Preovulatory Phase Research Articles

Effects of management and health on the use of activity monitoring for estrus detection in dairy cows

The aim was to investigate 1) the relationship between the physical activity index created for each cow by activity monitoring systems and the identification of the preovulatory follicular phase, and 2) the influence of various production, health, and cow factors on the relationship between physical activity and estrous behavior. Eighty-nine spring calving cows, on pasture, were monitored during the breeding season using the neck-mounted estrous activity monitor Heatime (SCR Engineers Ltd., Netanya, Israel). Milk samples were collected twice weekly for progesterone assay to characterize resumption of reproductive activity. Reproductive tract health was assessed weekly by ultrasonography and vaginal mucus scoring. Body condition score and milk yield were assessed every 2 wk. Heatime identified 72% of preovulatory follicular phases from which 145 inseminations resulted in 69 conceptions; 32% of activity clusters were associated with high-progesterone states (i.e., false positives). By inclusion of a 6 to 8-h duration threshold and maintaining the borderline peak activity threshold, this was improved to 87.5% with 21.3% false positives. Mean (± standard error of the mean) peak activity and cluster duration (19.3±0.53 and 10.8±0.38, respectively) were highest for the second or subsequent preovulatory follicular phases followed, in descending order, by those during first preovulatory follicular phases (14.8±2.13 and 8.4±1.4, respectively) and high progesterone states (8.0±0.47 and 3.0±0.42, respectively). The odds of an activity cluster being in a preovulatory follicular phase rather than a high-progesterone phase improved by 29% for every 1-unit increase in peak activity and by 91% for every 2-h increase in duration. The probability of an activity cluster detecting a preovulatory follicular phase was improved if it was a second or subsequent follicular phase, if body condition score was 0.25 units higher, if milk yield was 10kg lower, and uterine infection was absent. Conception rate was influenced by insemination on the same day (52%) or day after a cluster (34.3%); inseminations were carried out using the a.m.-p.m. rule. Advances in the development of more accurate automatic monitoring of the preovulatory follicular phase will aid the timing of insemination and, thus, improve conception rates.

Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function, two different animal models were used. Experiment 1 compared Holstein-Friesian nonlactating heifers (n = 17) and lactating cows (n = 16) at three stages of preovulatory follicle development: 1) newly selected dominant follicle in the luteal phase (Selection), 2) follicular phase before the LH surge (Differentiation), and 3) preovulatory phase after the LH surge (Luteinization). Experiment 2 compared newly selected dominant follicles in the luteal phase in beef heifers fed a diet of 1.2 times maintenance (M, n = 8) or 0.4 M (n = 11). Lactating cows and 0.4 M beef heifers had higher concentrations of β-hydroxybutyrate, and lower concentrations of glucose, insulin, and IGF-I compared with dairy heifers and 1.2 M beef heifers, respectively. In lactating cows this altered metabolic environment was associated with reduced dominant follicle estradiol and progesterone synthesis during Differentiation and Luteinization, respectively, and in 0.4 M beef heifers with reduced dominant follicle estradiol synthesis. Using a combination of RNA sequencing, Ingenuity Pathway Analysis, and qRT-PCR validation, we identified several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, to be downregulated by the catabolic state. Based on this, we propose that the adverse metabolic environment caused by lactation or nutritional restriction decreases preovulatory follicle function mainly by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.

Sex Differences, Hormone Fluctuations, Ankle Stability, and Dynamic Postural Control

