of offspring

Concentrations of progesterone in blood plasma and tissue were studied in pregnant mice of strains BALB/cLac and C57BL/6J. Both intra- and interstrain mating and embryo transplantations were used as models of homo- and heterotopic pregnancy. On day 4 of heterotopic pregnancy, plasma progesterone concentrations of females of both strains were higher than those in females of both strains undergoing homotopic pregnancy. In addition, tissue progesterone content of hybrid embryos was higher than that of purebred embryos. Adrenocortical responses to social conflict as indicators of stress resistance were studied in progeny aged 2-3 months. There was a minimal increase in plasma glucocorticoid concentrations in heterotopic transplantants after 15 min pair-matching tests and 30 min crowding compared with those of other progeny, including purebred male mice of BALB/cLac and C57BL/6J strains, homotransplantants and reciprocal hybrids. Thus, genetic dissimilarity of mother and fetuses plays an important role in progesterone provision during pregnancy and also modifies development of progeny.     

Quantification of progesterone binding in mammary tissue of pregnant ewes

Progestin-binding sites in mammary tissue from 14 prepartum, multiparous ewes at 50, 80, 115, and 140 d of gestation were demonstrated by the binding of [3H] R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) to ovine mammary cytosol in the presence of sodium molybdate and excess cortisol. Homogenization extracted 89% of total mammary receptors (nuclear) into cytosol. Binding was specific for progestins and was of high affinity. The average dissociation constant for [3H] R5020 specifically bound to receptors extracted into mammary cytosol was 1.9 (+/- .4) X 10(-9) M (n = 14) and did not change significantly over the test period. However, binding capacities (fmol/mg cytosolic protein) differed according to stage of gestation with averages of 125 +/- 53, 149 +/- 26, 656 +/- 216, 57 +/- 22 at 50, 80, 115, and 140 d of pregnancy, respectively. Increased number of progestin-binding sites at 115 d of gestation (whether data are expressed per unit of tissue weight, DNA, or cytosolic protein) suggests that an increase per mammary epithelial cell may be necessary to produce the full lobuloaveolar proliferation observed at this stage of gestation.     

Regulation of endometrial endothelial cell proliferation by oestrogen and progesterone in the ovariectomized mouse

Although the endometrial epithelial and stromal cell response to oestrogen and progesterone is well characterized, relatively little is known about the endothelial cell response. The aim of this study was to investigate the time course of endometrial endothelial cell proliferation in response to a specific regimen of oestrogen and progesterone, and to compare it with the stromal and epithelial cell response in mouse endometrium. Adult female mice were ovariectomized to induce endometrial regression. After 7 days, hormonal treatments were given according to the following regimen: days 1-3: 100 ng oestradiol; days 4-6: 10 ng oestradiol and 500 microg progesterone; and day 7: 100 ng oestradiol and 500 microg progesterone. On each day of hormonal treatment, mice (n = 5) were injected with bromodeoxyuridine and perfusion fixed 4 h later with buffered formalin. Proliferating endometrial cells were detected by monoclonal antibody against bromodeoxyuridine, and endothelial cells were detected by antibody to CD31. At day 7 after ovariectomy few proliferating cells were found in the endometrium. After 1 day of oestrogen treatment, significant proliferation was detected in the endothelial cells (0.0% versus 16.1 +/- 1.2%, P < 0.001). In contrast to the rapid response of the vasculature, glandular epithelial proliferation increased only after 2 days of oestrogen treatment (7.6 +/-1.3% versus 18.8 +/- 2.4%, P < 0.05). Progesterone with low dose oestrogen treatment tended to reduce epithelial and endothelial cell proliferation compared with the effect of high dose oestradiol alone. A combination of progesterone with high dose oestrogen induced higher rates of endothelial cell proliferation than did any other treatment (20.8 +/- 3.2%). These results demonstrate that oestrogen induces rapid proliferation of endometrial endothelial cells, indicating that vascular growth apparently precedes endometrial tissue remodelling. These data also demonstrate that the proliferative response of endometrial endothelial cells to oestrogen and progesterone is different from that of either epithelial or stromal cells.     

Milk progesterone in postpartum and pregnant cows as a monitor of reproductive status.

Milk samples were analyzed for progesterone content by a petroleum ehter extraction and competitive protein binding assay validated for milk. In one experiment, 11 cows were sampled twice daily for 24 days beginning with an observed estrus 15 to 45 days postpartum, and again 19, 21, 23, and 25 days after breeding. Progesterone values during the estrous cycle paralleled those for blood plasma but were slightly higher at estrus (1.49 ng/ml milk) and maximum (9 ng/ml) on days 11 to 16 of the estrous cycle. After breeding, cows later diagnosed pregnant averaged 7.12 ng/ml while those later found to be nonpregnant averaged 2.36 ng/ml. All diagnoses of pregnancy were correct. In a separate experiment there was no difference between milk from front and rear quarters, but progesterone was highest in last milk, intermediate in composite milk, and lowest in first milk.     

