Identification of Prolificacy-Related Differentially Expressed Proteins from Sheep (Ovis aries) Hypothalamus by Comparative Proteomics.

Reproduction, as a physiologically complex process, can significantly affect the development of the sheep industry. However, a lack of overall understanding to sheep fecundity has long blocked the progress in sheep breeding and husbandry. In the present study, the aim is to identify differentially expressed proteins (DEPs) from hypothalamus in sheep without FecB mutation in two comparison groups: polytocous (PF) versus monotocous (MF) sheep at follicular phase and polytocous (PL) versus monotocous (ML) sheep at luteal phase. Totally 5058 proteins are identified in sheep hypothalamus, where 22 in PF versus MF, and 39 proteins in PL versus ML are differentially expressed, respectively. A functional analysis is then conducted including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis to reveal the potential roles of these DEPs. The proteins ENSOARP00000020097, ENSOARP00000006714, growth hormone (GH), histone deacetylase 4 (HDAC4), and 5'-3' exoribonuclease 2 (XRN2) in PF versus MF, and bcl-2-associated athanogene 4 (BAG4), insulin-like growth factor-1 receptor (IGF1R), hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), and transthyretin (TTR) in PL versus ML appear to modulate reproduction, presumably by influencing the activities of gonadotropin-releasing hormone (GnRH). This study provides an alternative method to identify DEPs associated with sheep prolificacy from the hypothalamus. The mass spectrometry data are available via ProteomeXchange with identifier PXD013822.

Amino acids profile within peripheral blood and follicular fluid based on high-performance liquid chromatography methods may explain differences in folliculogenesis between short-term under/over-fed treatments during luteal phase of Hu sheep.

The nutritional alteration of amino acids (AAs) profile in physiological fluid was poorly characterized in livestock. After oestrus synchronization, 24 ewes were randomly assigned to two groups based on the nutrient requirement recommended for maintenance (M): the feed-supplemented group (S, 1.5 × M, N = 12) and feed-restricted group (R, 0.5 × M, N = 12) on days 6-12 of their oestrous cycle, which occurred shortly before ovulation. The concentration of 30 AAs in peripheral blood (PB) and follicular fluid (FF) was quantified to calculate the PB-to-FF concentration gap for each AA and determine its correlation with metabolites and hormones in PB and FF. Results showed that the feed restriction enlarged the oestrous cycle length, decreased the number of follicles 2.5-3.5 mm, increased the number of follicles >3.5 mm and augmented the volume of follicles >2.5 mm. Nineteen AAs from PB were significantly different between the groups. The phosphoethanolamine (PEtN) and ration of essential AAs to nonessential AAs (EAA/NEAA) in FF significantly (p < 0.05) increased and decreased in the R group, respectively. Most AAs, except aspartate (Asp) and carnosine (Car) in the R group and alanine (aAla) in both groups, were significantly lower within FF than those within PB. The correlation of AAs with FSH and progesterone (P4 ) was more significant than that of AAs with other endocrine milieu characteristics. In conclusion, our results revealed that the influence of short-term nutritional manipulation during luteal phase on folliculogenesis might not be due to the variation of intrafollicular AAs profile but rather attribute to the peripheral blood AAs profile alteration.

Leukotriene production profiles and actions in the bovine endometrium during the oestrous cycle.

We have previously shown the influence of leukotrienes (LTs) on reproductive functions in vivo: LTB4 is luteotrophic and supports corpus luteum function inducing PGE2 and progesterone (P4) secretion, whereas LTC4 is luteolytic and stimulates PGF2α secretion in cattle. The aim of this study was to examine expression and production profiles of LTs and their actions in the endometrium. LT receptors (LTB4R for LTB4 and CysLTR2 for LTC4), 5-lipoxygenase (LO), 12-LO synthase (LTCS) and LTA4 hydrolase (LTAH) mRNA and protein expression, as well as LT production were measured in bovine endometrial tissue during the luteal phases of the oestrous cycle. The action of LTs on uterine function was studied by measuring the level of PGs after stimulating uterine slices with LTs on Days 8-10 of the cycle. Expression of 5-LO and LTB4R mRNA and protein were highest on Days 2-4 of the cycle, while CysLTR2 and LTCS were highest on Days 16-18 (P<0.05). LTB4 concentration was highest on Days 2-4 of the cycle, whereas the greatest LTC4 level was on Days 16-18 (P<0.05). Both LTB4 and C4 increased the content of PGE2 and F2α in endometrial slices at a dose of 10(-7)M (P<0.05). In summary, mRNA expression and activation of receptors for LTB4 and production occur in the first part of the cycle, whereas LTC4 and its receptors predominate at the end of the cycle. The 12-LO and 5-LO pathways are complementary routes of LT production in the bovine uterus.