Hormonal fluctuation as a risk factor in anterior cruciate ligament injury has been investigated with conflicting results. However, the influence of hormone fluctuations on ankle laxity and function has not been thoroughly examined. To examine the potential hormone contributions to ankle laxity and dynamic postural control during the preovulatory and postovulatory phases of the menstrual cycle using an ankle arthrometer and the Star Excursion Balance Test in healthy women. The cohort group consisted of male control participants. Cohort study. Research laboratory. Twenty healthy women (age = 23.8 ± 6.50 years, height = 163.88 ± 8.28 cm, mass = 63.08 ± 12.38 kg) and 20 healthy men (age = 23.90 ± 4.15 years, height = 177.07 ± 7.60 cm, mass = 80.57 ± 12.20 kg). Ankle stability was assessed with anterior-posterior and inversion-eversion loading. Dynamic postural control was assessed with the posteromedial reaching distance of the Star Excursion Balance Test. Female participants used ovulation kits for 3 months to determine the time of ovulation; during their preovulatory and postovulatory phases, they were tested in the laboratory with an ankle arthrometer and the Star Excursion Balance Test. Male participants were tested on similar dates as controls. For each dependent variable, a time by side by sex repeated-measures analysis of variance was performed. Statistical significance was set a priori at P < .05. For anterior-posterior laxity, a side main effect was noted (F₁,₃₈ = 10.93, P = .002). For inversion-eversion laxity, a sex main effect was seen (F₁,₃₈ = 10.75, P = .002). For the posteromedial reaching task, a sex main effect was demonstrated (F₁,₃₈ = 8.72, P = .005). No influences of time on the dependent variables were evident. Although women presented with more ankle inversion-eversion laxity and less dynamic postural control, hormonal fluctuations during the menstrual cycle (preovulatory compared with postovulatory) did not affect ankle laxity or dynamic postural control, 2 factors that are associated with ankle instability.

Localization of adiponectin and its receptor and its possible roles in the ovary of a vespertilionid bat, Scotophilus heathi

The aim of this study was to evaluate the seasonal variation in serum adiponectin levels and ovarian expression of adiponectin and its receptor in the Scotophilus heathi bat and their relationship to the changes in the body fat mass, serum insulin and luteinizing hormone (LH) levels, and ovarian activity. A very high level of circulating adiponectin was found during recrudescence, whereas a very low level of circulating adiponectin was observed during pre-ovulatory period. The increased circulating adiponectin level coincided with fat deposition, insulin resistance and hyperinsulinemia (HI) in S. heathi. Immunocytochemical study in the ovary of bat showed localization of adiponectin mainly in thecal-interstitial cells (TICs), and adiponectin receptor 1 (AdipoR1) in the granulosa cells of growing follicles, but showed no staining in atretic follicle. Seasonal changes in ovarian adiponectin and AdipoR1 levels showed two peaks (during recrudescence and ovulatory phases) coinciding with two periods of follicular development. Adiponectin in the absence of LH, as occur during recrudescence, stimulates androstenedione (A4) synthesis in vitro by up-regulating the insulin receptor (IR). Adiponectin in presence of LH, as occur during pre-ovulatory phase, inhibits A4 synthesis in vitro by down-regulating androgen receptor (AR). Further, the in vitro study showed that adiponectin, in presence of LH, also promotes luteinizing hormone receptor (LH-R) and steroidogenic acute regulatory protein (StAR) level in the ovary, which might help in development of ovulatory follicles. In brief, adiponectin in the absence of LH induces increased A4 synthesis and consequently the condition of delayed ovulation, whereas in the presence LH suppresses both synthesis and action of A4 and thus induces preovulatory condition in the ovary of S. heathi.

