Preovulatory Period Research Articles

P-667 The RANK-RANKL-OPG System - A new player in Oocyte Maturation

Abstract Study question Does the RANK-RANKL-OPG system play a role in oocyte maturation and ovulation? Summary answer RANKL, a new ovulatory gene expressed by cumulus and mural granulosa cells activates RANK receptors in oocytes, promoting oocyte maturation through oocyte-granulosa cell communication. What is known already The receptor activator of nuclear factor-kB (RANK), its ligand RANKL, and the soluble decoy osteoprotegerin (OPG) system are known due to their role in bone biology, the immune system, and other tissues. It was recently shown that RANKL was a highly upregulated gene in our ovulation-associated gene library of cumulus cells (CCs) from mature and non-mature oocytes. However, its ligand, RANK was undetected in both samples. The goal of this study was to elucidate the expression and function of the RANK-RANKL-OPG system in the ovary during the preovulatory period, and ovulation. Study design, size, duration In vitro and in vivo experiments using mice and human oocytes and granulosa cells. Participants/materials, setting, methods RANKL regulation experiments involve granulosa cells isolated from follicular fluid as well as mice oocytes. Analysis included cell culture, qPCR, and western blot. To examine RANK-RANKL-OPG function upon ovulation and oocyte maturation, super-ovulated mice and human oocytes were used. Main results and the role of chance We demonstrated for the first time that RANK mRNA is expressed in murine oocytes. Moreover, RANKL expression was demonstrated in human MGCs and CCs and is regulated by hCG/LH and PGE2. The addition of RANKL peptide, to the murine and human oocyte during IVM procedure, induces the maturation of GV oocytes. OPG (natural RANKL inhibitor) injection to mice during super-ovulation reduced oocyte maturation rate. Limitations, reasons for caution Animal model results may not fully translate to humans. Additional studies are needed to confirm the effects of RANKL/RANK-OPG system on fertility and long-term health. Wider implications of the findings Our observations suggest that RANKL expressed by granulosa cells acts on the RANK receptor found in the oocyte and plays an important role in oocyte-granulosa cell communication activating oocyte maturation. This research contributes to new insights in understanding pre-ovulatory processes and may improve the procedure of in vitro maturation (IVM). Trial registration number not applicable

Expression dynamics of adipokines during induced ovulation in the bovine follicles and early corpus luteum.

The study aimed to assess the local gene expression of adipokine members, namely vaspin, adiponectin, visfatin, resistin and their associated receptors - heat shock 70 protein 5 (HSPA5), adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) - in bovine follicles during the preovulatory period and early corpus luteum development. Follicles were collected before gonadotropin-releasing hormone (GnRH) treatment (0 h) and at 4, 10, 20, 25 and 60 h after GnRH application through transvaginal ovariectomy (n = 5 samples/group). Relative mRNA expression levels were quantified using real-time reverse transcription polymerase chain reaction (RT-qPCR). Vaspin exhibited high mRNA levels immediately 4 h after GnRH application, followed by a significant decrease. Adiponectin mRNA levels were elevated at 25 h after GnRH treatment. AdipoR2 exhibited late-stage upregulation, displaying increased expression at 20, 25 and 60 h following GnRH application. Visfatin showed upregulation at 20 h post-GnRH application. In conclusion, the observed changes in adipokine family members within preovulatory follicles, following experimentally induced ovulation, may constitute crucial components of the local mechanisms regulating final follicle growth and development.

Hysterolaparoscopy as a comprehensive diagnostic and therapeutic tool in modern ART

The aim of the study is to study Hysterolaparoscopy as a comprehensive diagnostic and therapeutic tool in modern art. The scheduled investigations were done to determine fitness for surgery. Patient was admitted one day prior to the procedure, and preanesthetic checkup was done. Hysterolaparoscopy was scheduled in preovulatory period between day 5 and day 10 of cycle and done under general anesthesia. Diagnostic laparoscopy was performed using a 30-degree deflection angle telescope powered with a fiber-optic cable for light source. Panoramic view of the abdomen was obtained a stepwise evaluation was made. Hysterolaproscopy may appear to be invasive, but is more beneficial, as diagnosis and therapeutic interventions can be done at the same sitting. Furthermore, it is possible to identify patients who will need ART at the earliest, thus expediting treatment. This helps in preventing further compromise in ovarian reserve and progression of disease and thus reducing financial burden to the couples.

