Early Luteal Phase Research Articles

Interleukin-1β system in the oviducts of pigs during the oestrous cycle and early pregnancy

Interleukin-1β (IL-1β) acts throughout the IL-1β system, which contains IL-1β and the IL-1β receptor (IL-1R), accessory protein (IL-1RacP), and receptor antagonist (IL-1Ra). In pigs, the expression of the members of the IL-1β system was documented in uterine tissues during the oestrous cycle and early pregnancy, as well as in embryos harvested during the peri-implantation period. In the oviducts of non-gravid and gravid pigs, the expression of the IL-1β system is unknown. Thus, in this study, the expression of the IL-1β system was examined in porcine oviducts harvested on days 2–3 to 18–20 of the oestrous cycle and on days 2–3 to 15–16 of pregnancy. The expression of IL-1β, IL-1R and IL-1RacP mRNAs in oviducts increased during the mid-luteal phase of the oestrous cycle, whereas the expression of IL-1Ra mRNA increased only during the early luteal phase, e.g., on days 2–3 of the oestrous cycle. Low expression of IL-1β and IL-1Ra mRNAs was observed during the follicular phase of the oestrous cycle. In gravid pigs, the expression of IL-1β, IL-1Ra and IL-1R mRNAs decreased (P < 0.05) from days 2–3 to 15–16 of pregnancy, whereas IL-1RacP mRNA expression did not change in pregnant pigs (P > 0.05). Significantly greater expression of the IL-1β system mRNAs was demonstrated in oviducts harvested on days 2–3 of pregnancy vs. the respective days of the oestrous cycle. On days 2–3 of pregnancy, compared to respective days of the oestrous cycle, the quantity of IL-1β protein was decreased (P < 0.05) in the ampulla and isthmus, while the quantity of IL-1Ra (only in the ampulla) and IL-1RacP proteins (in the ampulla and isthmus) were increased. The concentration of IL-1β in oviductal flushings did not change (P > 0.05) in non-pregnant pigs, and it was greater (P < 0.05) on days 2–3 of pregnancy vs. the respective days of the oestrous cycle. Therefore, the presence of embryos in oviducts on days 2–3 after mating may influence the oviductal expression of the members of the IL-1β system, determining the action of IL-1β, which may be considered to be the earliest sign of pregnancy in pigs.

Incubation of boar spermatozoa in viscous media by addition of methylcellulose improves sperm quality and penetration rates during in vitro fertilization

This work was designed to study whether viscous media can improve the in vitro sperm functionality in pigs by using methylcellulose as a thickener. Viscosity of porcine oviductal fluid (POF) was compared with culture medium (Tyrode's) supplemented with methylcellulose (MET 0, 0.5 and 1% w/v). Spermatozoa were incubated in the different media (0, 1 and 2 h) and sperm motion parameters, lipid membrane disorder, plasma membrane integrity and reactive oxygen species (ROS) formation were assessed. Fertilization results were assessed i) preincubating spermatozoa in the viscous media followed by gamete coculture in a non-viscous medium; and ii) gamete coculture in the viscous media. Viscosity of POF from early luteal phase was higher than late follicular phase. Medium without methylcellulose presented constant viscosity with increased shear rate, while viscosity of the POF and media with methylcellulose was reduced by increased shear rates. Methylcellulose improved sperm linearity, straightness and the proportion of fast-linear spermatozoa. Moreover, methylcellulose increased the rate of viable spermatozoa with intact acrosome and low lipid disorder, reducing the ROS generation. Preincubation in viscous media increased the penetration rate and the mean number of spermatozoa bound to the zona pellucida (both with 0.5 and 1% MET) and reduced monospermy with 1% MET. On the other hand fertilization in the viscous media reduced penetration rate and increased monospermy. The efficiency of the IVF system was not improved with the use of viscous media. The results show the relevance of increasing viscosity thus making the in vitro media more comparable to physiological conditions.

