Gonadotropin-releasing Hormone Treatment Research Articles

Activation of Pyk2 by CaM kinase II in cultured hypothalamic neurons and gonadotroph cells.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons and stimulates a GnRH receptor in gonadotroph cells and GnRH neurons. The GnRH receptor belongs to the G-protein-coupled receptors, and stimulation of the GnRH receptor activates extracellular signal-regulated protein kinase (ERK). We reported previously that the δ2 isoform of Ca2+ /calmodulin-dependent protein kinase II (CaM kinase IIδ2) was involved in GnRH-induced ERK activation in cultured GnRH neurons (GT1-7 cells). Recently, we found that GnRH treatment of GT1-7 cells activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in ERK activation. In the current study, we examined the possibility that CaM kinase IIδ2 might activate Pyk2. Knockdown of CaM kinase IIδ2 and KN93, an inhibitor of CaM kinases, inhibited the GnRH-induced activation of Pyk2. In the case of cultured gonadotroph cells (αT3-1 cells), knockdown of CaM kinase IIβ'e inhibited GnRH-induced Pyk2 activation. In addition, our inhibitor studies indicated that Pyk2 and CaM kinase II were involved in the GnRH-induced shedding of proHB-EGF in GT1-7 cells. These results suggested that CaM kinase II activated the ERK pathway through Pyk2 activation and HB-EGF production in response to GnRH.

Hydrometra in dairy goats: Ultrasonic variables and therapeutic protocols evaluated during the reproductive season

Hydrometra is characterized by the accumulation of fluid within the uterus due to the persistence of corpus luteum. The diagnosis of this disorder occurs with an ultrasonic exam. This study evaluated uterine drainage and fertility rates in goats after the use of d-cloprostenol in association or not with Gonadotropin-releasing hormone (GnRH) treatment. Twenty Saanen goats, diagnosed with hydrometra, received three 37.5-μg doses of d-cloprostenol laterovulvarly at 10-day intervals. On D5, the goats were assigned into two groups receiving 1 mL of GnRH or saline solution intramuscularly. Ultrasonography (US) was performed from D0 to D25. An US approach was used to rank hydrometra in scores. The pregnancy rate was assessed 45 and 90 days after the end of treatment. The uterine fluid was totally drained after the first and second administration of d-cloprostenol in 50% and 95% of the goats, respectively. In one female, full emptying of the uterus occurred only after D20. US performed at 45 and 90 days after the end of treatment indicated there was a pregnancy rate of 45.0% and 55.0%, respectively. Fertility did not differ between the GnRH-treated and control goats. Those goats not pregnant at 45 days had a follicular cyst, hydrosalpinx or hydrometra. At 90 days, no change was observed in the hydrosalpinx, and four goats had hydrometra. The use of three doses of d-cloprostenol 10 days apart was efficient for induction of draining the contents of the uterus, resulting in a relatively acceptable pregnancy rate. This treatment associated with the US approach can be important when applied in the field.

Huge abdominal and perineal aggressive angiomyxoma: A misdiagnosed case report and literature review

Aggressive angiomyxoma (AA) is a distinctive soft tissue tumor with a high risk of local recurrence. Clinicians must be aware of this rare tumor pre-operatively. Excision is the preferred method of AA treatment. The case report presents a case of a 36-year-old woman who was difficulty in walking due to a non-painful tumor in the abdomen and perineum. She was misdiagnosed as abdomen neurofibroma for more than 10 years, and an operation was performed in 1997. However, the tumor was incompletely resected because its huge volume accompanies with extensive infiltration and bleeding. The tumors in her abdomen and perineum were growing gradually, and the latter became a large lump which impeded her daily life. In 2008, the perineal tumor was incompletely resected, which weighed 10725 g. The severe hemorrhage had been ceased by Gonadotropin-Releasing Hormone treatment. She is alive till now. Details of the history and operative procedures are presented. An AA diagnosis was made by microscopy immunohistochemically. Long-time misdiagnosis and improper treatment are the important reasons for making it impossible to be radically resected. Pathological and immunohistochemical examination are important for avoiding misdiagnosis. For this case, there is a remaining tumor in her abdomen. A special project including further follow-up and treatment will be taken out.

Efficacy and safety of human chorionic gonadotropin for treatment of cryptorchidism: A meta-analysis of randomised controlled trials.