Altered expression of angiogenic factors in dominant preovulatory follicles of dairy cattle treated with ACTH

Studies in cows have reported that ovulation, steroidogenesis and angiogenesis are affected by stress and consequently fertility decreases. The purpose of this study was to evaluate the effects of ACTH administration during the preovulatory period on the expression of growth factors (CD-31, PDGF-A, PDGF-B, VEGFA-164, VEGFA-164b, VEGF-R1 and VEGF-R2) associated with the angiogenic process by immunohistochemistry in cows (n = 14). Results evidenced the expression of these growth factors in theca and granulosa cells from antral, atretic and dominant preovulatory follicles of ACTH-treated cows, suggesting that, under stress conditions, their expression continues to be required. VEGFA-164, VEGF-R1 and VEGF-R2 expression was greater in theca cells of dominant preovulatory follicles of the ACTH-treated group than in those of the control group. CD-31 protein expression was lower in the dominant preovulatory follicles of the ACTH-treated group than in those of the control group. PDGF-A and PDGF-B expression did not differ between groups, either in granulosa or in theca cells. These results suggest that VEGFA-164, its receptors and CD-31 are actors in the normal cycle of the ovaries and could have greater pathophysiological importance in the altered angiogenic process and other events that occur during anovulation and stress conditions. This dysregulation reinforces the importance of the angiogenic process in the pathophysiology of cystic ovarian disease in cows. This is the first report on the expression and localization of components of the VEGF and PDGF systems and CD-31 in cells from dominant preovulatory follicles after ACTH administration.

ACTH impairs the migratory and secretory profile of mononuclear cells during proestrus in cattle

The aim was to evaluate the effect of ACTH on the mechanisms involved in peripheral blood mononuclear cells (PBMCs) infiltration into the ovary during dairy cattle proestrus. Regarding this, proper expression pattern of adhesion molecules must take place both in PBMCs and in endothelial cells. Argentinian Holstein cows (n = 12) were treated with 100 IU of ACTH every 12 h for 4 days before ovulation when ovariectomy was performed (day 18). Blood samples were taken on day 15 (0 h) and immediately before (72 h) and after (74 h) the last ACTH administration. In PBMCs, flow cytometry was performed to analyze CD44, CD11b and CD62-L expression along with gene expression of chemokines' receptors. Interleukin (IL)-4 and tumor necrosis factor-α (TNF-α) production was analyzed by flow cytometry after exposing PBMCs to autologous follicular fluid. In ovarian blood vessels, expression of the vascular endothelium cell adhesion-1 (VCAM-1) and the platelet endothelial cell adhesion molecule-1 was evaluated by immunohistochemistry. In T-lymphocytes, the expression of CD44 and CD11b was lower at 72 h in ACTH-treated cows (P < 0.05). In monocytes, the expression of CD11b and CD62-L was lower at 72 h in ACTH-treated cows (P < 0.05). Also, the percentage of IL-4+ cells was higher in ACTH-treated cows, meanwhile, the percentage TNF-α+ cells was lower in ACTH-treated cows (P < 0.05). Finally, in the vessels associated with the preovulatory follicle VCAM-1 immunoexpression was lower in ACTH-treated cows (P < 0.05). Here, we present novel insights into the effect of stress during the preovulatory period on the inflammatory pathway necessary for ovulation.

Does Phase of the Menstrual Cycle Affect Balance and Postural Control?

. The aim of the study is to examine the effects of menstrual cycle phases (MCP) on balance and postural control. The study was carried out with 63 volunteer women. Digital ovulation kits and, a Menstrual Cycle Regularity Questionnaire (MCRQ) to detect menstrual cycle regularity and duration, Premenstrual Syndrome Questionnaire (PMSQ) to question the presence of premenstrual syndrome, Menstruation Attitude Questionnaire (MAQ) to assess menstrual attitudes, International Physical Activity Questionnaire Short Form (IPAQ-SF) to question physical activity level was used. Balance and postural oscillation were evaluated with Balance Master balance and performance test device and Tekscan MatScan™ Pressure Mat System, respectively. All evaluations were repeated twice, in the preovulatory period and the postovulatory period. When the evaluations of the preovulatory period and the postovulatory period were compared, there was %3 increase in the percent weight-bearing of the non-dominant extremity (p = 0.01) and %2.5 decrease in the percent weight-bearing of the dominant limb in the postovulatory period (p = 0.01). %8 increase in functional reach distances was detected in the postovulatory period (p < 0.01). It was determined that there was %7.4 decrease in the oscillation rate of the center of gravity in the static stance with eyes open and %9 decrease in the static stance with eyes closed in the postovulatory period (p = 0.35, p = 0.18, respectively). It has been determined that the balance and postural control of young women are negatively affected in the preovulatory period and the function improved from the preovulatory period to the postovulatory period.