Effect of investigational kisspeptin/metastin analog, TAK-683, on luteinizing hormone secretion at different stages of the luteal phase in goats

This study aimed to examine the response of luteinizing hormone (LH) secretion and ovarian steroid profile to TAK-683, an investigational metastin/kisspeptin analog, through treatment during different stages of the luteal phase in goats. Nine cycling Shiba goats (4.4 ± 2.3 years old) were assigned to early luteal phase (ELP, n = 4), mid-luteal phase (MLP, n = 4), and control (n = 5) groups. The ELP and MLP groups were administered 50 µg of TAK-683 intravenously on either day 5 or between days 7–14 after ovulation, respectively. The control group received vehicle between days 7–14 after ovulation. Blood samples were collected at 10-min (2–6 h), 2-h (6–24 h), and 24-h (24–96 h) intervals after treatment. Significant increases in plasma LH concentration were detected during the periods of 3 to 5 h and 2 to 5 h in the ELP and MLP groups, respectively. Estradiol concentrations continuously increased with the rise of basal LH secretion after TAK-683 treatment in two goats of the ELP group with a surge-like release of LH, but not in the goats without LH surge, i.e. the MLP and control group ones. Plasma progesterone concentration and the lengths of estrous cycle in all groups did not change significantly from the time before and after treatment. Present findings indicate that the responses of LH and ovarian steroids to treatment with TAK-683 depend on the stage of the luteal phase of the estrous cycle. We suggest that the stimulatory effects of TAK-683 on LH secretion are reduced in the process leading to the mid-luteal phase in cycling goats.

Cortisol awakening response is blunted and pain perception is increased during menses in cyclic women

Background and aimsThe incidence of menstrual symptoms is reported to be as high as 90% in cyclic women. These symptoms, including anxiety and pain, might be associated with cortisol, as its receptors are widely distributed in the brain areas associated with behavior. Therefore, the current study aimed to assess the cortisol awakening response (CAR) throughout the menstrual cycle and correlate it with pain perception and trait anxiety. Materials and methodsCAR was assessed by measuring salivary cortisol at 0, 15, 30, and 60min following awakening in the same women (n=59, age 22.2±0.37years) at various stages of the menstrual cycle (menses, midcycle, luteal and premenstrual phases). Progesterone and estradiol concentrations were also determined in saliva samples to assess cyclic changes. Self-reported pain, trait anxiety, and menstrual symptoms were assessed by visual analog scale (VAS), state-trait anxiety inventory (STAI-T), and the Daily Record of Severity of Problems (DRSP), respectively. ResultsEstradiol was significantly elevated during the midcycle period and remained high during the early luteal phase (p<0.05). Progesterone was increased during the luteal phase (p<0.05). Post-awakening cortisol values increased during midcycle, luteal phase, and premenstrual phase (p<0.05, classical CAR), but not during the menses (p>0.05, blunted or flat CAR). Positive and significant correlations were found between cortisol and estradiol (R2=0.322; p=0.000), cortisol and progesterone (R2=0.156; p=0.000), and estradiol and progesterone (R2=0.349; p=0.001). Premenstrual symptom scores were higher in the menses and premenstrual phases than in the midcycle and luteal phases (p<0.001). Pain perception was the highest during the menses followed by the premenstrual phase (p<0.01). ConclusionsCAR was blunted during the menses, suggesting that cortisol might play a phase-specific role in the regulation of the cycle. Additionally, premenstrual symptoms, including pain, were more severe when ovarian steroid levels reduced (i.e., menses and the premenstrual phase).