Ovulation detection through salivary levels of sialic acidand glycosaminoglycans

Background: One in ten couples of reproductive age encounter some level of infertility. Identification of the period of ovulation in humans is critical in the treatment of infertility. Success in in vitro fertilization and embryo transfer has been associated with the exact time of ovulation. Saliva is a unique diagnostic fluid, the composition of which immediately reflects the sympathetic nervous system, parasympathetic nervous system, hypothalamic- pitutaryadrenal axis and immune system response to stress. The study aims at evaluating the changes in salivary sialic acid and Glycosaminoglycans in the regular menstrual cycle. Thus, the presence of these carbohydrates in the ovulatory saliva makes the possibility to develop a biomarker for the detection of ovulation by noninvasive methods. Subjects, materials and methods: Randomly, seventy five volunteer females were recruited and divided into 5 groups; each contains 15 subjects as follow: Nine years old females and postmenopausal females as control groups, pre-ovulatory period, ovulatory period and post-ovulatory period females as experimental groups. Each female, of the experimental groups, underwent sonographic examination to estimate her period regarding ovulation. Unstimulated whole saliva was collected using the spitting method. Colorimetric procedure was used for total sialic acid determination and for Glycosaminoglycans quantitative determination, the method of ELISA was used. Results: The concentration of sialic acid was significantly decreased in saliva of females in the ovulatory phase of the menstrual cycle; whereas, a significant increase in salivary sialic acid concentration was in the post-ovulatory phase. Glycosaminoglycan concentration showed a gradual increase from the pre-ovulatory phase then ovulatory to reach its maximum in the post-ovulatory phase with a significant difference between the pre-ovulatory and post-ovulatory phases. A significant correlation was not found between sialic acid and Glycosaminoglycans in different study groups. Conclusions: On the basis of the results arrived at, the study concluded that there are remarkable cyclic variations in sialic acid and glycosaminoglycans during the menstrual cycle but in conclusion, glycosaminoglycans and sialic acid salivary levels cannot be used for the precise prediction of ovulation.

The elasticity of cervical-vaginal secretions is abnormal in polycystic ovary syndrome: Case report of five PCOS women

Polycystic Ovary Syndrome (PCOS) is a leading cause of infertility. We studied the rheological properties of cervical-vaginal secretions in five PCOS patients during variable treatment intervals with metformin. Five ovulatory normal women for a total of thirteen cycles served as control patients. All subjects collected daily cervical-vaginal fluid(CVF) samples with an aspirator, and CVF samples were characterized by a flow metric, die swell ratio (B), which measured CVF elasticity. Ovulatory cycles were indexed to the day of positive urine or serum LH (luteinizing hormone). CVF B levels from ovulatory normal women had a characteristic concave parabolic pattern of a minimum near the day of ovulation with higher values outside the periovulatory phase. In contrast, the five PCOS patients with or without metformin treatment had no periodicity of CVF B, and their CVF B levels were typically less compared to those in the early preovulatory and luteal phases of normal patients.

Doppler sonography of the uterine and ovarian arteries during a superovulatory program in horses

The aim of the present study was to investigate the effects of a gonadotropin treatment to induce superovulation on ovarian and uterine blood flow and its relationship with steroid hormone levels and ovarian response in mares, using color Doppler sonography. Each of six mares were examined sonographically in five cycles for 3 d (t1 to t3) during the follicular development phase (FDP) beginning at a follicle size of ≥ 22 mm, and for 4 d (D-4 to D-1; D0 = Ovulation) in the preovulatory phase (POP). After each examination, total estrogens (Etot) and progesterone (P4) levels were determined in peripheral plasma. Cycles 1, 3, and 5 (c1, c3, c5) were unstimulated cycles (USC); in c2 and c4, the mares were stimulated (SC) with eFSH and inseminated when in estrus at 12 and 24 h after hCG administration. Embryo recovery was performed 6.5 d post ovulation. Cycle 5 c5 was an unstimulated cycle with hCG treatment, insemination, and embryo recovery. Ovarian and uterine blood flow was quantified by the blood flow volume (BFV) and the pulsatility index (PI) in ovarian and uterine arteries. The mean number of ovulations and developing CL was 1.3 ± 0.4 in USC and 4.4 ± 3.1 in stimulated cycles (SC) with no difference (P ≥ 0.05) between the ovaries within mares. No difference (P > 0.05) was observed in utBFV and utPI during FDP between USC and SC, but during POP, utPI was lower (P < 0.05) and utBFV higher (P < 0.001) in SC than USC. The ovBFV was higher (P < 0.01) and ovPI lower (P < 0.05) in SC compared to USC. All uterine and ovarian blood flow parameters were related to the number of developing follicles in SC. Parameters utPI (r = −0.67; P < 0.001) and ovPI (r = −0.53; P < 0.001) were negatively correlated with the number of ovulations on t3, and with the number of collected embryos on t3 (utPI: r = −0.81; P < 0.001), D-4 (utPI: r = −0.64; P < 0.0001), and D-1 (ovPI: r = −0.41; P < 0.01). P4 levels were not positively correlated with utBFV (P > 0.05), but Etot concentrations (D-4: r = 0.790; D3: r = 0.639; P < 0.001; D-1: r = 0.48; P < 0.001) and ovBFV from D-4 to D-1 (r = 0.64; P < 0.001) in SC were. The results of the present study show that in mares treatment with gonadotropins to induce superovulation is associated with a marked increase in uterine and ovarian perfusion, concurrent with the development of multiple follicles and an increase in Etot levels. The increased blood flow seems to be related to the effectiveness of ovarian response to stimulation.

