Placental development during early pregnancy in sheep: nuclear estrogen and progesterone receptor mRNA expression in the utero-placental compartments.

Sex steroid hormones are major regulators of uterine and placental growth and functions, as well as many other biological processes. To examine the mRNA expression of nuclear estrogen (ESR1 and 2) and progesterone (PGRAB and B) receptors in different compartments of the uterus and placenta, tissues were collected in experiment 1 on days 16, 20, and 28 after natural mating (NAT) and on day 10 after estrus (nonpregnant controls [NP]); and in experiment 2 on day 22 of NAT, and pregnancies established after transfer of embryos generated through mating of FSH-treated ewes (NAT-ET), in vitro fertilization (IVF), or in vitro activation (parthenotes). In experiment 1, ESR1 expression in endometrial stroma (ES), endometrial glands (EGs), and myometrial blood vessels (MBVs), ESR2 in endometrial blood vessels (EBV), PGRAB in ES, and PGRB in ES, EG, and MBV was greater in pregnant than NP ewes depending on the day of pregnancy. The day of pregnancy affected the expression of ESR1 in MBV, ESR2 in EBV and MBV, and PGRAB in ES. In experiment 2, ESR1, PGRAB, and PGRB in EG, but not in other compartments, was greater in NAT-ET than NAT, and PGRB was greater for NAT-ET than IVF. These data demonstrate that ESR and PGR expression differ in pregnant versus NP ewes in selected compartments and was affected by pregnancy stage or embryo origin in selected utero-placental compartments. Thus, sex steroid hormone mRNA expression is differentially regulated in a spatiotemporal manner in the uterus and placenta and is affected by the application of assisted reproductive technology in sheep.

Placental development during early pregnancy in sheep: Progesterone and estrogen receptor protein expression.

The aim of this study was to evaluate the pattern of protein expression of the steroid receptor isoforms of nuclear progesterone receptors (PGR) A and B, and estrogen receptors (ESR1 and 2) in utero-placental compartments during early pregnancy. Utero-placental tissues were collected from days 14-30 (n = 4 ewes/day), and uterine tissues were collected from non-pregnant ewes on day 10 after estrus (n = 4). Cross sections of formalin-fixed and paraffin embedded tissues were immunofluorescently stained to detect PGRAB, PGRB, ESR1 and ESR2, followed by image generation of entire cross-sections of uterine and utero-placental tissues, confocal imaging of individual uterine and utero-placental compartments, and image and statistical analyses. PGRAB, PGRB, ESR1 and ESR2 were detected in several compartments of uterine and utero-placental tissues. Quantitative image analysis of staining intensity demonstrated that compared to non-pregnant controls 1) expression of PGRAB and PGRB was less in luminal epithelium and endometrial glands from day 14-16 till 30; 2) PGRAB expression tended to be greater in endometrial and myometrial blood vessels on days 28 and/or 30; 3) PGRB expression in myometrum was lower on days 16 and 28; 4) ESR1 in endometrial stroma was lower in all days of pregnancy; 5) ESR2 expression was similar in all compartments and not affected by pregnancy stage; and 6) in FM, expression of steroid receptors was similar. Thus, we have demonstrated spatial and temporal expression of nuclear PGR and ESR isoforms in utero-placental compartments during early pregnancy.

Glucose-induced increase in circulating progenitor cells is blunted in polycystic amenorrhoeic subjects.

BACKGROUND: Glucose-induced kinetics of bone marrow-derived stem cells in healthy females is presently unknown. The objectives of this study were to determine whether circulating levels of CD133(+), CD34(+) and CD133(+)CD34(+) cells increase in response to glucose load in healthy females and whether the kinetics is altered in amenorrhoeic women. The other objective of the work was to compare the endothelial differentiation potential of peripheral blood-derived endothelial progenitor cells (EPCs) from healthy versus amenorrhoeic women. METHODS: In this case-control study, 44 amenorrhoeic subjects and 36 age-matched females with no menstrual disturbance were recruited at Apollo Hospitals, a Tertiary health care center in Chennai, India. Circulating bone marrow-derived stem cells were measured by two color direct flow cytometry. Cultured progenitor cells were characterized at Day 7 and 14 for expression of endothelial markers and production of nitric oxide (NO) via immunofluoroscence. RESULTS: The amenorrhoeic subjects were insulin resistant with homeostatic model of assessment of insulin resistance values of 3.33 ± 0.3 versus 1.75 ± 0.148 observed for controls (P< 0.0001). Among the amenorrhoeic subjects, 38 subjects had polycystic ovaries with no signs of hyperandrogenism. Fasting levels of CD133(+), CD34(+) and CD133(+)CD34(+) cells were reduced in amenorrhoeic subjects (P< 0.001). There was a 1.5 to 2-fold increase in the circulating levels of these cells in response to 75 g oral glucose challenge at 1 and 2 h post-load conditions in controls, which was significantly blunted for CD133(+) (P< 0.001) and CD133(+)CD34(+) (P< 0.001) cells in amenorrhoeic subjects. A positive correlation was observed between estrogen and fasting CD133(+) (r= 0.205, P= 0.070), CD34(+) (r= 0.249, P= 0.027) and CD133(+)CD34(+) (r= 0.217, P= 0.055) cell counts. Additionally, fasting counts for CD34(+) and CD133(+)CD34(+) cells positively correlated with FSH and inversely correlated with LH and C-peptide in the polycystic group. Cultured cells from polycystic subjects exhibited reduced adherence to fibronectin and expressed lower levels of endothelial nitric-oxide synthase and NO. CONCLUSIONS: Oral glucose-induced increase in circulating numbers of CD133(+) and CD133(+)CD34(+) cells and endothelial differentiation potential of peripheral blood-derived EPCs is attenuated in insulin resistant amenorrhoeic subjects.

Glucose challenge increases circulating progenitor cells in Asian Indian male subjects with normal glucose tolerance which is compromised in subjects with pre-diabetes: A pilot study.

BACKGROUND: Haematopoietic stem cells undergo mobilization from bone marrow to blood in response to physiological stimuli such as ischemia and tissue injury. The aim of study was to determine the kinetics of circulating CD34+ and CD133+CD34+ progenitor cells in response to 75 g glucose load in subjects with normal and impaired glucose metabolism. METHODS: Asian Indian male subjects (n = 50) with no prior history of glucose imbalance were subjected to 2 hour oral glucose tolerance test (OGTT). 24 subjects had normal glucose tolerance (NGT), 17 subjects had impaired glucose tolerance (IGT) and 9 had impaired fasting glucose (IFG). The IGT and IFG subjects were grouped together as pre-diabetes group (n = 26). Progenitor cell counts in peripheral circulation at fasting and 2 hour post glucose challenge were measured using direct two-color flow cytometry. RESULTS: The pre-diabetes group was more insulin resistant (p < 0.0001) as measured by homeostasis assessment model (HOMA-IR) compared to NGT group. A 2.5-fold increase in CD34+ cells (p = 0.003) and CD133+CD34+ (p = 0.019) cells was seen 2 hours post glucose challenge in the NGT group. This increase for both the cell types was attenuated in subjects with IGT. CD34+ cell counts in response to glucose challenge inversely correlated with neutrophil counts (ρ = -0.330, p = 0.019), while post load counts of CD133+CD34+ cells inversely correlated with serum creatinine (ρ = -0.312, p = 0.023). CONCLUSION: There is a 2.5-fold increase in the circulating levels of haematopoietic stem cells in response to glucose challenge in healthy Asian Indian male subjects which is attenuated in subjects with pre-diabetes.