80 EFFECT OF BOVINE OVIDUCTAL FLUID ON DNA METHYLATION OF BOVINE BLASTOCYSTS PRODUCED IN VITRO

During the transit through the oviduct, the early embryo undergoes an epigenetic reprogramming of its genome, which induces changes in DNA methylation pattern. Given that epigenetic modifications are susceptible to environmental influence, the oviducal milieu may affects DNA methylation marks in the developing embryo. The aim of this study was to evaluate whether bovine oviducal fluid (OF) exerts an effect on methylation status of genomic regions at different time points of embryo development. In vitro-produced zygotes were cultured in SOF + 3 mg mL−1 BSA (control, C) or in SOF + 1.25% OF at 3 different time points: until 98 h post-insemination (hpi) (OF1–16: 1–16 cell), 52 hpi (OF1–8: 1–8 cell), or from 52 until 98 hpi (OF8–16: 8–16 cell). The OF used was acquired from Embryocloud (Murcia, Spain) from cow oviducts at the early luteal phase (Day 1–4). After, embryo culture took place in control medium up to Day 8. For all the groups, the speed of development was considered, and normal developing embryos that reached ≥6 cells at 52 hpi and ≥16 cells at 98 hpi were selected and separately cultured from slow developing embryos. Cleavage (52 hpi) and blastocyst yield (Day 7–8) were analysed by ANOVA (8 replicates). Expanding blastocysts (Day 7–8) from the normal developing groups were collected for bisulfite sequencing analysis. The DNA bisulfite conversion was performed with a MethylEdge Bisulfite Conversion System kit (Promega, WI, USA) in groups of 20 blastocysts obtained from 5 replicates. Methylation status was analysed on regions localised in 4 developmental important genes (MTERF2, ABCA7, OLFM1, and GMDS) and within 2 LINE L1 elements located on chromosomes 9 (L9) and 29 (L29). Methylation percentages (10 sequenced clones/group) were compared using statistical z-test. No significant differences were found on cleavage rate (C: 89.7 ± 1.0, OF1–16: 84.9 ± 1.7; OF1–8: 85.4 ± 1.9; OF8–16: 89.1 ± 1.9%) and blastocyst yield between normal developing embryos (C: 36.8 ± 5.3; OF1–16: 34.7 ± 3.7; OF1–8: 41.0 ± 3.8; OF8–16: 43.9 ± 5.1%). Blastocysts derived from all OF groups showed the CpG region of MTERF2 hypomethylated compared with C group (20.0, 26.2, and 32.9% v. 56.2%, respectively; P &lt; 0.001). The CpG sequence of ABCA7 exhibited significant hypomethylation in embryos from OF1–16 group compared with OF1–8, OF8–16, and C groups (31.1 v. 56.8, 57.9, and 65.8%, respectively; P &lt; 0.001). Although the methylation of the CpG region within OLFM1 did not differ between OF1–16 and C groups (24.1 v. 19.4%, respectively), embryos from OF1–8 group showed a highly methylated region (47.1%) compared with OF1–16 and C groups (P &lt; 0.001). The CpG sequence on L9 showed a high methylation level in blastocysts derived from OF1–16 group compared with OF8–16 and C groups (36.4 v. 14.5 and 20.0%, respectively; P &lt; 0.05). There were no differences in methylation marks between groups examined for CpG regions of GMDS and L29. These results indicated that embryos exhibit a temporal sensitivity to OF at early embryonic stages, which is reflected by DNA methylation changes of specific genes at blastocyst stage. This is the first report describing that OF could modify specific epigenetic marks of the bovine embryonic genome.

Luteal function during the estrous cycle in arginine-treated ewes fed different planes of nutrition.

Functions of corpus luteum (CL) are influenced by numerous factors including hormones, growth and angiogenic factors, nutritional plane and dietary supplements such as arginine (Arg), a semi-essential amino acid and precursor for proteins, polyamines and nitric oxide (NO). The aim of this study was to determine if Arg supplementation to ewes fed different planes of nutrition influences: (1) progesterone (P4) concentrations in serum and luteal tissue, (2) luteal vascularity, cell proliferation, endothelial NO synthase (eNOS) and receptor (R) soluble guanylate cyclase β protein and mRNA expression and (3) luteal mRNA expression for selected angiogenic factors during the estrous cycle. Ewes (n = 111) were categorized by weight and randomly assigned to one of three nutritional planes: maintenance control (C), overfed (2× C) and underfed (0.6× C) beginning 60 days prior to onset of estrus. After estrus synchronization, ewes from each nutritional plane were assigned randomly to one of two treatments: Arg or saline. Serum and CL were collected at the early, mid and late luteal phases. The results demonstrated that: (1) nutritional plane affected ovulation rates, luteal vascularity, cell proliferation and NOS3, GUCY1B3, vascular endothelial growth factor (VEGF) and VEGFR2 mRNA expression, (2) Arg affected luteal vascularity, cell proliferation and NOS3, GUCY1B3, VEGF and VEGFR2 mRNA expression and (3) luteal vascularity, cell proliferation and the VEGF and NO systems depend on the stage of the estrous cycle. These data indicate that plane of nutrition and/or Arg supplementation can alter vascularization and expression of selected angiogenic factors in luteal tissue during the estrous cycle in sheep.

Stomatin-like protein 2 is involved in endometrial stromal cell proliferation and differentiation during decidualization in mice and humans

The molecular mechanisms underlying endometrial stromal cell proliferation and differentiation (decidualization) are still not fully understood. This study revealed that increased Slp-2 expression is a significant factor modulating endometrial stromal cell proliferation and decidualization in both mice and humans. Our results showed a significant difference in the mRNA and protein levels between the implantation site and inter-implantation site on day 5 and day 6 of pregnancy in mice (all P < 0.05). Strong Slp-2 immunostaining was mainly localized within the decidual zone of mice through the post-implantation period. Mice with artificially induced deciduoma showed significantly higher expression of Slp-2 compared with uninduced controls (P < 0.005). Human stromal cells in the middle and late-secretory phases demonstrated significantly (all P < 0.05) upregulated SLP-2, compared with cells in the proliferative phase and early secretory phases. Further analyses of the SLP-2 gene knocked down revealed a significant (P < 0.005) repression of both the decidualization marker gene's expression (decidual/trophoblast prolactin-related protein in mice, insulin-like growth factor binding protein and prolactin in human) and the cell proliferation in in vitro-induced decidualized primary endometrial stromal cells in mice and humans.