Pathological postpartum breast engorgement: prediction, prevention, and resolution.

BACKGROUND: Severe breast engorgement can cause substantial discomfort for mothers and interfere with an infant's ability to feed at the breast. This study explored the possibility of prediction of pathological postpartum breast engorgement in lactating women in relation to intense breast engorgement at the end of the luteal phase of the menstrual cycle and the possibility of prevention and resolution of postpartum breast engorgement with expression with a breast pump of colostrum before the appearance of transitional milk. SUBJECTS AND METHODS: The first group included 70 women with pathological postpartum breast engorgement. The second group included 52 postpartum women, with 24 women having colostrum extracted by the breast pump from each breast once or twice for a duration of 20-25 minutes sequentially in the first 2-3 days after delivery in addition to the removal of colostrum by the baby, before engorgement developed. Twenty-eight women had colostrum removed only by the baby. The degree of breast engorgement was assessed using the previously published Robson four-level scale. RESULTS: Of the 70 patients with severe postpartum engorgement studied in the first group, 90% showed intense breast engorgement in the late luteal phase of the menstrual cycle. Expression of colostrum milk in the first experimental group from each breast eliminated excessive breast engorgement in breastfeeding mothers. CONCLUSIONS: Presence of intense breast engorgement at the end of the luteal phase of the menstrual cycle may be one of the most important indicators useful for predicting severe postpartum breast engorgement. Extraction of colostrum before the appearance of transitional milk lowers the risk of excessive engorgement in breastfeeding women.

Genistein and daidzein affect in vitro steroidogenesis but not gene expression of steroidogenic enzymes in adrenals of pigs.

Phytoestrogens (PEs), including genistein and daidzein, are plant-derived substances that mimic or antagonize estrogen action in animals. The majority of studies investigated the effects of PEs on reproduction in humans and laboratory animals. The mechanisms of phytoestrogen action on reproductive processes in domesticated animals, including pigs, are garnering increasing attention. However, very few in vivo and in vitro studies investigating the effects of PEs on adrenal glands have been carried out on models other than humans and rats. The aim of the present study was to determine whether the effects of genistein and daidzein on adrenal in vitro steroidogenesis are accompanied by changes in expression of genes encoding key steroidogenic enzymes in porcine adrenocortical cells. The following genes were analyzed: cholesterol side-chain cleavage enzyme (P450scc, CYP11A1 gene), 3ß-hydroxysteroid dehydrogenase (3ß-HSD, HSD3B1 gene), 17α-hydroxylase/C17-20 lyase (P450c17, CYP17A1 gene) and 21-hydroxylase (P450c21, CYP21A2 gene). Porcine adrenocortical cells collected from both luteal- and follicular-phase gilts were exposed for eight hours to genistein (10 µM), or daidzein (10 µM), in the absence or presence of ACTH (5 nM). Genistein and daidzein inhibited basal and ACTH-stimulated secretion of cortisol and corticosterone and stimulated secretion of androstenedione. PEs did not affect the expression of CYP11A1, HSD3B1, CYP17A1 and CYP21A2 in the adrenocortical cells of luteal- and follicular-phase gilts. It can be concluded that the influence of PEs on steroid secretion in porcine adrenal glands is not mediated by changes in the expression of genes encoding major steroidogenic enzymes. More studies are needed to elucidate the intracellular mechanisms leading to the PE-induced changes in adrenal steroidogenesis in pigs.