Pre- and Postovulatory Auditory Brainstem Response in Normal Women

Studies with ovarian hormones on auditory brainstem response (ABR) have conflicting reports although women have ABR shorter than men. This study compared ABR between pre- and postovulatory phases of menstrual cycle in consenting 40 healthy female volunteers (age 19±2.35years). The study was conducted under standard laboratory conditions (room temperature 26±2°C) using Neuropack (Nihon Kohden machine, NM-420S; H636, Japan) and earphone dynamic receiver ELEGA (Type DR-531; no.237, Japan) in sound proof room. Ears were stimulated simultaneously using standard protocol. ABR was recorded in pre- and postovulatory phases. Ovulation was estimated by measuring basal body temperature. The ABR wave latencies (WLs) I, II, III, IV, V and inter-peak latencies (IPLs) I-III, III-V, I-V were compared between the two phases of menstrual cycle using paired t test. The postovulatory phase had shorter WL V (5.71±0.18ms vs. 5.81±0.19ms, p<0.01), IPL III-V (1.89±0.16ms vs. 1.94±0.19ms, p<0.05), and I-V (3.88±0.16ms vs. 3.95±0.18ms, p<0.05) than in preovulatory. Other WLs and IPLs showed decreasing trend in postovulatory phase. ABR is better in postovulatory phase as compared to preovulatory phase probably due to progesterone hormone which might be involved in modulating auditory hearing pathway at postovulatory phase.

Premenstrual asthma and leukotriene variations in the menstrual cycle

Several authors have reported an increase in leukotriene C4 in the premenstrual phase in women with severe premenstrual asthma, indicating that antileukotrienes could be used in treatment. To analyse the role of leukotrienes in premenstrual asthma. A questionnaire on respiratory symptoms and peak flow during one complete menstrual cycle was given to women of fertile age to define them as asthmatics who suffered from premenstrual asthma or not. Premenstrual asthma (PMA) was defined as a clinical or functional deterioration (≥20%) in the premenstrual phase compared with the preovulatory phase. Blood samples to measure leukotriene C4 were taken during the preovulatory and premenstrual phases. Blood samples were taken in 62 asthmatic women, 34 of whom (54.3%) presented PMA criteria, all with a premenstrual deterioration of between 20 and 40%. There was no difference in leukotriene C4 levels between the preovulatory and premenstrual phases in the women who suffered from PMA (1.50ng/mL vs. 1.31ng/mL; p=0.32) and those who did not (1.40ng/mL vs. 1.29ng/mL; p=0.62). Neither were there any differences in leukotriene levels between women with or without PMA. The results were similar for each category of asthma severity. Our data show that leukotriene C4 does not appear to be involved in the pathogenesis of premenstrual asthma, or support the use of anti-leukotrienes in the specific treatment of premenstrual asthma, at least in women with a moderate premenstrual deterioration. No differences appeared in any of the categories of asthma severity.