Progesterone supplementation in the early luteal phase after artificial insemination improves conception rates in high-producing dairy cows

This study examines the possible effects on the reproductive performance of high-producing dairy cows of progesterone (P4) given in the early luteal phase (1.55 g of P4), from Days 3 to 5 post-artificial insemination (AI) as compared with the time of pregnancy recognition, from Days 15 to 17 post-AI. Cows in their third day post-AI were alternately assigned on a weekly rotational basis to the following groups: control, no treatment (C: n = 351), P4 treatment started 15 days after AI (P4-D15: n = 261), or P4 treatment started 3 days after AI (P4-D3: n = 203). Based on odds ratios, cows in P4-D3 were 1.71 times more likely to conceive than control cows (P = 0.004), whereas cows in P4-D15 showed a 1.4-fold greater risk approaching significance of becoming pregnant compared with control cows (P = 0.06). Differences were not observed between treatments. In nonpregnant cows, the given treatment (D3 vs. D15) had no effect on subsequent return to estrus or AI interval and neither were any effects of treatment observed on early fetal loss rates. In contrast, in pregnant cows, the relative risk of twin pregnancy was 2.5 times higher for those in P4-D15 (P = 0.02) than the remaining cows. These findings indicate the efficacy of P4 supplementation after AI. However, when given at the time of pregnancy recognition rather than in the early luteal phase, this treatment increases the twin pregnancy rate.

Expression pattern of HIF1alpha and vasohibins during follicle maturation and corpus luteum function in the bovine ovary.

The aim of this study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A) and vasohibin family members (VASH1 and VASH2) during different stages of ovarian function in cow. Experiment 1: Antral follicle classification occurred by follicle size and estradiol-17beta (E2) concentration in the follicular fluid into 5 groups (<0.5, 0.5-5, 5-40, 40-180 and >180 E2 ng/ml). Experiment 2: Corpora lutea (CL) were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16 and >18 (after regression) of oestrous cycle and of pregnancy (months 1-2, 3-4, 6-7, >8). Experiment 3: Cows on days 8-12 were injected with a prostaglandin F2alpha (PGF) analogue and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64hr after PGF injection. Expression of mRNA was measured by qPCR, steroid hormone concentration by EIA and localization by immunohistochemistry. HIF1A mRNA expression in our study increases significantly in follicles during final maturation. The highest HIF1A mRNA expression was detected during the early luteal phase, followed by a significant decrease afterwards. In contrast, the mRNA of vasohibins in small follicle was high, followed by a continuous and significant downregulation in preovulatory follicles. The obtained results show a remarkable inverse expression and localization pattern of HIF1A and vasohibins during different stages of ovarian function in cow. These results lead to the assumption that the examined factors are involved in the local mechanisms regulating angiogenesis and that the interactions between proangiogenic (HIF1A) and antiangiogenic (vasohibins) factors impact all stages of bovine ovary function.

Elevated circulating micro-ribonucleic acid (miRNA)-200b and miRNA-429 levels in anovulatory women

To study the role of micro-RNA (miRNA)-200b and miRNA-429 in human ovulation and to measure their expression levels in ovulatory and anovulatory patients. Micro-RNA-200b and miRNA-429 expression analysis in human serum and granulosa cells at different phases of the ovulation cycle in normal cycling women and women undergoing assisted reproductive technology cycles. University-affiliated hospital and academic research laboratory. Forty women (7 normally ovulating, 15 normally ovulating with pure male infertility factor, and 18 with polycystic ovary syndrome) were included in this study. None. The expression profile of circulating miRNAs and granulosa cells was assessed by means of real-time quantitative reverse transcription-polymerase chain reaction analysis. We identified miRNA-200b and miRNA-429 in the sera of all women tested. These miRNA expression levels were elevated during the early follicular phase of the cycle compared with serum levels during the early luteal phase. Anovulatory women, diagnosed with polycystic ovary syndrome, expressed significantly higher levels of miRNA-200b and miRNA-429 compared with spontaneously ovulating women. Ovulation induction with exogenous gonadotropins during an IVF cycle reduced these levels to the levels measured in normal ovulating women. Our findings suggest an involvement of miRNA-200b and miRNA-429 in the pituitary regulation of human ovulation. Although it is unclear whether this altered miRNA expression profile is a cause or a result of anovulation, the levels of these molecules in the serum of anovulatory women may serve as serum biomarkers for the ovulation process.