Although human chorionic gonadotropin (hCG) has long been employed in the management of cryptorchidism, its safety and efficacy is still controversial. Hence, in the present study, we conducted a meta-analysis of the treatment of cryptorchidism using hCG. We searched the Medline, Embase, CINAHL, EBSCO, The Cochrane Library, China National Knowledge Infrastructure and WanFang databases. Data were extracted by two reviewers using the designed extraction form. Data up to July 2015 were obtained using the terms 'cryptorchidism', 'chorionic gonadotropin' and 'randomised controlled trials'. All the publications were downloaded, and the respective authors were contacted for any further details and clarifications, if deemed necessary. The data analysis included randomised controlled trials that compared hCG with other hormone treatments offered to prepubescent males presenting with cryptorchidism. Testicular descent rate was used as the final positive outcome of the treatments offered. The software Review Manager (RevMan 5.3, The Cochrane Collaboration, London, UK) was used to review the management and data analysis. Risk ratios (RRs) with 95% confidence intervals (CIs) were pooled with a fixed effect model if no heterogeneity was present. A total of seven trials satisfied the selection criteria. The overall quality of the studies downloaded from various databases was low. Data from these seven studies were divided into three subgroups depending on the design of the trials: Two studies compared hCG with a placebo, and three studies compared hCG with gonadotropin-releasing hormone (GnRH) in unilateral cryptorchidism, whereas two other studies compared hCG with GnRH in bilateral cryptorchidism. Analysis of these trials revealed no significant differences between the effectiveness of hCG treatment and GnRH treatment in bilateral (RR 0.05, 95% CI (-0.29-0.40), two trials, n = 104, P = 0.76) as well as unilateral cryptorchidism (RR 0.04, 95% CI (-0.12, 0.21), three trials, n = 81, P = 0.61). A meta-analysis of these studies showed that hCG treatment is not superior to placebo (RR 7.74, 95% CI (0.14-425.72), two trials, n = 31, P = 0.32). A meta-analysis of the seven studies led us to conclude that hCG treatment is no more effective than placebo, and there were no significant differences in the effectiveness of hCG versus GnRH treatment.

Influence of pretreatment of insulin on phosphorylation of extracellular receptor kinase by gonadotropin releasing hormone in cultured mouse granulosa gells

Background: To investigate the influence of pretreatment of insulin on the phosphorylation of extracellular receptor kinase (ERK) by gonadotropin releasing hormone (GnRH) in cultured mouse granulosa cells. Materials and Methods: The granulosa cells from the mouse were collected simultaneously from the ovaries of gonadotropin-primed ICR female mice. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of the receptors of GnRH and insulin. The granulosa cells were cultured with ten nM of insulin, ten nM of GnRH, and ten nM of insulin pretreatment before ten nM of GnRH. Western blotting was used for analysis of phosphorylation of ERK. Student t-test and Dunnett’s comparison test were used for statistical analysis and statistical significance was defined as p Results: The authors confirmed the presence of receptors of GnRH and insulin in the mouse granulosa cells by RTPCR. Both insulin and GnRH could activate ERK phosphorylation. Pretreatment of insulin for 30 minutes before GnRH treatment inhibited ERK phosphorylation by GnRH. Conclusion: Insulin might have a negative effect of GnRH regulation of mouse ovarian physiology by inhibition of GnRH activation of ERK. Based on these study results, the authors demonstrated the role of insulin as a delicate modulator of reproductive function.

Response of lactating dairy cows with or without purulent vaginal discharge to gonadotropin-releasing hormone and prostaglandin F2α.