Preovulatory LH and FSH profiles of aluteal cycles

Interovulatory intervals are significantly shortened in aluteal cycles (Coffman et al, Theriogenology. 2014;82:1241-45). We hypothesized that the early return to estrus and ovulation would result from a marked elevation in gonadotropins. Therefore, the aim of this study was to characterize the LH and FSH blood profiles and follicle development of cycling mares during the preovulatory period of aluteal cycles. Eight cycling mares were studied during one control (luteal) cycle (CTRL) and one aluteal cycle (AL), arranged in a crossover design. With the aid of serial ultrasound exams, mares detected in estrus and with ≥ 35 mm follicles were treated with deslorelin acetate (1.8 mg intramuscularly). At detected ovulation (Day 0), mares were randomly allotted to a CTRL or an AL cycle as previously described: administration of 500 mcg of cloprostenol twice daily on Days 0, 1, and 2 and once daily on Days 3 and 4. Blood samples for RIA determination of plasma LH and FSH concentrations were collected daily. Areas under the curve (AUC) for concentrations of plasma LH and FSH from samples collected starting six days preceding ovulation (Day 0) were calculated with the linear interpolation trapezoidal method and compared between groups using a t-test. Interovulatory intervals (IOI) were compared using a paired t-test and a Sign test was used to compare the number of dominant follicles (≥ 28 mm), ovulations and ovulationfailures. The significance level was set as p ≤ 0.05 and a trend as 0.05 < p ≤ 0.1. Total AUC concentrations (± SEM) of LH for AL and CTRL groups were 41.57 ± 8.72 ng·day·mL-1 and 16.69 ± 4.17 ng·day·mL-1, respectively (p = 0.012) and FSH for AL and CTRL groups were 68.35 ± 11.44 ng·day·mL-1 and 45.60 ± 5.69 ng·day·mL-1, respectively (p = 0.079). Mean (± SD) IOI of AL cycles was significantly shorter than that of CTRL cycles (12.88 ± 1.35 days vs. 21.38 ± 1.30 days, p < 0.0001). A significant number of mares (5/8, 62.5%) presented an increase in the number (median ± IQR) of dominant follicles during AL cycles (2.5 ± 1.75 vs. 1.5 ± 1; p = 0.025). In contrast, no significant difference in the median (± IQR) number of ovulations was observed between AL and CTRL groups (1 ± 0 vs. 1 ± 0.75, respectively; p = 0.563). While all AL and CTRL cycles were ovulatory, there was a trend for AL mares to have remaining presumptive preovulatory follicles failing to ovulate (3/8 versus 0/8, respectively; p = 0.083). In conclusion, aluteal cycles were characterized by elevated concentrations of blood LH and FSH during the preovulatory period. These elevated gonadotropins were likely associated with the increase in the number of dominant follicles that became presumptive preovulatory follicles in aluteal cycles.

Thyrotropin-releasing hormone stimulates NR4A3 expression in the pituitary thyrotrophs of proestrus rats.

The secretion of several hypothalamic peptide hormones is activated during the preovulatory period. Hypothalamic thyrotropin-releasing hormone (TRH) is one such hormone with reproductive and/or metabolic significance. However, it remains unclear whether thyroid-stimulating hormone (TSH)-producing thyrotrophs are produced during the preovulatory period. We previously found a transient increase in the expression of the nuclear receptor NR4A3, a well-known immediate early gene, in the proestrus afternoon in the anterior pituitary glands of rats. To investigate the relationship between TRH secretion and pituitary NR4A3 expression during proestrus, we used proestrus and thyroidectomized rats to identify NR4A3-expressing cells and examined the regulation of Nr4a3 gene expression via the hypothalamus-pituitary-thyroid (HPT) axis. The percentage of NR4A3-expressing cells increased in thyrotrophs at 14:00 h of proestrus. Incubation of rat primary pituitary cells with TRH transiently stimulated Nr4a3 expression. Thyroidectomy to attenuate the negative feedback effects led to increased serum TSH levels and Nr4a3 gene expression in the anterior pituitary, whereas thyroxine (T4) administration conversely suppressed Nr4a3 expression. Additionally, the administration of T4 or TRH antibodies significantly suppressed the increase in Nr4a3 expression at 14:00 h of proestrus. These results demonstrate that pituitary NR4A3 expression is regulated by the HPT axis, and that TRH stimulates thyrotrophs and induces NR4A3 expression during the proestrus afternoon. This suggests the potential involvement of NR4A3 in the regulation of the HPT axis during pre- and post-ovulatory periods.