Dynamique de l’index de vascularisation et de flux dans la phase préovulatoire de l’hyperstimulation ovarienne contrôlée. Relation avec le taux de conception

ObjectivesTo study the evolution of subendometrial vascularization flow index (VFI) in controlled ovarian hyperstimulation (COH+IIU) cycles and compare its dynamic changes in pregnant and non pregnant women. Patients and methodsThis is a retrospective study on 61 couples. To determine the profile of IVF just before ovulation, patients had 3D-Power Doppler angiography (3D-PDA) in this precise preovulatory phase. ResultsWe observed a decreasing profile of VFI in 52% (32/61) of the main group cycles, in 45% (14/31) of the subgroup with spontaneous peak of LH (luteinizing hormone), and in 60% (18/30) of the hCG (human chorionic gonadotropin) trigger subgroup. There were ten conceptions, (10/61, 16.4%), including eight (8/32, 30%) that were associated with a decreasing profile of VFI versus two (2/29, 7%) that were associated with an increasing profile (NS). In the detection of LH peak subgroup, we observed five conceptions (5/14, 36%) associated with a decreasing profile versus one conception (1/17, 6%) associated with an increasing profile of VFI (OR: 8.3; confidence interval [CI] 95%: 0.8–444; P=0.06 in favor of a of decreasing VFI profile). In the hCG subgroup, there were three conceptions in the decreasing VFI profile (3/18, 17%) and one conception with increasing VFI (1/12, 17%, P=0.6). Discussion and conclusionIn controlled ovarian hyperstimulation, a decreasing VFI profile is more common compared to an increasing one. Such au profile seems to be associated with a higher conception rate in cycles with spontaneous LH surge.

The Effects of Kisspeptin-10 on Reproductive Hormone Release Show Sexual Dimorphism in Humans

Kisspeptin peptides are critical in human reproductive physiology and are potential therapies for infertility. Kisspeptin-10 stimulates gonadotropin release in both male and female rodents. However, few studies have investigated the effects of kisspeptin-10 on gonadotropin release in humans, and none have investigated the effect in women. If kisspeptin is to be useful for treating reproductive disease, its effects in both men and women must be established. To compare the effects of kisspeptin-10 administration on reproductive hormone release in healthy men and women. Intravenous bolus kisspeptin-10 was administered to men and women (n = 4-5 per group). Subcutaneous bolus and i.v. infusion of kisspeptin-10 was also administered to female women (n = 4-5 per group). Circulating reproductive hormones were measured. In healthy men, serum LH and FSH were elevated after i.v. bolus kisspeptin-10, at doses as low as 0.3 and 1.0 nmol/kg, respectively. In healthy women during the follicular phase of the menstrual cycle, no alterations in serum gonadotropins were observed after i.v. bolus, s.c. bolus, or i.v. infusion of kisspeptin-10 at maximal doses of 10 nmol/kg, 32 nmol/kg, and 720 pmol/kg/min, respectively. In women during the preovulatory phase, serum LH and FSH were elevated after i.v. bolus kisspeptin-10 (10 nmol/kg). Kisspeptin-10 stimulates gonadotropin release in men as well as women during the preovulatory phase of menstrual cycle but fails to stimulate gonadotropin release in women during the follicular phase. The sexual dimorphism of the responsiveness of healthy men and women to kisspeptin-10 administration has important clinical implications for the potential of kisspeptin-10 to treat disorders of reproduction.

The state-of-the-art of emergency contraception with the cutting edge drug.

The objective of this study is to evaluate and elucidated the potential of selective progesterone receptor modulators (SPRMs) to be an effective emergency contraception (EC). The data are extracted from the literature through the MEDLINE database service from 2000–2010. The SPRMs are in fact progesterone receptor ligands that could bind to progesterone receptor (PR) and exert antagonistic, agonistic or mixed agonist-antagonistic effects. These SPRMs are mifepristone, onapristone, asoprisnil, ulipristal, proellex among other compounds. Currently developed SPRMs may exert contraceptive effects by inhibiting ovulation and retarding endometrial synchronization. Low-doses of progesterone antagonists retard endometrial maturation without affecting ovulation. Mifepristone being a SPRM is effective for prevention of pregnancy but with prostaglandin acts as an excellent abortifacient; yet could not compete with levonorgestrel as EC. However, a single dose of 30 mg ulipristal acetate, another SPRM with similar effectiveness and side effect profiles as 1.5 mg levonorgestrel EC, has shown wider ‘window of effect’ by inhibition of the LH peak even if administered at the advanced pre-ovulatory phase, a time when use of levonorgestrel EC is no longer effective. Thus, ulipristal acetate goes one-step ahead of levonorgestrel in the field of emergency contraception treatment. Further studies are needed to explore the potential of other SPRMs to be cutting edge emergency contraceptive drugs.