Purulent vaginal discharge (PVD) is a common uterine disease in dairy cattle that has negative effects on reproductive performance. Reproductive management programs that synchronize ovulation use gonadotropin-releasing hormone (GnRH) to induce ovulation and prostaglandin F2α (PGF2α) to induce luteolysis. The objectives of this study were to evaluate ovarian response to treatment with GnRH and the odds of bearing a corpus luteum or being inseminated in dairy cows with or without PVD. Another objective was to determine the hazard of insemination after administration of PGF2α in dairy cows with or without PVD. Primiparous (n = 291) and multiparous (n = 402) cows were evaluated for PVD using a Metricheck device at 46 ± 3 and 35 ± 3 days in milk (DIM) (study day 0), respectively. On study day 14, primiparous (n = 107) and multiparous (n = 197) cows were treated with GnRH and subsequent ovulation was recorded. Primiparous (n = 178) and multiparous (n = 368) cows not inseminated by study day 21 were administered PGF2α and response to PGF2α treatment was determined by detection of estrus. Furthermore, cows were categorized by the presence of a CL or being inseminated by study days 14, 21, and 35. Overall prevalence of PVD was 28.5% and 13.4% for primiparous and multiparous cows, respectively. Projected 305-d milk yield was less (P < 0.01) in PVD+ multiparous cows compared with PVD- multiparous cows, however, no (P = 0.26) difference was detected between primiparous PVD+ and PVD- cows. Ovulatory response to GnRH treatment was 51.8% and 47.8% for primiparous and multiparous cows, respectively. Primiparous PVD- cows tended (P = 0.06) to be less likely to ovulate to GnRH than primiparous PVD+ cows, whereas multiparous PVD+ cows were less (P = 0.04) likely to ovulate to GnRH than PVD- multiparous cows. The odds of bearing a corpus luteum or being inseminated by study days 14, 21, or 35 was not associated with PVD in primiparous cows. In contrast, the odds of bearing a corpus luteum or being inseminated by study days 14 and 21 was (P ≤ 0.03) associated with PVD in multiparous cows, but not (P = 0.11) on study day 35. Hazard of insemination after PGF2α was not (P ≥ 0.38) associated with PVD in primiparous or multiparous cows. Purulent vaginal discharge is associated with response to treatment with GnRH in dairy cattle. Purulent vaginal discharge might negatively affect reproductive management programs that use GnRH to induce ovulation.

LH and testosterone secretions in response to GnRH challenge in pubertal Japanese Black beef bulls with normal and abnormal semen.

Plasma luteinizing hormone (LH) and testosterone concentrations were examined in Japanese Black beef bulls with normal and abnormal semen in response to gonadotropin releasing hormone (GnRH) challenge at the start (10 months) and completion (20 months) of puberty. Bulls with normal semen had higher testosterone concentrations after GnRH treatment at 20 months than they did at 10 months, while LH concentrations did not differ between the two age groups. LH and testosterone concentrations were not different between bulls with normal and abnormal semen at 20 months. Thus, testosterone secretions in response to the GnRH challenge were higher for bulls with normal semen at pubertal completion compared to bulls at the start of puberty, but responsiveness of LH to GnRH and of testosterone to the LH increment was not altered in bulls with abnormal semen.

Differential response of AMH to GnRH agonist among individuals: the effect on ovarian stimulation outcomes.

The purpose of this study is to investigate whether individual response of anti-Mullerian hormone (AMH) to gonadotropin-releasing hormone (GnRH) treatment is associated with difference in ovarian stimulation outcomes. The retrospective study included 1058 non-polycystic ovary syndrome (PCOS) women undergoing long agonist protocol in a single in vitro fertilization unit from January 1, 2016, through December 31, 2016. Patients were grouped according to AMH changes from day 3 to the day of stimulation (group 1, change < 1ng/ml, n = 714; group 2, decrease ≥ 1ng/ml, n = 143; group 3, increase ≥ 1ng/ml, n = 201). A generalized linear model including Poisson distribution and log link function was used to evaluate the association between AMH response and the number of oocytes retrieved. Group 2 was characterized by higher basal AMH level and increased AMH to AFC ratio in comparison with two other groups. However, the number of oocytes and ovarian sensitivity index in group 2 was significantly lower than group 3. Adjusted for age, BMI, ovarian reserve markers, and stimulation parameters, the population marginal means (95% confidence interval) of oocyte number in groups 1 through 3 were 9.51 (9.17, 9.86), 8.04 (7.54, 8.58), and 10.65 (10.15, 11.18), respectively. For patients from group 2 and group 3, basal AMH is no longer significantly associated with oocyte yield. AMH change in response to GnRH agonist varies among individuals; for those undergoing significant changes in AMH following GnRH agonist treatment, basal AMH may not be a reliable marker for ovarian response in long agonist protocol.