Immature oocyte retrieval and in vitro oocyte maturation at different phases of the menstrual cycle in women with cancer who require urgent gonadotoxic treatment

To evaluate the feasibility and the efficacy of invitro maturation (IVM) when immature oocyte collection was performed in the early follicular, late follicular, or luteal phases in women with cancer who require urgent chemotherapy. Retrospective cohort study. University teaching hospital. One-hundred and sixty-four women with cancer undergoing IVM treatment for fertility preservation. Oocyte retrieval, IVM, cryopreservation. Medians (interquartile range) of oocytes collected, maturation rates after 48hours of culture, and metaphase II oocytes cryopreserved. The analysis included a total of 192 cycles grouped into early follicular phase (n = 46), late follicular phase (n = 107), or luteal phase (n = 39). Embryo cryopreservation was performed in 82 cycles, and oocyte cryopreservation in 105 cycles. Between the early follicular, late follicular, and luteal phases, no statistically significant differences were found in the number of oocytes collected (8.5 [4-15.8], 8 [5-14], and 7 [4-9], respectively), the maturation rates after 48hours of culture (53.5% [39.8-77], 58% [44-82], and 50% [33-67], respectively), or the number of oocytes cryopreserved (3 [0-7.3], 3 [0-7], and 3 [1-5.5], respectively). Similarly, the fertilization rates (77 [62.8-92.5], 75 [60-100], and 63.5 [50-75], respectively) and number of embryos cryopreserved (3 [2-5.8], 3 [0.5-5], and 2 [1-3], respectively) were not statistically significantly different among the groups. Our study confirms the feasibility of IVM collection at any time during the menstrual cycle. Treatment with IVM is an alternative method when chemotherapy cannot be delayed or ovarian stimulation is contraindicated. The long-term outcomes remain to be studied.

The impact of diet and arginine supplementation on pancreatic mass, digestive enzyme activity, and insulin-containing cell cluster morphology during the estrous cycle in sheep

To determine the effect of feed intake and arginine treatment during different stages of the estrous cycle on pancreatic mass, digestive enzyme activity, and histological measurements, ewes (n = 120) were randomly allocated to 1 of 3 dietary groups; control (CON; 2.14-Mcal metabolizable energy/kg), underfed (UF; 0.6 × CON), or overfed (OF; 2 × CON) over 2 yr. Estrus was synchronized using a controlled internal drug release device for 14 d. At controlled internal drug release withdrawal, ewes from each dietary group were assigned to 1 of 2 treatments; Arg (L-Arg HCl, 155-μmol/kg BW) or Sal (approximately 10-mL saline). Treatments were administered 3 times daily via jugular catheter and continued until slaughter on d (day) 5 and 10 of the second estrus cycle (early luteal phase, n = 41 and mid-luteal phase, n = 39; yr 1) and d 15 of the first estrus cycle (late luteal phase, n = 40; yr 2). A blood sample collected from jugular catheters for serum insulin analysis before slaughter. The pancreas was then removed, trimmed of mesentery and fat, weighed, and a sample snap-frozen until enzyme analysis. Additional pancreatic samples were fixed in 10% formalin solution for histological examination of size and distribution of insulin-containing cell clusters. Data were analyzed as a completely randomized design with a factorial arrangement of treatments. Diet, treatment, and diet × treatment were blocked by yr and included in the model with initial BW used as a covariate. Day of the estrous cycle was initially included in the model but later removed as no effects (P > 0.10) were observed for any pancreatic variables tested. Overfed ewes had the greatest (P < 0.001) change in BW, final BW, change in BCS, and final BCS. A diet × treatment interaction was observed for change in BW and final BW (P ≤ 0.004). Overfed and CON had increased (P < 0.001) pancreas weight (g) compared with UF ewes. Protein concentration (g/pancreas) was the lowest (P < 0.001) in UF ewes, whereas protein content (mg/kg BW) was greater (P = 0.03) in UF than OF ewes. Activity of α-amylase (U/g, kU/pancreas, U/kg of BW, and U/g protein) and trypsin (U/pancreas) was greater (P ≤ 0.003) in OF than UF ewes. Serum insulin was the greatest (P < 0.001) in OF ewes. No effects were observed for pancreatic insulin-containing cell clusters. This study demonstrated that plane of nutrition affected several measurements of pancreatic function; however, the dosage of Arg used did not influence pancreatic function.