Results of Ultrasound Studies of the Growth Dynamics of Dominant, Subdominant Follicles and Determination of Estradiol Concentration in the Preovulatory Period in Cows

Results of Ultrasound Studies of the Growth Dynamics of Dominant, Subdominant Follicles and Determination of Estradiol Concentration in the Preovulatory Period in Cows

5d CIDR-Heatsynch improves the circulatory estradiol levels, estrus expression and conception rate in anestrus buffalo (Bubalus bubalis)

The present study aimed to investigate whether increasing estradiol (E2) during preovulatory period would increase estrous expression, luteal profiles and conception rate in 5d CIDR based timed AI protocol. A total 156 anestrus buffalo allocated (78 per group) to either 5d CIDR-Cosynch (d-5: CIDR + GnRH; d0: PGF2α+CIDR removal; 72 h post-CIDR removal: GnRH) or 5d CIDR-Heatsynch (d-5: CIDR + GnRH; d0: PGF2α+CIDR removal; 24 h post-CIDR removal: estradiol benzoate) group. All the buffaloes inseminated at 72 and 84 h post-CIDR removal. A subset of buffalo (n = 58) were subjected to examination of the follicle diameter and luteal profile during protocol, post-AI on days 5 and 12. The buffalo in 5d CIDR-Heatsynch had greater (p < .05) E2 concentrations, estrus induction and increasing trend (p < .08) for conception rate (57.7% vs. 43.6%) than 5d CIDR-Cosynch. The percentage of pregnant buffalo that exhibited estrus signs was greater (p < .01) in 5d CIDR-Heatsynch than 5d CIDR-Cosynch. Positive correlation (p < .01) was observed between POF and E2 concentrations; POF and CL diameter, CL diameter and P4 concentrations. Estrus response and P4 concentrations were indicators of probability of pregnancy. In conclusion, 5d CIDR-Heatsynch tended to improve conception rate. The estrus expression and P4 concentrations (d5 and 12 post-first-AI) is the indicator of probability of pregnancy in buffalo.

Features of the sonographic picture in female veterans of reproductive age who suffered injuries during combat actions

The objective: to study the features of the sonographic picture of the pelvic organs in women of reproductive age who took part in combat operations and suffered a concussion. Materials and methods. Ultrasound examination of the pelvic organs with Doppler study on the 5th and 21st days of the menstrual cycle was performed in 457 women of reproductive age who took part in combat operations and suffered a concussion. 246 patients of theses persons with a history of trauma without post-concussion syndrome were included in the comparison group, 211 women with consequences of concussion in the form of post-concussion syndrome – in the main group. The control group included 30 healthy civilian women of reproductive age. The concentration of progesterone in blood serum was studied in all subjects on the 5th and 21st days of the menstrual cycle. The average age of women was 37.08±4.23 years. The period of stay in the combat zone was 60.26±42.21 months, the time since the traumatic brain injury (concussion) was 18.8±9.2 months. Results. In patients of the main group without hyperplastic processes of the endometrium the M-echo thickness on the 21st day of the menstrual cycle was 1.12 times less (p&lt;0.001) than in the control group, and 1.15 times less in patients in the comparison group (p&lt;0.001). In 20.71 % of women with a contusion during combat operations, there is an inferior secretory transformation of the endometrium with increased echogenicity only in the peripheral parts of the M-echo with a hypoechoic zone in the center, in 13.81 % of patients there are no secretory changes in the endometrium on echograms, which manifested by the preservation on the 21st day of its three-layer construction, typical for the preovulatory period, with a hypoechoic functional layer. Intrauterine synechiae are formed in 10.95 % of women who suffered contusions during combat operationss, hyperplastic endometrial processes – 30.24 % of women, 12.3 8% of which had hyperplasia, 17.86 % –endometrial polyps.In patients who suffered a contusion during combat operations a decrease in the vascularization of the corpus luteum with a significant increase in the resistance index of blood flow in the corpus luteum by 1.27 times and a decrease in the maximum systolic velocity by 1.19 times was, in the subjects of the main group compared to women of the comparison group – 1.07 (p&lt;0.01) and 1.05 (p&lt;0.01) times, respectively. On the 21st day of the menstrual cycle in women who took part in combat operations and had concussion, a correlation between the level of serum progesterone and the index of blood flow resistance in the corpus luteum – r=-0.76, p&lt;0.05, as well as maximum systolic blood flow velocity - r=0.70, p&lt;0.05 was established. Conclusions. According to the ultrasound examination of the pelvic organs for women of reproductive age who took part in combat operations and suffered a concussion, ultrasound changes in the structure of the tissues of the pelvic organs and their vascularization are typical, in particular, the thickening of the M-echo on the 21st day of the menstrual cycle, and in a third of cases – the presence of hyperplastic changes of the endometrium.