Comparison of mRNA for IGFs and their binding proteins in the oviduct during the peri-oestrous period between dairy heifers and lactating cows

The oviduct provides the environment to support gamete maturation, fertilisation and early embryo development. As there is a high incidence of early embryonic death in lactating dairy cows, this study compared expression of IGF family members in the oviduct between lactating Holstein-Friesian dairy cows (n=16, 81±2.4 days in milk) and nulliparous heifers (n=16, age 1.6±0.07 years) at three stages of the oestrous cycle: A) newly selected dominant follicle in the luteal phase, B) follicular phase before the LH surge and C) pre-ovulatory phase after the LH surge. Expression of IGF1, IGF2, IGF binding protein 2 (IGFBP2), IGFBP3 and IGFBP6 mRNA was determined in the ampulla of the oviduct. Oviduct side (ipsilateral or contralateral) with respect to the dominant follicle did not affect gene expression. Expression of IGF1 and all three IGFBPs increased significantly between the luteal and the pre-ovulatory phases, with no further significant alteration post-LH surge. Concentrations of circulating IGF1 were higher in heifers than in cows, as was the mRNA expression of IGF1, IGFBP3 and IGFBP6. The pre-LH surge rise in IGFBP2 mRNA was only observed in heifers. IGF2 expression was not influenced by either age or stage of cycle. These three IGFBPs are generally considered to inhibit IGF action. These results indicate tight regulation of IGF bioavailability in the oviductal environment around oestrus, with pronounced differences between cows and heifers, which are likely to influence early embryonic development. Further studies are required to assess the implications for embryo survival.

Interaction of Potential Intrinsic and Extrinsic Risk Factors in ACL Injured Recreational Female Skiers

The aim of this study was to investigate the interaction of potential intrinsic and extrinsic risk factors in ACL injured recreational female skiers. 93 female recreational skiers who had suffered a non-contact ACL injury and 93 age-matched controls completed a self-reported questionnaire relating to intrinsic risk factors (menstrual history, BMI, previous knee injuries, self reported weekly sports participation) and extrinsic risk factors (type of ski used, time of last binding adjustment, snow condition, weather and slope difficulty). A logistic regression model revealed the following independent ACL injury risk factors for female recreational skiers: icy snow conditions (odds ratio, 24.33; 95% confidence interval, 6.8-86.5, P<0.001), skiing during snowfall (odds ratio, 16.63; 95% confidence interval, 1.8-152.1, P=0.013), use of traditional skis (odds ratio, 10.49; 95% confidence interval, 2.0-54.5, P=0.005), and preovulatory phase of menstrual cycle (odds ratio, 2.59; 95% confidence interval, 1.2-5.5, P=0.013). In conclusion, ACL injuries in female recreational skiers are the result of a complex interaction of intrinsic and extrinsic risk factors.