Vitex agnus-castus extracts for female reproductive disorders: a systematic review of clinical trials.

Vitex agnus-castus L. (chaste tree; chasteberry) is a popular herbal treatment, predominantly used for a range of female reproductive conditions in Anglo-American and European practice. The objective of this systematic review was to evaluate the evidence for the efficacy and safety of Vitex extracts from randomised, controlled trials investigating women's health.Eight databases were searched using Latin and common names for Vitex and phytotherapeutic preparations of the herb as a sole agent, together with filters for randomised, controlled trials or clinical trials. Methodological quality was assessed according to the Cochrane risk of bias and Jadad scales, as well as the proposed elaboration of CONSORT for reporting trials on herbal interventions.Thirteen randomised, controlled trials were identified and twelve are included in this review, of which eight investigated premenstrual syndrome, two premenstrual dysphoric disorder, and two latent hyperprolactinaemia. For premenstrual syndrome, seven of eight trials found Vitex extracts to be superior to placebo (5 of 6 studies), pyridoxine (1), and magnesium oxide (1). In premenstrual dysphoric disorder, one study reported Vitex to be equivalent to fluoxetine, while in the other, fluoxetine outperformed Vitex. In latent hyperprolactinaemia, one trial reported it to be superior to placebo for reducing TRH-stimulated prolactin secretion, normalising a shortened luteal phase, increasing mid-luteal progesterone and 17ß-oestradiol levels, while the other found Vitex comparable to bromocriptine for reducing serum prolactin levels and ameliorating cyclic mastalgia. Adverse events with Vitex were mild and generally infrequent. The methodological quality of the included studies varied, but was generally moderate-to-high. Limitations include small sample sizes in some studies, heterogeneity of conditions being treated, and a range of reference treatments.Despite some methodological limitations, the results from randomised, controlled trials to date suggest benefits for Vitex extracts in the treatment of premenstrual syndrome, premenstrual dysphoric disorder and latent hyperprolactinaemia. Further research is recommended, and greater transparency in reporting for future trials.

Effects of cycle stage on regionalised galanin, galanin receptors 1-3, GNRH and GNRH receptor mRNA expression in the ovine hypothalamus.

The neurotransmitter galanin has been implicated in the steroidogenic regulation of reproduction based on work mainly conducted in rodents. This study investigated the temporal changes in the expression of galanin and its three receptor isoforms and GNRH and GNRHR mRNA in specific hypothalamic nuclei known to be involved in the regulation of reproductive cyclicity, namely the medial pre-optic area (mPOA), the rostral mPOA/organum vasculosum of the lamina terminalis, the paraventricular nucleus and the arcuate nucleus using an ovine model. Following synchronisation of their oestrous cycles, tissues were collected from ewes at five time points: the early follicular, mid follicular (MF) and late follicular phases and the early luteal and mid luteal phases. The results indicated significant differences in regional expression of most of the genes studied, with galanin mRNA expression being highest during the MF phase at the start of the GNRH/LH surge and the expression of the three galanin receptor (GalR) isoforms and GNRH and its receptor highest during the luteal phase. These findings are consistent with a role for galanin in the positive feedback effects of oestradiol (E(2)) on GNRH secretion and a role for progesterone induced changes in the pattern of expression of GalRs in the regulation of the timing of E(2)'s positive feedback through increased sensitivity of galanin-sensitive systems to secreted galanin.

Effect of different levels of short-term feed intake on folliculogenesis and follicular fluid and plasma concentrations of lactate dehydrogenase, glucose, and hormones in Hu sheep during the luteal phase.