Exogenous ACTH stimulus during the preovulatory period alters patterns of leukocyte recruitment in the ovary of dairy cows

Before ovulation, the ovary exhibits signs of local inflammation. However, the effects of adrenocorticotropin (ACTH) on the complexity of this inflammatory response are not yet well described. Thus, the aim of this study was to evaluate the effects of ACTH administered to dairy cows during the preovulatory period on the local distribution of different subsets of leukocytes infiltrated in the ovary, along with the gene expression of relevant chemokines (C–C motif chemokine ligand-2 (CCL2), C-X-C motif chemokine ligand-8 (CXCL8), CCL25 and CXCL1) involved in leukocyte chemotaxis and blood perfusion on the follicular wall of dominant follicles. Also, the direct effect of ACTH on chemokine gene expression was addressed in cultured antral follicular walls. For this purpose, both an in vivo and an in vitro experiment were performed. For the in vivo experiment, exogenous ACTH (100 IU) was administered intramuscularly to Holstein cows (n = 12) during proestrus every 12 h for four days before ovulation, when ovariectomy was performed (day 18). Daily ovarian Doppler ultrasonography was used to evaluate the percentage of irrigated area, the pulsatility index and the resistance index in the dominant follicles. The distribution of monocytes-macrophages (CD14), T- (CD2) and B-lymphocytes (CD79a) and granulocytes (CH138A) in the ovary was analyzed by immunohistochemistry. In follicular wall samples, gene expression of CCL2, CXCL8, CXCL1 and CCL25 was evaluated, whereas IL-17A expression was analyzed by Western blot. The total number of CD14, CD79a and CD2 infiltrated cells was lower in the ACTH-treated group than in the control group (p < 0.05). Chemokine gene expression showed lower mRNA of CCL2, CCL25 and CXCL1 (p < 0.05) in the ACTH-treated group. Meanwhile, IL-17A protein expression and hemodynamic parameters were similar between groups (p > 0.05). In the in vitro assay, antral follicular walls were stimulated with ACTH to corroborate the gene expression profile of chemokines. mRNA expression of CCL2 tended to be lower in the stimulated follicular walls (p = 0.092). Our results suggest that exogenous ACTH stimulus during the preovulatory period reduces the number of infiltrated leukocytes in the bovine ovary and this could be due to a lower chemotaxis capacity of the ovary.

Redox Status and Hematological Variables in Melatonin-Treated Ewes during Early Pregnancy under Heat Stress.

Simple SummaryHeat stress induces oxidative stress that negatively affects the fertility rate in farm animals, as it may disrupt normal follicular development and preimplantation embryogenesis. Although indigenous sheep breeds may exhibit adaptation to a certain level in high summer temperatures, their reproductive competence is still compromised under extreme severe heat stress conditions. Thus, the administration of melatonin is suggested, as an antioxidant regime, for improving the fertility rate and redox balance in heat-stressed ewes. The influence of environmental thermal stress in conjunction with melatonin treatment on hematological variables and cortisol secretion was also investigated. In this respect, melatonin seems to enhance the progressive adaptation of indigenous-breed ewes to environmental heat stress.The preovulatory follicles and preimplantation stage embryos are found to be rather sensitive to heat stress due to their low potential for scavenging reactive oxygen species (ROS). The aim of the present study was to assess the impact of melatonin administration on redox status and hematological variables during the preovulatory period and early stages of embryogenesis in heat-stressed ewes in vivo. Forty Karagouniko-breed ewes were divided in two groups, the melatonin (M, n = 20) group and control (C, n = 20) one. All animals were subjected to heat stress throughout the study, which lasted forty days (D0 to D40). In M group, melatonin implants were administered on D0. Then, oestrous synchronization was applied (D19-D33). On D34, six rams were introduced into the ewe flock for mating. Ultrasonographic examination was conducted on D73 for pregnancy diagnosis. The temperature humidity index (THI), the rectal temperature (RT), and the number of breaths per minute (BR) were evaluated twice daily. Redox biomarkers, namely total antioxidant capacity (TAC), reduced glutathione (GSH), and thiobarbituric acid reactive substances (TBARS), were assayed in blood samples collected on D0, D33, and D40. In addition, packed cell volume (PCV), white blood cells (WBCs), leukocyte differential count, and cortisol assessment were conducted in blood samples on D33 and D40. The results indicated improved fertility rate and mean number of lambs born per ewe due to improved redox status (p < 0.05) in ewes that received melatonin implants 34 days approximately before the onset of oestrus. The PCV decreased in both groups between the two time-points (p < 0.05). However, the NEU/LYMPH ratio decreased (p < 0.05) only in group M. The low cortisol levels and the decreased NEU/LYMPH ratio in both groups support the hypothesis that ewes of the indigenous Karagouniko breed may exhibit adaptation to environmental thermal stress. The administration of melatonin as an antioxidant regime may improve the reproductive competence of heat stressed ewes and may also enhance their ability to adapt at high ambient temperatures.