The effect of day of the estrous cycle, location of ovulatory structure, and progesterone on in vitro bovine endometrial secretions.

Endometrial tissue was collected by biopsy from mature Holstein cows either on d 0 (estrus) or on d 9, 14, or 18 of the estrous cycle to determine the effects of day of the cycle, uterine horn, and in vitro progesterone on endometrial protein secretion. Tissue was incubated for 24 h in supplemented media containing 14C amino acids and either 0, 4.7, or 47 ng of progesterone/ml. Media were analyzed for total protein, radiolabeled protein, and profile of protein released during incubation. Endometrial tissue at d 0 released more protein than did tissue collected on all other days. Radiolabeled proteins were greater on d 0 and 18 than on d 9 and 14. Endometrium from the uterine horn contralateral to the corpus luteum released more radiolabeled protein than endometrium from the uterine horn ipsilateral to the corpus luteum. Seventeen protein bands were identified by electrophoresis. Proximity of the uterine horn to the site of ovulation affected the distribution of specific bands 21,400, 55,000, 74,600, and 88,100 molecular weight. The proportion of released proteins represented by proteins of molecular weights 12,700, 19,100, 21,400, 32,000, and 66,500 was affected either by day of the estrous cycle, proximity of uterine horn to the site of ovulation, or progesterone concentration. The results show that day of the estrous cycle and uterine horn not only alter overall endometrial protein secretion and synthetic activity but also have specific effects on distribution of individual proteins.     

Dioscorea improves the morphometric and mechanical properties of bone in ovariectomised rats

BACKGROUND: The aim of this work was to determine the effects of oral administration of dioscorea on the morphometric and mechanical properties of the femur in ovariectomised (OVX) rats. Female Wistar rats that had undergone surgery for ovariectomy were used as a model of menopause and osteoporosis. Four weeks after surgery the animals were given oral dioscorea (0, 250, 750 or 1500 mg kg^?1 day^?1) for 27 days, then the porosity, mineral fraction, stiffness and toughness of the femur and the ultimate force needed to break the femur were measured. RESULTS: Ovariectomy resulted in an increase in the total volume, dry weight and porosity but a decrease in the mineral fraction of femora. Subsequent chronic administration of dioscorea reversed the effect on porosity and increased the ultimate force of the femur in OVX rats but did not affect the bone properties of sham‐operated rats. CONCLUSION: These results suggest that chronic administration of dioscorea may enhance bone strength and provide insight into the role of dioscorea in bone remodeling and osteoporosis during the menopause. However, the benefit is not clear for the reproductive female. Copyright © 2008 Society of Chemical Industry     

Temporal and spatial associations of oestrogen receptor alpha and progesterone receptor in the endometrium of cyclic and early pregnant mares

Uterine function is primarily controlled by the combined actions of oestrogen and progesterone working through their cognate nuclear receptors. The mechanism of establishment of pregnancy in the mare is of interest because it involves prolonged pre-attachment and conceptus migration phases, and both invasive and non-invasive placental cell types, and as such has been an important comparative model. This study characterised regulation of oestrogen (ER) and progesterone (PR) receptors in the endometrium of the mare during the oestrous cycle and early pregnancy. Endometrial tissues collected during the oestrous cycle and early pregnancy were analysed for steady-state levels of ER and PR mRNA and protein. Steady-state levels of ER and PR mRNA were highest on days 0, 17 and 20 in cyclic mares and lowest on days 11 and 14. A day-by-status interaction was detected, indicating that day 17 and day 20 pregnant mares exhibited low levels of ER and PR compared with the corresponding days of the oestrous cycle. In situ hybridisation analyses showed receptor mRNA localisation primarily in the luminal epithelium (LE), glandular epithelium (GE) and stroma around oestrus. During dioestrus and early pregnancy, receptors were not detected in the LE, and were lower in the stroma and deeper GE. Changes in hybridisation intensity in these cell types were consistent with changes in mRNA levels detected by slot-blot hybridisation. ER and PR proteins were detected in the nuclei of LE, GE and stromal cells. Consistent with results from in situ hybridisation, levels of ER and PR immunoreactivity were higher around oestrus, declined to low levels during dioestrus and remained low during early pregnancy. Results described here for temporal and spatial changes in steroid receptor gene expression in mares show the greatest similarities with those described for cattle and sheep.     

Quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels

This study describes a digital technique for uterine morphometry and its application to endometrial structure during the bovine oestrous cycle. Neither the number nor the size of uterine gland ducts changed during the cycle but a reduction in total endometrial area from days 0 to 8 after oestrus led to an increase in the proportion of the endometrium occupied by gland ducts (gland duct density). This effect on day 8 was maintained to day 16. When endometrial morphology was related to circulating progesterone concentrations on days 5 and 8 of the luteal phase, no relationships were found on day 5, but on day 8, a high progesterone concentration was associated with an increased number of gland ducts. Furthermore, in animals slaughtered on day 8, a high progesterone concentration on day 5 was associated with decreased gland duct size, though a simultaneous decrease in endometrial area led to an increase in gland duct density. The results suggest that contrary to expectation, endometrial glands do not grow and regress during the oestrous cycle, although cyclic changes in endometrial area controlled by progesterone lead to changes in gland duct density.     