Priming anoestrous Corriedale ewes with progesterone and gonadotrophin-releasing hormone causes cervical tissue remodelling due to metalloproteinase-2 (MMP-2) activity

The aim was to obtain experimental evidence of cervical collagen degradation in anoestrous Corriedale adult ewes induced to ovulate with progesterone (P) and gonadotrophin-releasing hormone (GnRH), at the expected time of induced ovulation and early luteal phase. In Experiment 1, anoestrous ewes were treated with P for 10 days (P, n = 4), with nine micro-doses of GnRH followed by a GnRH bolus injection (GnRH, n = 4) or with P plus GnRH treatments (P+GnRH, n = 3), and cervices were obtained either without treatment (A, n = 4), when P was removed, or 24 h after the GnRH bolus injection. In Experiment 2, cervices were obtained 1 (group P+GnRH, n = 5) or 5 (P+GnRH 5, n = 6) days after the GnRH bolus injection with P pretreatment. MMP-2 activity was detected in all samples; however, MMP-9 activity was only detected in 15% of the samples. The activity of the latent (L) form of MMP-2 in the cranial zone of group A was higher than in the cranial zone of groups P, GnRH and P+GnRH, and was also higher than that in the caudal zone of the same group (P &lt; 0.05). The collagen concentration was lower in group P+GnRH 1 than in group P+GnRH 5 (P &lt; 0.04). The activity of the activated (A) form of MMP-2 and the A/L MMP-2 ratio were higher in group P+GnRH 1 than in group P+GnRH 5 (P &lt; 0.05). Data suggest that the L form of MMP-2 was expressed mainly in a constitutive form in the cervix of anoestrous ewes and that an oestrogen-dependent activation mechanisms due to the GnRH treatment may be responsible for the lowest collagen content at the moment of the induced ovulation. This work provides evidence about cervical collagen remodelling in anoestrous ewes treated with P + GnRH.

P.1.h.051 - Are smarter rats less social? − ‘empathy’ and pro-social behavior in relation to cognitive performance

In this study, we evaluated the changes in leptin and ghrelin concentrations, eating behavior, depression, and impulsivity and their correlations within the luteal phase among women with premenstrual dysphoric disorder (PMDD).In 63 women with PMDD and 53 healthy controls, we prospectively evaluated serum levels of leptin and ghrelin, Body Mass Index(BMI), and self-reported sweet cravings, cognitive restraint, uncontrolled eating, emotional eating, depression, and impulsivity during the early luteal (EL) and late luteal (LL) phases.Compared with the controls, the women with PMDD had higher BMI, higher leptin concentrations in the EL and LL phase, and leptin concentrations increased from the EL to the LL phase. However, there is no significant difference in ghrelin. Women with PMDD increased sweet cravings and uncontrolled eating from EL to LL phase. No significant correlation was observed between the EL-LL changes in leptin or ghrelin concentrations and those in eating behaviors. Both depression and impulsivity correlated with sweet craving and uncontrolled eating. Depression mediated the association between PMDD and uncontrolled eating. The BMI of women with PMDD positively correlated with their EL-LL change in leptin, and LL depression levels and emotional eating.Young women with PMDD had higher leptin concentrations and BMI in the luteal phase. The LL leptin level was not the primary factor responsible for the increased uncontrolled eating of PMDD. Whether the increased eating and depression in the LL phase contribute to the risk of obesity or hyperleptinemia among women with PMDD need to be evaluated in the future.