Hormone regulation of thrombospondin-1 mRNA in porcine granulosa cells in vitro

Thrombospondin-1 (THBS1) is involved in the process of angiogenesis and is down-regulated by insulin-like growth factor 1 (IGF1) in porcine granulosa cells (GC), but what other hormones regulate GC THBS1 and its role in follicular growth is unclear. Thus, six experiments were conducted to determine the influence of other hormones on THBS1 gene expression in porcine GC, and to determine if THBS1 mRNA changes during follicular development. For Exp. 1–5, small (1–5 mm) follicles from ovaries of abattoir gilts were aspirated, GC collected and treated with FSH, IGF1, fibroblast growth factor 9 (FGF9), Sonic hedgehog (SHH), estradiol, cortisol, and/or prostaglandin E2 (PGE2). FSH, IGF1 and FGF9 each decreased (P < 0.05) THBS1 mRNA abundance. Alone, PGE2 increased (P < 0.05) THBS1 mRNA abundance. PGE2 significantly attenuated the FSH-induced inhibition of THBS1 mRNA expression. Estradiol, cortisol, and SHH had no effect on THBS1 mRNA abundance. In Exp. 6, small (1–3 mm), medium (4–6 mm) and large (7–14 mm) follicles were aspirated to measure abundance of THBS1 mRNA in GC which did not differ (P > 0.10) between small and medium-sized follicles but was threefold greater (P < 0.05) in large compared to small or medium follicles. We hypothesize that the inhibitory effects of FSH, IGF1 and FGF9 on the antiangiogenic gene THBS1 could contribute to promoting angiogenesis in the developing follicle, while stimulation of THBS1 mRNA by PGE2 may help reduce angiogenesis during the preovulatory period when PGE2 and THBS1 mRNA are at their greatest levels.

Impact of preovulatory estradiol concentrations on subsequent luteal function in beef cattle

It has been hypothesized that circulating concentrations of estradiol during the preovulatory period, can impact subsequent progesterone concentrations. Ovulation was synchronized in nonlactating beef cows (n = 53). Cows that exhibited estrus before gonadotrophin-releasing hormone (GnRH)-induced ovulation (d 0) had greater (p<.01) peak concentrations of estradiol compared with cows that did not express estrus (11.5 ± 0.8 vs. 6.2 ± 0.6 pg/mL), respectively, but there was no difference in ovulatory follicle size (p= .80) or interval from GnRH2 to ovulation (p=.23). Circulating concentrations of progesterone during luteal formation (d 3–7; p=.70 and p=.77) or mid-luteal phase (d 8–14; p=.39 and p=.12) were not affected by elevated periovulatory estradiol or an interaction with day. To investigate the direct influence of estradiol on luteal function, ovulation (d 0) was synchronized in nonlactating beef cows and cows were allocated to three groups (control, n = 5; vehicle injection, n = 4; or an estradiol antagonist (Fulvestrant; ICI 182,780), n = 4. Intrafollicular injection of vehicle (100 µL) or an estradiol antagonist (25 μg Fulvestrant in 100 µL) into the largest follicle occurred on d –2. Concentrations of estradiol increased (p<.0001) from d –2 to 0 but did not differ among groups (p>.50). Furthermore, plasma concentrations of progesterone on d 0 through 20 were not affected by treatment (p=.86). These results indicate that elevated preovulatory estradiol before ovulation was not required to prepare granulosa cells for luteinization or subsequent luteal progesterone secretion but did tend to impact luteal lifespan.

MC2R/MRAP2 activation could affect bovine ovarian steroidogenesis potential after ACTH treatment

Stressors activate the hypothalamic–pituitary–adrenal (HPA) axis, reducing fertility by interfering with the mechanisms that regulate the timing of events within the follicular phase of the estrous cycle. In the HPA axis, melanocortin 2 receptor (MC2R) mediates responses to adrenocorticotropic hormone (ACTH) in concert with melanocortin receptor accessory protein 2 (MRAP2). The aims of the present study were: (1) to evaluate the effects of ACTH administered in cows in the preovulatory period on the expression of the MC2R/MRAP2 complex in the dominant follicle; and (2) to analyze the involvement of Extracellular signal Regulated Kinase 1 (ERK1) signaling in the activation of MC2R and the expression of key enzymes involved in the biosynthesis of glucocorticoids (GCs) in the dominant follicle. To this end, 100 IU ACTH was administered to Holstein cows from a local dairy farm during pro-estrus every 12 h for four days until ovariectomy, which was performed before ovulation. Protein immunostaining of MC2R was higher in the dominant follicles of ACTH-treated cows (p < 0.05). Also, Western blot analysis showed higher activation of the ERK1 signaling pathway in ACTH-treated cows (p < 0.05). Finally, immunohistochemistry performed in the dominant follicles of ACTH-treated cows detected higher expression of CYP17A1 and CYP21A2 (p < 0.05). These results suggest that the bovine ovary is able to respond locally to ACTH as a consequence of stress altering the expression of relevant steroidogenic enzymes. The results also confirm that the complete GC biosynthesis pathway is present in bovine dominant follicle and therefore GCs could be produced locally.