62 Will Gonadotropin-Releasing Hormone Treatment Hasten the Onset of Puberty in Peripubertal Heifers?

Treatment with gonadotropin-releasing hormone (GnRH) has been used to induce ovulation in prepubertal heifers. The objective of this study was to evaluate whether peripubertal heifers will continue to ovulate at regular intervals (i.e. attain puberty) after GnRH treatment. Prepubertal crossbred Hereford heifers, 11.0 ± 0.5 months of age, 344 ± 26 kg of body weight, and at random stages of ovarian follicular wave status, were assigned to 2 groups (age- and weight-matched) and given GnRH (n = 24) or no treatment (Control, n = 22) on Day 0. Ovarian ultrasonography was performed every second day from Day 0 to 44 to record the size of 2 largest follicles and the corpus luteum (CL). Thereafter, examinations were done every 4 days until the third ovulation or Day 100, whichever came first. The first and second interovulatory intervals (IOI) were categorized as short (&lt;12 days), normal (16-24 days), or long (&gt;24 days), and the onset of the puberty was defined by the occurrence of 2 consecutive short or normal IOI (i.e. 3 uninterrupted ovulations). Proportional data were compared among groups by chi-squared test. Single-point measurements were compared by analysis of variance and multiple comparisons were made using Tukey’s test. Two heifers (one in each group) failed to ovulate during the experiment. The diameter of the largest follicle on Day 0 did not differ (P = 0.31) between the GnRH and Control groups (12.6 ± 0.37 mm and 13.1 ± 0.29 mm, respectively). The proportion of heifers that ovulated by Day 4 after treatment was higher in the GnRH than in the Control group (9/24 v. 1/22; P &lt; 0.01). However, the proportion of heifers in the GnRH v. Control groups that ovulated twice (19/24 v. 18/22; P = 0.60) or 3 times (9/24 v. 9/22; P = 0.81) did not differ. The age at first ovulation tended to be younger in the GnRH group than in the Control (12 ± 0.97 v. 13 ± 0.90 months; P = 0.07), but the effect was attributed only to those heifers that ovulated in response to GnRH treatment (n = 9). The age at first ovulation was 11.2 ± 0.50, 12.7 ± 0.73, and 12.6 ± 0.90 months in GnRH-responders, non-responders, and the control group, respectively (P &lt; 0.0001). One heifer that ovulated in response to GnRH treatment failed to ovulate again during the study period (Day 100). In 87.5% (7/8) of the heifers that responded to GnRH treatment and ovulated at least twice, the first IOI was long (55.5 ± 8.3 days). When data were combined between the GnRH non-responders and the control group (n = 35), the first IOI was short (8.9 ± 0.4 days) in 77% of the heifers, and the second IOI was of normal length (18.8 ± 0.9 days) in 93% of heifers. The age at the second ovulation in GnRH responders (12.7 ± 0.3 months) was similar (P = 0.82) to the age at first ovulation in GnRH non-responders (12.9 ± 0.1 months) and the control group (12.7 ± 0.2 months), indicating that GnRH treatment did not hasten the onset of continuous cyclicity. In conclusion, although GnRH treatment induced first ovulation in some peripubertal heifers, treatment did not hasten the onset of puberty. Research was supported by the Natural Sciences and Engineering Research Council of Canada.