This study investigated the effects of short-term food restriction or supplementation on folliculogenesis and plasma and intrafollicular metabolite and hormone concentrations. Ewes were randomly assigned to three groups: the control group received a maintenance diet (M) while the supplemented group and restricted group received 1.5×M and 0.5×M respectively on days 6-12 of their estrous cycle. Estrus was synchronized by intravaginal progestogen sponges for 12 days. On days 7-12, blood samples were taken. After slaughter, the ovarian follicles were classified and the follicular fluid was collected. Compared with restriction, supplementation shortened the estrous cycle length, decreased the number of follicles 2.5-3.5 mm and follicular fluid estradiol (E2) concentration, increased the number of follicles>3.5 mm and plasma glucose, insulin and glucagon concentrations, and augmented the volume of follicles>2.5 mm. Restricted ewes had higher intrafollicular insulin concentration, but it was similar to that of supplemented ewes. Compared with follicles≤2.5 mm, the intrafollicular glucose and E2 concentrations were increased and the testosterone, insulin, and glucagon concentrations and lactate dehydrogenase (LDH) activity were decreased in follicles>2.5 mm. Only in restricted ewes were intrafollicular LDH and testosterone concentrations in follicles≤2.5 mm not different from those in follicles≤2.5 mm. In conclusion, the mechanism by which short-term dietary restriction inhibits folliculogenesis may involve responses to intrafollicular increased E2, testosterone, and LDH levels in late-stage follicles. This may not be due to the variation of intrafollicular insulin level but rather due to decreased circulating levels of glucose, insulin, and glucagon.

Lower levels of prepulse inhibition in luteal phase cycling women in comparison with postmenopausal women.

Menopause denotes the end of the reproductive period in a woman's life and is characterized by gradually declining plasma levels of ovarian hormones. Mounting evidence suggests that prepulse inhibition (PPI) is sensitive to fluctuations in estradiol and progesterone. Deficits in PPI are associated with conditions characterized by increased levels of ovarian steroids, such as the mid-luteal phase of the menstrual cycle and the third trimester of pregnancy. The aim of the current study was to further elucidate ovarian steroid-related effects on PPI by examining 43 women with regular menstrual cycles, 20 healthy postmenopausal women without hormone replacement treatment (HRT) and 21 healthy postmenopausal women with ongoing estradiol-only or estradiol and progesterone therapy (EPT). Cycling women were tested during the late luteal phase of the menstrual cycle while postmenopausal women were tested on any arbitrary day. The PPI was measured by electromyography. Cycling women exhibited lower levels of PPI than postmenopausal women (p<0.05). There were no differences in PPI between postmenopausal HRT users and non-users. However, postmenopausal women with estradiol serum concentrations in the cycling range had lower PPI than postmenopausal women with low estradiol concentrations (groupxPPI interaction, p<0.05). In conclusion, the results further suggest a role for the ovarian steroids in PPI regulation as PPI is increased in postmenopausal women in comparison to regularly menstruating women examined during the late luteal phase. Furthermore, postmenopausal women with estradiol levels in the cycling range had lower PPI than postmenopausal women with low estradiol levels.

Red clover (Trifolium pratense) isoflavones and serum homocysteine in premenopausal women: a pilot study.

BACKGROUND: There is limited information on the effect of isoflavones on homocysteine concentrations, a risk factor for a number of chronic diseases. METHODS: Twenty-three premenopausal women participated in a double-blind, randomized, parallel study for four menstrual cycles. Subjects consumed either placebo or purified red clover (Trifolium pratense) isoflavone (86 mg/day) tablets. Blood samples were collected weekly during cycles 1, 3, and 4 for determination of serum folate and total homocysteine concentrations. Dietary intake was monitored monthly. RESULTS: Concentrations of folate and homocysteine in serum did not change significantly in either group, and there were no significant differences observed between the follicular and luteal phases of the menstrual cycle. The participants' dietary records indicated that nutrient intake was constant, and compliance was confirmed by analysis of urinary isoflavone concentrations and tablet counts in returned containers. CONCLUSIONS: These results suggest that in the absence of any dietary modification, supplementation with purified isoflavones that are predominantly methoxylated has no effect on serum homocysteine or folate in premenopausal women.

Leptin and long form of leptin receptor genes expression in the hypothalamus and pituitary during the luteal phase and early pregnancy in pigs.