The Involvement of Lumican in Human Ovulatory Processes.

Based on a previous global transcriptome sequencing project, we hypothesized that Lumican (LUM) might play a role in ovulatory processes. We sought to determine LUM gene expression under various conditions in human preovulatory follicles. The in vitro expression of LUM mRNA in mural (MGCs) and cumulus (CGCs) granulosa cells was characterized using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical staining was used to identify human LUM expression in follicles at different developmental stages. Cell signaling studies were performed by treating human MGCs with human chorionic gonadotropin (hCG) and both, different stimulators and inhibitors to determine their effect on LUM expression by using qRT-PCR. Cell confluence studies were carried out to study the correlation between LUM expression and follicle cell proliferation. Follicular MGCs and CGCs of women undergoing in vitro fertilization (IVF) procedures due to endometriosis were analyzed for differences in LUM expression patterns by qRT-PCR. LUM mRNA expression was significantly higher in MGCs as compared to CGCs. In CGCs, LUM mRNA was higher in mature metaphase II (MII) oocytes than in germinal vesicle (GV) and metaphase I (MI) oocytes. LUM expression was significantly upregulated in response to hCG in cultured MGCs. Immunohistochemistry of human ovaries revealed LUM was mostly present in MGCs of large preovulatory and postovulatory follicles and absent from primordial follicles. Using pharmacological activators and inhibitors, we demonstrated that LUM induction by luteinizing hormone (LH)/hCG is carried through the mitogen-activated protein kinase (MEK) pathway. LUM expression was induced in high-density cell cultures in a confluence-dependent manner. MGCs from follicles of subjects with endometriosis exhibited reduced mRNA transcription levels compared to control subjects. Our study confirms that LUM is a newly discovered ovulatory gene. LUM might play an important role during the preovulatory period up until ovulation as well as in endometriosis infertility. A better understanding of LUM's role might provide potential new treatment paradigms for some types of female infertility.

ITRAQ-based proteomic analysis of bovine pre-ovulatory plasma and follicular fluid

Bovine follicular fluid (FF) creates a unique microenvironment in follicles necessary for follicle growth, oocyte maturation, and estradiol (E2) production. The objective of this study was to analyze changes in proteins in FF and plasma (PL) from animals with high E2 (HE2) or low E2 (LE2) during the preovulatory period. Beef cows were synchronized, and follicular dynamics and ovulatory response were monitored using transrectal ultrasonography. Nine cows were selected and slaughtered, blood samples were collected at slaughter and FF was aspirated from dominant follicles (DF; >10 mm). Abundant proteins (albumin, IgG, IgA, and alpha-1-antitrypsin) were depleted from both PL and FF. Peptides were labeled with iTRAQ reagents and quantified using 2-dimentional liquid chromatography ESI-based mass spectrometry. Estradiol was associated with protein changes in PL and FF. Protein expression changes between FF HE2 and FF LE2 were greater than between PL HE2 and PL LE2. There were 15 up-regulated proteins and 10 down-regulated proteins in FF HE2 compared to FF LE2, and 7 proteins up-regulated and 9 proteins down-regulated in PL HE2 compared to PL LE2. Several of the differentially expressed proteins function in follicle development and were mainly categorized under cellular process and metabolic process. Pathway analysis identified the up- and down-regulated proteins were predominantly associated with the complement and coagulation cascades. The data demonstrate E2 regulates a wide range of reproductive associated proteins in bovine PL and FF and can provide the basis for further investigation of specific processes involved in such regulation.