The Water Permeability Channels Aquaporins 1–4 Are Differentially Expressed in Granulosa and Theca Cells of the Preovulatory Follicle during Precise Stages of Human Ovulation

Changes in vascular permeability and expansion of the fluid-filled antrum are major events in the LH-induced ovulatory process. Our objective was to investigate the presence and expression levels of aquaporins (AQPs) in the granulosa and theca cell compartments of the follicle during defined phases of human ovulation. We conducted a prospective experimental study at the Department of Obstetrics and Gynaecology at a university hospital. Twenty-eight women underwent laparoscopic sterilization and at the same time follicle retrieval at four periovulatory phases. mRNA levels of AQP1-4 were measured in separated granulosa and theca cells from preovulatory phase, early ovulatory (EO) phase, late ovulatory phase, and postovulatory phase. Immunohistochemistry was done for AQP1-4 in intact human follicles. All four AQPs were expressed in both the theca and granulosa cells during ovulation. In granulosa cells, AQP1 levels increased in the late ovulatory and postovulatory phases. Expression of AQP2-3 followed a similar pattern with a marked increase in the EO phase, whereas AQP4 levels decreased from preovulatory to the EO phase. The presence of AQP1-4 in the human follicle was verified by immunohistochemistry. The results show for the first time the presence of AQP1-4 in human follicles during ovulation. The marked early rise in expression of AQP2 and AQP3 suggests a role during the process leading to follicular rupture, and the late rise of AQP1 suggests a role in corpus luteum formation.

Detection of Fatty Acids Profile in Human Saliva with Special Reference to Ovulation

Human saliva was analyzed by Gas-Chromatography (GC) for fatty acids study throughout menstrual cycle. Thefatty acid profiles from preovulatory, ovulatory and postovulatory phase’s sample were compared to establishany qualitative and quantitative differences that might have potential value in detection of ovulation in women.In the present study, 17 different types of fatty acids were identified. The fatty acids resembling as Oleic acid,Palmitic acid and acetic acid were comparatively higher in concentration than the other fatty acids. These threefatty acid were mostly present in ovulatory phase, to be higher when compared to follicular and luteal phases ofmenstrual cycle bear out the cyclic fluctuations in lipid levels do occur under the influence of both endogenousand exogenous sex hormones. The results imply that the variation in the fatty acids is unswervingly/ obliquelycorrelated to the phase of menstrual cycle.

Changes of Connexin43 Expression in Non‐pregnant Porcine Myometrium Correlate with Progesterone Concentration During Oestrous Cycle

Connexin43 (Cx43) is a major protein of myometrial gap junctions. The number of Cx43 gap junctions increase dramatically with the onset of labour in association with development of synchronized uterine contractions. The formation of myometrial gap junctions follows an increase in the oestrogen to progesterone ratio indicating an important role of steroid hormones in regulating Cx43 expression at term. However, no relationship has been established between the expression of Cx43 in the non-pregnant myometrium and concentration of steroid hormones during the oestrous cycle. Here, we used immunofluorescence and Western blotting to analyse the expression of Cx43 gap junctions in the myometrium of pre-pubertal pigs (n = 7) and mature pigs at pre-ovulatory (n = 7), luteal (n = 5) and late luteal (n = 3) stages of the oestrous cycle. The number of Cx43 gap junctions calculated per 1 mm(2) of the myometrial section was low in pre-pubertal pigs and significantly higher (p < 0.022) in pre-ovulatory animals. In relation to pre-ovulatory animals the number of myometrial gap junctions was significantly lower (p < 0.019) at the luteal phase and correlated with significantly higher (p < 0.005) concentration of endogenous progesterone. Phosphorylated isoforms of Cx43 protein were expressed in the myometrium of pre-pubertal pigs and mature animals at pre-ovulatory and late luteal phases, while they were down regulated at the luteal stage. These results indicate that changes of Cx43 expression in the porcine myometrium during the oestrous cycle may be regulated by progesterone concentration and may contribute to the modulation of uterine motility.

Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period

BackgroundFollicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation.MethodsFollicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry.ResultsThe concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml).Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively.ConclusionOur results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.

Human Ovarian Expression and Localization of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period.