1076 Global gene expression in the endometrium of primiparous dairy cows during the early-luteal phase of the estrous cycle

1076 Global gene expression in the endometrium of primiparous dairy cows during the early-luteal phase of the estrous cycle

Lipid droplets in the ovine uterus during the estrous cycle: Effects of nutrition, arginine, and FSH

The aim of this study was to evaluate lipid droplet (LD) expression in uteri of FSH-treated or nontreated sheep administered with arginine (Arg) or vehicle (saline, Sal) and fed a control (C), excess (overfed, O) or restricted (underfed, U) diet. In experiment 1, ewes from each diet were randomly assigned to Arg or Sal treatments administered three times daily starting on Day 0 of the first estrous cycle until blood sample and uterine tissue collection at the early- or mid-luteal phase of the second estrous cycle or the late-luteal phase of the first estrous cycle. In experiment 2, ewes were injected twice daily with FSH on Days 13 to 15 of the first estrous cycle, and blood samples and uterine tissue were collected at the early- and mid-luteal phases of the second estrous cycle. Cryopreserved in optimum cutting temperature (OCT) compound, cross-sections of uterine horn were stained with boron-dipyrromethene (BODIPY; marker of LDs) followed by 4′,6-diamidino-2-phenylindole (DAPI) staining and image analysis to determine the proportion (%) of area occupied by LD in luminal epithelium (LE) and endometrial glands (EGs). Control ewes maintained, O ewes gained, and U ewes lost body weight during the experiments. Serum progesterone concentration was not affected by nutritional plane or Arg treatment and was 5.5-fold greater in FSH- than Sal-treated ewes. LDs were detected in LE and superficial EG (close to LE) but not deep EG, or other uterine compartments, and area occupied by LD was greater in LE than in EG. In experiment 1, in LE and EG, area occupied by LDs was greater in C than in O or U; greater in Arg than in Sal; and greater at the late-, less at mid-, and least at early-luteal phases. In experiment 2, in LE and EG, area occupied by LDs was greater at mid- than in early-luteal phase. Comparison of data from FSH-treated and nontreated ewes (e.g., experiment 1 vs. experiment 2) demonstrated that FSH increased the area occupied by LD in LE and EG regardless of diet. Interactions between FSH treatment, stage of the estrous cycle, and plane of nutrition demonstrated that FSH increased the area occupied by LD in LE and EG at the mid-luteal phase in O and U. Thus, LDs are differentially distributed in uterine compartments, and area occupied by LD in endometrium is affected by nutritional plane, Arg or FSH, and stage of the estrous cycle. Such changes in dynamics of LD in the endometrium during the estrous cycle indicate their specific role in uterine functions.

Failure to establish and maintain a pregnancy in undernourished recipient ewes is associated with a poor endocrine milieu in the early luteal phase

Embryos from undernourished and control donor ewes were transferred to undernourished and control recipient ewes. Progesterone and metabolic hormones were investigated in recipient ewes to determine their association with pregnancy success. Forty-five donor and 52 recipient Rasa Aragonesa ewes were fed 1.5 (control group; donor n=20; recipient n=25) or 0.5 (low group; donor n=25; recipient n=27) times the daily requirements for maintenance from the onset of estrous synchronization treatment to embryo collection and transfer. The embryos were collected 7days after the onset of estrus (day 0), and two good-quality embryos were transferred into each recipient ewe. The percentage of pregnant ewes on day 18 and 40 did not differ between the two groups, although the recipient undernourished ewes tended to have greater late embryonic mortality (from days 18–40) than the control recipient ewes (P=0.11). No effect of the nutrition of the donor was found. Recipients that became pregnant had a higher ovulation rate than non-pregnant ewes (P=0.02). Undernourished ewes had lower plasma insulin concentrations than control ewes (P=0.03), and those that suffered late embryo mortality (from days 18–40) tended to have lower insulin and progesterone concentrations than their counterparts that remained pregnant (P=0.06 and P=0.07, respectively). In this study, pregnancy in control and undernourished recipient ewes was not associated with the origin of the embryo (undernourished and control donors). In conclusion, failure to establish and maintain a pregnancy was associated with lower progesterone and insulin levels one week after estrus in recipient ewes.

Expression and localization of angiopoietin family in corpus luteum during different stages of oestrous cycle and modulatory role of angiopoietins on steroidogenesis, angiogenesis and survivability of cultured buffalo luteal cells.

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P4 ) secretion and mRNA expression of phosphotidylinositide-3kinase-protein kinase B (PI3K-AKT), phosphoinositide-dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real-time RT-PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P4 secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p<.05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p<.05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P4 secretion was highest (p<.05) with 100ng/ml at 72hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p<.05) and BAD along with caspase 3 were lowest (p<.05) at 100ng/ml at 72hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P4 secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions in buffalo CL.