12 Ovulation Timing Following an Aromatase Inhibitor-Based Synchronization Protocol in Beef Heifers and Cows

The study was conducted to determine the effects of parity/lactation and the timing of gonadotropin-releasing hormone (GnRH) treatment on the efficacy of a non-steroidal aromatase inhibitor-based synchronization protocol in cattle. Results from previous studies confirmed drug-release from a new letrozole-impregnated intravaginal silicone device, which was used in the present study. Hereford-cross cows with suckling calves (41 to 65 days postpartum; n = 30) and sexually mature heifers (n = 30), at random stages of the oestrous cycle, were given a letrozole intravaginal device for 4 days followed by a luteolytic dose of prostaglandin F2α (PGF2α). Following PGF2α treatment, animals were assigned randomly to 3 groups and given GnRH (100 μg of gonadorelin) intramuscularly at 48 or 60 h, or no GnRH (n = 10 cows and 10 heifers per group). Ovaries were examined by transrectal ultrasonography every 8 h starting at the time of PGF2α treatment to record follicle diameter and ovulation. After ovulation, ultrasonography was done every 24 h until Day 10 (Day 0 = ovulation) to assess the corpus luteum (CL) diameter profile. The timing of ovulation, diameter of the preovulatory follicle, synchrony of ovulation, and Day-7 CL diameter were compared using two-way ANOVA, and CL diameter profiles were compared by two-way ANOVA for repeated measures. There was no treatment × parity/lactation status interaction for any endpoint. The ovulation rate within 96 h of PGF2α treatment was not different between heifers and cows (24/30 v. 27/30; P = 0.14) or treatment group (18/20, 18/20, and 15/20 in the 48 h, 60 h, and no GnRH groups, respectively; P = 0.18). The interval from PGF2α treatment to ovulation was not influenced by parity/lactation (83.1 ± 2.4 h) but was shortest in the GnRH 48 h group (mean ± SEM; 74.2 ± 2.7 h, 85.6 ± 4.8 and 89.2 ± 4.1, respectively; P &lt; 0.05). Similarly, the variation in the interval to ovulation (mean ± s.e.M of residuals) was not influenced by parity/lactation (16.0 ± 2.0 h), but was lower in the GnRH groups than the no-GnRH group (P &lt; 0.01), and tended to be lower (P = 0.1) in the GnRH 48-h v. 60-h group (10.0 ± 2.8, 14.2 ± 3.5, and 24.1 ± 3.1 h, respectively). The maximum diameter of the ovulatory follicle was larger for cows than heifers (17.0 ± 0.4 v. 15.1 ± 0.5; P &lt; 0.01), and was smaller in the GnRH groups than the no-GnRH group (15.3 ± 0.3, 15.4 ± 0.7 and 17.3 ± 0.5 mm, respectively; P &lt; 0.01). The diameter of the CL on Day 7 was larger for cows than heifers (22.3 ± 0.8 v. 20.2 ± 0.6 mm; P &lt; 0.05) and was influenced by treatment (21.9 ± 0.5, 19.5 ± 0.7, 22.3 ± 1.1 mm, respectively; P = 0.05). A tendency for a treatment effect on CL diameter profile (P = 0.1) was attributed to a smaller profile in the GnRH 60-h group. In conclusion, GnRH treatment 48 h after PGF2α treatment increased synchrony of ovulation without adverse effects on ovulating follicle diameter or resulting CL growth, and may be incorporated into a novel steroid-free oestrous synchronization protocol for use in beef heifers and lactating cows. Research was supported by the Alberta Livestock and Meat Agency and Vencofarma, Brazil.

Reproductive performance of lactating dairy cows after inducing ovulation using hCG in a five-day progesterone-based fixed-time AI protocol

This study compares the effects of inducing ovulation using human chorionic gonadotropin (hCG) or gonadotropin releasing hormone (GnRH) at the end of a 5-day progesterone(P4)-based protocol for fixed-time artificial insemination (FTAI) in lactating dairy cows on ovarian dynamics and fertility. A P4 intravaginal device (PRID) was fitted for five days and GnRH administered upon PRID insertion and a double dose (24 h apart) of prostaglandin F2α upon PRID removal. Cows received either GnRH (GnRH group; n = 98), 1000 IU hCG (hCG-1 group; n = 97), or 3000 IU hCG (hCG-2 group; n = 104) 36 h after PRID removal and were inseminated 50–56 h after PRID removal. Based on Tukey-Kramer tests, cows in hCG-1 and hCG-2 showed a larger follicle diameter at AI than cows in GnRH (P < 0.05). HCG-2 treatment increased corpus luteum (CL) size on Day 7 post-AI compared with the GnRH and hCG-1 treatments (P < 0.05). Based on odds ratios, the likelihood of ovulation failure decreased with increasing follicle diameter at AI (P = 0.002). Cows in hCG-1 or hCG-2 that did not become pregnant were more likely to return to estrus than cows in GnRH (P < 0.01). A larger CL size post-AI was related to an increased conception rate at FTAI (P = 0.003). Cows given hCG-2 treatment showed a 1.9-fold cumulative pregnancy rate after two rounds of AI compared to cows receiving GnRH. Our results indicate that hCG treatment hCG treatment used to induce ovulation at the end of a short protocol for FTAI improves follicular/luteal dynamics compared to GnRH treatment. Of the two hCG treatments tested, better results were obtained with the 3000 IU dose.

Clinical characteristics of 138 Chinese female patients with idiopathic hypogonadotropic hypogonadism.