Ovarian Reserve Condition in Women with Menstrual Dysfunction in the Puberty

The concept of «ovarian reserve» in recent years has been widely discussed in the literature. This is a functional reserve of the ovaries, reflecting the size of the follicular pool of the ovaries and the quality of the oocytes in them, that is, it characterizes the ability of the ovaries to develop a healthy follicle with a full egg. Determination of ovarian reserve allows you to more accurately assess the reproductive potential of women and prevent pathological conditions and diseases that reduce fertility.The objective: аccording to laboratory and instrumental methods of research, to give a comprehensive assessment of the condition of the ovarian reserve in women with menstrual dysfunction in the puberty.Materials and methods. The main group of women (n=90) with a pathology of menstrual function in the puberty, according to the nature of the violations, was divided into 3 subgroups (n=30): the first – women with primary oligomenorrhea in the anamnesis, the second – with late age menarche, the third – with puberty bleeding. The control group consisted of women (n=30) with the correct rhythm of menstruation in puberty. The age range of the subjects is from 19 to 32 years. The biochemical and echographic markers of the ovarian reserve were studied.Results. In a clinical study, it was found that in women with menstrual dysfunction in the puberty, the concentration of antimuller hormone (AMH) in the blood serum and the size of the ovaries (total volume) are greater due to the greater number of small follicles compared to women in the control group. A direct correlation between the parameters of AMH and the number of follicles and the inverse between AMH and the diameter of the follicles indicate normal ovarian reserve. This pattern was observed in women with a primary history of oligomenorrhea and late menarche. In women with pubertal hemorrhages, the concentration of AMH in the blood did not significantly differ from the control group, the total volume of both ovaries was greater than in women with a normal rhythm of menstruation (p&lt;0,05), but the total number of follicles did not differ significantly. The correlation between the total volume of both ovaries and the total number of follicles was lost, which may indicate an increase in ovarian volume due to the stromal component.Conclusion. A comprehensive analysis of the level of AMH in serum, the total volume of the ovaries and the number of follicles in the preovulatory period allows us to estimate the ovulatory reserve in women with menstrual dysfunction in the pubertal period. And although the indicated parameters in the examined women are significantly higher than in women with the correct rhythm of menstruation (p&lt;0,05), they remain within the reference values, which indicates the preservation of their ovarian reserve. Today, for screening the ovarian reserve, determining the concentration of AMH in the blood serum has several significant advantages over other methods, because this hormone is a marker that begins to change with age before other indicators, which is of great prognostic value in the treatment of various disorders of the reproductive system, premature extinction its function and infertility.

Особливості гормонального і цитокінового статусу в жінок, які мали порушення менструальної функції у пубертатний період

Deviations in the formation of reproductive function, and subsequently menstrual irregularities from the age of menarche, ovarian dysfunction and steroidogenesis disorders can be clinically manifested in remote periods after their direct action. The nature and depth of morphofunctional disorders in this case depends on the degree of maturity of the reproductive system, the initial functional state of the regulation centers (hypothalamus, pituitary gland) and ovaries, and the duration of the action of adverse factors. The accumulated specific research experience indicates that in addition to the pituitary gonadotropin hormones, cytokines (interleukins), which can simulate ovarian function and play an important role in ovulation, are of great importance in the normal functioning of the ovaries. The objective: аccording to laboratory and instrumental methods of research, to study the characteristics of hormonal status and the relationship of ovarian hormones with interleukins (IL) in women with menstrual dysfunction in the puberty. Materials and methods. According to the nature of the violations, the main group of women (n=90) who had a pathology of menstrual function in the puberty period was divided into 3 subgroups (n=30): the first – women with primary oligomenorrhea in the anamnesis, the second – with a late age menarche, the third – with puberty bleeding. The control group included women (n=30) with the correct rhythm of menstruation in puberty. Results. A clinical study found a decrease in the concentration of progesterone in women with a history of primary oligomenorrhea, a decrease in estradiol in the preovulatory period in women with late menarche. When assessing the concentration of follicle-stimulating hormone in the subgroups, an increase was noted in patients with puberty bleeding compared with the control group, due to the low level of antimuller hormone in the blood serum. The correlation between the indicators of these hormones and interleukins (IL-4, IL-8) indicates the influence of cytokines on folliculogenesis and the formation of the corpus luteum. Ovarian reserve parameters are not reduced. Conclusion. An analysis of the hormonal and cytokine status in women with menstrual dysfunction in the puberty allows us to draw an analogy between the processes of ovulation and the inflammatory response. The cytokines produced by certain immune cells are signaling molecules that affect cell proliferation and apoptosis of ovarian cells, folliculogenesis, hormone secretion and thus play an important role in ovulation. Therefore, the immune system may be an additional local regulator of ovarian function. Estradiol and progesterone are of great importance in the secretory transformation of the endometrium, especially during the «implantation window». Keywords: puberty, menarche, oligomenorrhea, pubertal bleeding, folliculogenesis, progesterone, estradiol, interleukins, pituitary gland, anti-varial antibodies.