Leptin is a polypeptide that plays a key role in the regulation of energy homeostasis and is also linked, among others, to mechanisms controlling reproductive processes. Data concerning the involvement of leptin in controlling reproductive functions at the level of hypothalamus and pituitary in the pig are limited. Therefore, in the present study, an expression of genes coding for leptin and long-form leptin receptor (Ob-Rb) was determined by a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) in the discrete areas of porcine hypothalamus (medial basal hypothalamus - MBH, preoptic area - POA, stalk median eminence - SME) and pituitary (anterior - AP and posterior/neural - NP parts) during the luteal phase of the cycle (days 10-12 and 14-16) and two early stages of pregnancy (days 14-16 and 30-32). Leptin gene expression in MBH was found to be higher in the mid- than in the late-luteal phase, whereas in other structures studied it remained unchanged during these periods. More pronounced differences were noted in expression of Ob-Rb gene, which was increased in MBH, AP and NP during the late-luteal phase in comparison to the mid-luteal one, whilst the relationship in the POA was reversed. In turn, during pregnancy, leptin gene expression in all tested areas of hypothalamus as well as Ob-Rb mRNA content in MBH were higher on days 30-32 than on days 14-16. In contrast, in the anterior pituitary, Ob-Rb gene expression was more pronounced on days 14-16 than during later stage of pregnancy. Comparison of leptin and Ob-Rb mRNA content in studied structures between the mid-luteal phase and days 14-16 of pregnancy revealed inhibition of leptin gene expression in almost all examined tissues (MBH, POA, SME, NP) during early pregnancy whereas Ob-Rb gene expression was inhibited in POA but stimulated in both parts of the pituitary during this stage. In summary, obtained results suggest an involvement of leptin in the regulation of hypothalamic-pituitary axis activity during both the luteal phase of the cycle and early pregnancy in pigs.

Effective stimulation of daily LH secretion by the combined treatment with melatonin and naloxone in luteal-phase ewes.

This study was designed to test the hypothesis that melatonin would intensify daily LH release after central blockade of the opiate receptors in sexually active ewes. The intracerebroventricular infusions of vehicle (control), melatonin, naloxone and melatonin in combination with naloxone were made in ewes in the luteal phase of the estrous cycle, from 2:00 P.M. to 5:00 P.M. Blood samples were collected from 11:00 A.M. to 8:00 P.M. at 10-min intervals. The mean plasma LH concentrations were measured before, during and after the infusions. The frequency and amplitude of LH pulses were determined during the whole experimental period. The LH concentrations recorded during melatonin or naloxone infusions were significantly higher than the concomitant concentration in vehicle-infused animals. The mean LH pulse amplitude in melatonin- and naloxone-treated ewes was also significantly higher than in controls. The LH concentration measured during the combined infusion of melatonin and naloxone was significantly higher than that during vehicle infusion. The LH concentration recorded in turn after the treatment was significantly higher than the concomitant concentrations in vehicle-, melatonin- and naloxone-infused animals. The mean LH pulse amplitude in this group was significantly higher than in the vehicle-infused group. These results indicate that blockade of the opiate receptors within the CNS facilitated effective stimulation of daily LH secretion by exogenous melatonin. In conclusion, a relationship between melatonin and endogenous opioid peptides may be crucial in enabling melatonin to exhibit stimulatory action on LH secretion during the luteal phase of the estrous cycle in ewes.

ACTH treatment disrupts ovarian IGF-I and steroid hormone production.