ObjectiveTo evaluate the clinical features of Chinese women with idiopathic hypogonadotropic hypogonadism (IHH).MethodsWe retrospectively reviewed the clinical characteristics, laboratory and imaging findings, therapeutic management and fertility outcomes of 138 women with IHH. All patients had been treated and followed up at an academic medical centre during 1990–2016.ResultsAmong the 138 patients, 82 patients (59.4%) were diagnosed with normosmic IHH and 56 patients (40.6%) were diagnosed with Kallmann syndrome (KS). The patients with IHH experienced occasional menses (4.3%), spontaneous thelarche (45.7%) or spontaneous pubarche (50.7%). Women with thelarche had a higher percentage of pubarche (P < 0.001) and higher gonadotropin concentrations (P < 0.01). Olfactory bulb/sulci abnormalities were found during the magnetic resonance imaging (MRI) of all patients with KS. Most patients with IHH had osteopenia and low bone age. Among the 16 women who received gonadotropin-releasing hormone treatment, ovulation induction or assisted reproductive technology, the clinical pregnancy rate was 81.3% and the live birth rate was 68.8%.ConclusionsThe present study revealed that the phenotypic spectrum of women with IHH is broader than typical primary amenorrhoea with no secondary sexual development, including occasional menses, spontaneous thelarche or pubarche. MRI of the olfactory system can facilitate the diagnosis of KS. Pregnancy can be achieved after receiving appropriate treatment.

Oxytocin intranasal administration as a new hope for hypogonadotropic hypogonadism patients

Hypogonadotropic hypogonadism (HH) is a form of hypogonadism which also known as secondary or central hypogonadism. Congenital HH can occur due to defect in gonadotropin releasing hormone (GnRH) neurons, upstream regulators of GnRH neurons or pituitary gonadotropic cells. Testosterone or gonadotropins therapy are widely used to treat HH patients, however both have undesirable effects and GnRH treatment for HH patients is time and cost consuming. Direct delivery of therapeutics to the brain via the nasal route is located in the center of attention during the last decade and trial application of intranasal oxytocin as a method of enhancing social interactions are reported. It has been delineated that oxytocin stimulates GnRH release from the rat hypothalamic explants and intranasal applied oxytocin up-regulates GnRH expression in the male rat hypothalamus. Therefore application of intranasal oxytocin might be a new strategy to cure HH patients.

Progesterone concentration, pregnancy and calving rate in Simmental dairy cows after oestrus synchronisation and hCG treatment during the early luteal phase.

Early embryonic development may be negatively affected by insufficient progesterone (P4) production. Therefore, the aim of our study was to increase P4 by gonadotropin-releasing hormone (GnRH) and/or human chorionic gonadotropin (hCG) treatments after inducing oestrus by prostaglandin (PG) treatment. Lactating Simmental dairy cows (n = 110), between 1 to 5 lactations, with an average milk production of 6,500 1/305 days, at 40-80 days postpartum were used and grouped as follows: (1) PG + GnRH treatment at AI (GnRH group), (2) PG + hCG treatment at day 7 after AI (hCG group), (3) PG + GnRH at AI + hCG treatment at day 7 after AI (GnRH/hCG group), and (4) spontaneous oestrus (C: control group). All animals were double inseminated (at the time of oestrus detection and 12 ± 2 h thereafter). Blood serum and milk samples were collected at the day of observed oestrus (day 0), and 14, 21 and 28 days after AI. Serum P4 was determined using a commercial radioimmunoassay (RIA) test (INEP, Zemun), and milk P4 was determined using enzyme-linked immunoassay (ELISA) test (NIV Novi Sad). Pregnancy status was confirmed by ultrasonography between days 28 and 35 after AI. Differences of serum or milk P4 medians, pregnancy (and calving) rate were determined using Dunn's Multiple Comparison Tests and Z test, respectively. Serum P4 medians were significantly higher at days 14, 21 and 28 after AI in the hCG-treated animals, indicating increased luteal activity, with a similar tendency in whole milk P4 values. Treatment with hCG during the early luteal phase significantly contributed to the maintenance of gestation at days 28-35 after AI, and also increased the calving rate in Simmental dairy cows.

The effect of circulating progesterone on magnitude of the GnRH-induced LH surge: Are there any differences between Bos indicus and Bos taurus heifers?