Analysis of uterine gene expression in interleukin-15 knockout mice reveals uterine natural killer cells do not play a major role in decidualization and associated angiogenesis

During decidualization, uterine natural killer (uNK) cells are the most abundant immune cell types found in the uterus. Although it is well known that they play key roles in spiral arteriole modification and the maintenance of decidual integrity seen after mid-pregnancy, their roles in the differentiation of decidual cells and accompanying angiogenesis during the process of decidualization is less well characterized. To address this, we used whole-genome Illumina BeadChip analysis to compare the gene expression profiles in implantation segments of the uterus during decidualization on day 7.5 of pregnancy between wild-type and uNK cell-deficient (interleukin-15-knockout) mice. We found almost 300 differentially expressed genes and verified the differential expression of ~60 using quantitative RT-PCR. Notably, there was a lack of differential expression of genes involved in decidualization and angiogenesis and this was also verified by quantitative RT-PCR. Similar endothelial cell densities and proliferation indices were also found in the endometrium between the implantation site tissues of wild-type and knockout mice undergoing decidualization. Overall, the results of this study reveal that uNK cells likely do not play a major role in decidualization and accompanying angiogenesis during implantation. In addition, the study identifies a large number of genes whose expression in implantation-site uterine tissue during decidualization depends on interleukin-15 expression in mice.     

Enzymeimmunoassay of oestradiol,testosterone and progesterone in urine samples from female mice before and after insemination

ELISA measurements of 17beta-oestradiol, testosterone and progesterone were determined for urine samples collected non-invasively from female mice. Initial samples were collected during 5 successive days while mature female mice were isolated and cyclic. Subsequently, female mice were inseminated and additional urine samples were collected during days 2-6 after observation of copulatory plugs. Measurements of oestradiol and testosterone showed variance over days within individuals and did not significantly differ in measurements taken before or after insemination. Progesterone concentrations were significantly higher after insemination compared with before mating. In a second sample of inseminated females, urinary progesterone was measured during days 2-18 of pregnancy. Most females showed high urinary progesterone up to day 10 of pregnancy and lower concentrations during the remainder of gestation. These results indicate that urinary progesterone reflects established systemic increases of this hormone during pregnancy.     

铁皮石斛对孕鼠及仔鼠胚胎发育的影响

目的观察铁皮石斛对妊娠大鼠及胚胎发育的影响。方法将妊娠SD大鼠分为溶剂对照组及625、1250、2500 mg/kg铁皮石斛组。于妊娠的第7～16 d经口给予孕鼠不同剂量的铁皮石斛。妊娠第20 d颈椎脱臼法处死孕鼠并解剖,取出子宫称重,检查活胎、吸收胎和死胎数,逐个记录活胎鼠的体重、身长、胎盘重量。每窝检查约一半存活胎鼠的内脏,其余存活胎鼠检查骨骼。结果 3种剂量对妊娠大鼠无明显毒性。其中2500 mg/kg组胎鼠体重与对照组比较有统计学差异(P0.05),胎鼠其余各项指标与对照组比较,差异均无统计学意义(P0.05)。结论铁皮石斛对妊娠母鼠无明显影响,未见致胎鼠畸形作用,可引起胎鼠体重增加。     

The bone‐protective effect of a Taiwanese yam (Dioscorea alata L. cv. Tainung No. 2) in ovariectomised female BALB/C mice

BACKGROUND: Yam products have been marketed for treating postmenopausal syndromes. This study investigated the effects of Dioscorea alata L. cv. Tainung No. 2 (TNG yam) on the bone density of ovariectomised (OVX) female BALB/c mice and the mechanism whereby TNG yam exerted this effect. Sham and OVX control groups were fed a control diet while remaining OVX mice were randomly allocated into experimental diets, i.e. yam (630 g TNG powder kg^?1), E2 (20 mg 17β‐oestradiol kg^?1), or genistein (2 g genistein kg^?1) diet. After 12 weeks of feeding, the uterine weight, indices of bone mass and caecal short chain fatty acids were determined. RESULTS: Neither a yam nor genistein diet restored the OVX‐induced uterine atrophy as did the E2 diet. The femoral and lumbar bone mineral density (BMD) of mice fed the yam diet was greater than those of the sham group, respectively (P < 0.05 vs OVX control), while the lumbar BMD of yam and sham groups were similar (P > 0.05 vs sham). The femoral ash and calcium content in the yam group was significantly greater than that in the OVX control group, respectively (P < 0.05 vs OVX control). The total short chain fatty acid content in the caecum, only enhanced in the yam group, was not correlated with the calcium content of either bone or the plasma calcium level. CONCLUSION: TNG yam prevented loss of BMD and improved bone calcium status without stimulating uterine hypertrophy in OVX BALB/c mice. TNG yam may be beneficial for postmenopausal women for preventing bone loss. Copyright © 2008 Society of Chemical Industry