Hyper-adrenal activity and increased glucocorticoid hormone release are associated with disruptions in reproductive function and adverse effects on the ovary. The aim of this investigation was to determine whether elevated glucocorticoid hormone levels can influence ovarian IGF-I synthesis and action in vivo. To elevate endogenous glucocorticoid levels, gilts were treated with ACTH during the luteal phase of the oestrous cycle (days 9-13) while the control group received saline. The gilts were subsequently ovariectomized on either day 14 or day 18 of the oestrous cycle. Follicular fluid (FF) was collected from individual follicles; IGF-I and steroid hormone concentrations were determined by radioimmunoassay, and IGF-binding protein (IGFBP) expression was assessed by Western ligand blotting. Granulosa cells were also recovered and placed in culture to determine IGF-I, progesterone (P(4)) and oestradiol-17beta (E(2)) production levels. The cells were cultured in serum-free medium for 5 days and supplemented with: (a) media alone, (b) IGF-I, (c) FSH and androstenedione (A(4)), or (d) IGF-I with FSH and A(4). The FF from ACTH-treated gilts was characterized by elevated (P<0.05) cortisol levels on day 14 and lower (P<0.05) E(2) values on both day 14 and day 18. Lower (P<0.05) IGF-I concentrations were also measured in the FF of ACTH-treated gilts collected on day 18. This altered hormone profile in FF was associated with impaired IGF-I and steroid hormone synthesis by granulosa cells. IGF-stimulated P(4) production (P<0.01) by cells recovered from ACTH-treated gilts on day 14 was lower (P<0.05). By day 18, IGF-I, P(4) and E(2) production by cells from the ACTH group were all significantly (P<0. 05) lower. These results demonstrate that increased glucocorticoid concentrations can disrupt subsequent ovarian IGF-I synthesis and IGF action in vivo and can, potentially, impair follicle maturation.

Regulation of ovarian hyperluteinization.

Adult female rats with neonatally damaged posterior hypothalamus, made by a transversal cut, were investigated. Plasma levels of prolactin (PRL), gonadotropic hormones (GTH) and female gonadal steroids (GS) were determined by radioimmunoassay. The animals were sacrificed, at the ages of 4 and 6 months and their hypothalamus, pituitary gland, ovary and uterus were examined using light microscopy. The results can be summarized as follows: body mass of animals, with damaged posterior hypothalamus, was significantly reduced. Masses of luteinized ovaries were increased and uterine tissues decreased. Serum levels of PRL were significantly increased and luteinizing hormone (LH) decreased. Ultrastructural changes in the corpora lutea (CL), previously described, showed clear signs of their reduced capacities to produce GS, both estradiol (Oe) and progesterone (Pg) per total ovarian mass. However, prostaglandin 2 alfa (PGF2alpha) known as a luteolytic factor, was also diminished in the evidently retarded endometrium. As a result of decreased plasma values of LH, Pg and PGF2 alpha, luteolysis of CL in hyperluteinized ovaries did not occur, and their new generations were accumulated during subsequent cycles. The character of interruption and recovery of aminergic and peptidergic neurons, involved in regulation of hypothalamic-pituitary gonadal axis and feed-back effects of steroid hormones, require further studies.

Localization of interleukin-1 type I receptor and interleukin-1 beta in human endometrium throughout the menstrual cycle.

Previous studies in the human suggest that the interleukin-1 (IL-1) system, may be an important paracrine/autocrine mediator in local intercellular interaction in endometrial tissue. In this study we have determined that IL-1 receptor type I (IL-1R tI) is expressed at the messenger RNA (mRNA) and protein levels in glandular cells and its ligand, IL-1 beta has been localized by immunohistochemical methods in endothelial cells and isolated stromal cells in the human endometrium throughout the menstrual cycle. IL-1R tI mRNA was detected in glandular epithelium using both specific complementary DNA and complementary RNA 32P-labeled probes. Human glandular epithelium contains a 5.1-kilobase mRNA transcript throughout the complete menstrual cycle. Quantitative densitometric analysis of slot blot hybridization signals shows an increase of IL-1R tI mRNA in both early and mid-late secretory phases in comparison with the proliferative phase (P < 0.05). IL-1R tI protein was localized in endometrial glandular epithelial cells using both indirect immunofluorescence and avidin-biotin-peroxidase methods. However, more intense staining for IL-1R tI was observed in lumenal epithelial cells compared with the staining present deep in the endometrial glands. Using the same methods, IL-1 beta was detected in endothelial cells of spiral vessels and isolated stromal cells throughout the menstrual cycle, and an increased staining from proliferative to secretory phase was observed. The detection of IL-1R tI in the human endometrial epithelium and its ligand, IL-1 beta, in isolated stromal cells and endothelial cells, is another example of possible communication between the immune and reproductive systems with special relevance to human implantation.