Expression and localization of fibroblast growth factor (FGF) family in corpus luteum during different stages of estrous cycle and synergistic role of FGF2 and vascular endothelial growth factor (VEGF) on steroidogenesis, angiogenesis and survivability of cultured buffalo luteal cells

The aim of this study was to document the expression and localization of fibroblast growth factor (FGF) family members comprising of fibroblast growth factor (FGF1, FGF2, FGF7, FGF10), and their receptors (FGFR1, FGFR2, FGFR3, FGFR4, FGFR2IIIB, FGFR2IIIC) in buffalo corpus luteum (CL) obtained at different stages of the estrous cycle. In addition, the synergistic role of FGF2 and/or vascular endothelial growth factor (VEGF) on P4 secretion and mRNA expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 (CYP11A1), 3-beta-hydroxysteroid dehydrogenase (3βHSD), proliferating cell nuclear antigen (PCNA), BCL-2 associated X protein (BAX) and von willebrand factor (vWF) were studied in luteal cell culture obtained from mid-luteal phase (MLP) of estrous cycle in buffalo. Real-time PCR (qPCR), western blot, and immunohistochemistry were used to investigate mRNA and protein expressions, and the localization of examined factors whereas P4 secretion was assessed by RIA. The mRNA and protein expression of FGF1 and FGFR1 were maximum (P<0.05) during MLP whereas FGF2 was maximum (P<0.05) during early luteal phase (ELP). FGF7, FGF10, FGFR2, FGFR3, FGFR4, FGFR2IIIb, and FGFR2IIIc mRNA and protein expression did not change among luteal phases. FGF family members were localized in cytoplasm of luteal cells as well as in endothelial cells. P4 secretion in luteal cells treated with FGF2 or VEGF alone showed the maximum values (P<0.05) with the highest dose at 72h. P4 secretion was found to be greater (P<0.05) in luteal cells treated with FGF2+VEGF compared to FGF2 or at 72h of incubation. The mRNA expression of all factors were maximum (P<0.05) whereas BAX was minimum (P<0.05) at highest dose cultured for 72h of luteal cells subjected with either protein alone or in combination. Summarizing, the present findings explore the synergistic role of FGF2 and VEGF on steroidogenesis, angiogenesis, cell viability through an autocrine and paracrine actions in buffalo CL.

Effects of periovulatory gonadotrophin treatment on luteal function and endometrial expression of selected genes in cyclic pony mares

Progestin concentration in plasma during the early luteal phase is crucial for endometrial function and conceptus development. We hypothesized that periovulatory gonadotrophin treatment via support of luteal function affects endometrial gene expression in horses. Effect of age was analyzed as well. Shetland mares (n = 8, age 4–25 years) were assigned to the following treatments during five consecutive cycles in alternating order following a cross-over design: treatment hCG/-: preovulatory injection of hCG, but no gonadotrophin injection at detection of ovulation, treatment -/hCG: no preovulatory gonadodrophin injection, but injection of hCG at detection of ovulation, treatment eCG/-: preovulatory injection of eCG, but no gonadotrophin injection at detection of ovulation, treatment -/eCG: no preovulatory gonadotrophin injection, but injection of eCG at detection of ovulation, treatment control: no treatment. Concentration of progestin was analyzed by ELISA from the day of ovulation until Day 10. On Day 10, endometrial cells were collected transvaginally by cytobrush technique. Expression of mRNA of cyclooxygenase-2 (COX-2), prostaglandin F2α-synthase, prostaglandin E-synthase, progesterone receptor (PR), estradiol receptor (E2R), acyl-CoA-dehydrogenase (ACAD), uteroglobin (UGB), uteroferrin, and uterocalin was analyzed by RT qPCR. Immunohistological staining of endometrial tissue, obtained via biopsy, was performed for COX-2, PR and UGB. The P4 concentration was influenced by day of cycle (P < 0.01), but not by treatment. No effects of age on gene expression were determined. Neither of the periovulatory gonadotrophin treatments nor age influenced mRNA expression of the genes of interest. Treatment did also not affect immunohistological staining of the endometrium. In contrast, age affected the percentage of PR positive stromal cells (e.g. mare 1 (4 years): 65.5 ± 2.6, mare 2 (24 years): 82.7 ± 2.2%, P < 0.05) and COX-2 positive stained ciliated cells (e.g. mare 1: 15.8 ± 2.9, mare 2: 33.4 ± 6.0%, P < 0.05). In conclusion, no effects of periovulatory gonadotrophin treatment and age on endometrial gene expression in luteal phase pony mares were reported. A lack of treatment effects on luteal function and expression of PRs in the endometrium can at least in part be explained by differences in the reproductive physiology between horses and ponies.