Alterations in the interaction between the follicle and the matrix must occur before follicular rupture and ovulation can take place. Such interactions are controlled in part by the delicate balance between the matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). We hypothesize that the TIMPs would increase prior to ovulation in order to keep MMP activity in check to provide localized degradation at the apex and prevent wholesale destruction of the follicle wall. In the present study we focused on expression and localization of TIMP3, which is the only TIMP bound to the extracellular matrix, during the periovulatory period in the human. Follicles were collected from patients undergoing elective surgery at different stages of the ovulatory process. Patients were divided into four groups (preovulatory, early ovulatory, late ovulatory, and post-ovulatory phase). Surgery was performed at the preovulatory stage without administering hCG. The remaining women received an injection of 250 µg hCG (Ovitrelle, s.c.) when the diameter of their dominant follicle was ≥14mm and ≤ 20mm on trans-vaginal ultrasound. These patients had surgery during the following time intervals after hCG-injection; early ovulatory phase 12h to ≤18h, late ovulatory phase >18h to ≤ 34h and the postovulatory phase 44h to 70h. For the localization studies, the intact follicle with the apical orientation was embedded and processed for immunohistochemistry (IHC) using a TIMP3 antibody. For the quantitative studies, the follicle was bisected and the granulosa and theca cells separated and collected for real time PCR. Real time PCR analysis for Timp3 mRNA in granulosa and theca cells revealed a significant increase in Timp3 gene expression in granulosa cells from the early to the late ovulatory stage (n=5). Thecal Timp3 expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa cells and theca cell layers. In the preovulatory follicle there was positive staining in the granulosa cells, no staining in the theca. In the early ovulatory stage there was positive staining in both the granulosa and theca interna. The staining was most intense in the granulosa and theca cells; interna and externa of 2 follicles which were in the late ovulatory group. There was staining of TIMP3 in the lumen of some of the blood vessels and it was particularly intense in one early ovulatory follicle that had a large vascular bed surrounding the follicle. Several primordial follicles were seen in the sections and these were positively stained by the TIMP3 antibody. By using human follicles collected throughout the periovulatory period, we have been able to validate our hypothesis that Timp3 gene expression increases as the follicle progresses toward ovulation. We have shown that localization of TIMP3 protein appears first in the granulosa cell layer and then steadily accumulates in the granulosa-thecal layers as ovulation approaches. This localization places TIMP3 in the appropriate cellular layer to control the degradation of the follicular wall by the MMPs. (poster)

Fetal Alcohol Exposure Increases Mammary Tumor Susceptibility and Alters Tumor Phenotype in Rats

Altered fetal programming because of a suboptimal in utero environment has been shown to increase susceptibility to many diseases later in life. This study examined the effect of alcohol exposure in utero on N-nitroso-N-methylurea (NMU)-induced mammary cancer risk during adulthood. Study 1: Pregnant Sprague Dawley rats were fed a liquid diet containing 6.7% ethanol (alcohol-fed), an isocaloric liquid diet (pair-fed), or rat chow ad libitum (ad lib-fed) from day 11 to 21 of gestation. At birth, female pups were cross-fostered to ad lib-fed control dams. Adult offspring were given an I.P. injection of NMU at a dose of 50 mg/kg body weight. Mammary glands were palpated for tumors twice a week, and rats were euthanized at 23 weeks postinjection. Study 2: To investigate the role of estradiol (E2), animals were exposed to the same in utero treatments but were not given NMU. Serum was collected during the preovulatory phase of the estrous cycle. At 16 weeks postinjection, overall tumor multiplicity was greater in the offspring from the alcohol-fed group compared to the control groups, indicating a decrease in tumor latency. At study termination, 70% of all animals possessed tumors. Alcohol-exposed animals developed more malignant tumors and more estrogen receptor-α-negative tumors relative to the control groups. In addition, IGF-binding protein-5 (IGFBP-5) mRNA and protein were decreased in tumors of alcohol-exposed animals. Study 2 showed that alcohol-fed animals had significantly increased circulating E2 when compared to either control group. These data indicate that alcohol exposure in utero increases susceptibility to mammary tumorigenesis in adulthood and suggest that alterations in the IGF and E2 systems may play a role in the underlying mechanism.