This study evaluated the effects of differing circulating progesterone (P4) levels on the luteinizing hormone (LH) surge profile following treatment with gonadotropin releasing hormone (GnRH) in B. indicus (Nelore, n = 13) and B. taurus (Holstein, n = 16) heifers. All heifers were synchronized with a hormonal protocol to induce either a Low or High circulating P4 environment at the time of GnRH treatment. Heifers were randomly assigned to a 2 × 2 factorial design composed by two genetic groups (B. indicus and B. taurus) and two levels of circulating P4 concentrations (Low or High). Blood samples were collected every 30 min from −30 to 210 min and at 270 min after GnRH treatment. As expected, mean P4 concentration was greater for cows in the High than in the Low P4 group (P = 0.0008) and in Bos indicus than in Bos taurus heifers (P = 0.06). Despite genetic group, the area under the curve of LH release was greater in the Low-P4 than in High-P4 concentration group (P < 0.0001). Interestingly, it appears that High P4 concentrations had a more pronounced effect on LH peak in B. indicus than in B. taurus heifers, as indicated by the interaction (P = 0.01) between genetic group and P4 levels. In conclusion, circulating P4 concentration have a great impact on the GnRH-induced LH surge of both B. indicus and B. taurus heifers, but it does not explain the much lower LH peak in B. indicus with low circulating P4. Thus, more studies are essential to uncover some of the underlying physiological factors other than circulating P4 that are limiting LH release following a GnRH treatment in B. indicus cattle.

Effects of GnRH, a progesterone-releasing device, and energy balance on an oestrus synchronisation program in anoestrous dairy cows.

The aim of this research was to study the roles of the day 0 energy balance and gonadotrophin-releasing hormone (GnRH) and progesterone levels on dominant follicle (DF) and corpus luteum (CL) development during the first 7 days of a gonadotrophin-prostaglandin-gonadotrophin (GPG) + progesterone (P4) program in anoestrous dairy cows. Cows (n = 81) were allocated to one of the three treatments: (1) GPG + P4 (days 0 and 9, 100 µg GnRH; day 0-7, intravaginal P4 device; day 7, 500 µg PGF2α ); (2) GPG (as for treatment 1 but excluding the P4 device) and (3) prostaglandin + GnRH + P4 (as for treatment 1, but excluding day 0 GnRH). DF and CL size, plasma concentrations of insulin, insulin-like growth factor-I (IGF-I) and non-esterified fatty acid (NEFA) were measured on days 0 and 7. The proportion of cows with a CL on day 7 was significantly different between groups (GPG: 78%, GPG+P4: 69%, PGF2α + GnRH + P4: 42%, P = 0.02). The CL volume on day 7 was significantly associated with treatment, treatment by time postpartum and plasma concentrations of insulin, IGF-I and NEFA. In cows without a CL present on day 0 of an oestrus synchronisation program, removal of the day 0 GnRH treatment led to reduced CL development; however, no effect of adding progesterone was found. In contrast, in cows with a CL present on day 0 inclusion of a progesterone device led to a higher CL volume, but removal of the first GnRH injection had no effect. Response to the treatment was affected by plasma concentrations of insulin, IGF-I and NEFA.

Comparison of three superovulation protocols with or without GnRH treatment at the time of artificial insemination on ovarian response and embryo quality in Thai native heifers.

To optimize the superovulation protocol in Thai native cattle, the present research was designed to (1) compare three different protocols designed to induce superstimulation and (2) study the effect of gonadotropin-releasing hormone (GnRH) administration at insemination time (to induce ovulation) on ovarian follicular activities in terms of the number of large follicles, corpora lutea (CLs) and unovulated follicles, and the number and quality of ova/embryos recovered in Thai native heifers. Initially, the estrous cycles of animals (n = 36) at unknown stages were synchronized by two prostaglandin F2α (PGF2α) injections at an interval of 12days. Follicular development of heifers was randomly superstimulated with one of three different treatment protocols: treatment A-a total of 100mg of pituitary-derived FSH (pFSH; Folltropin®-V) administered in eight decreasing doses; treatment B-a single dose of 100mg pFSH dissolved in 30% (w/v) polyvinylpyrrolidone; or treatment C-ablation of all follicles ≥5mm with a single dose of pFSH. All heifers received PGF2α 48h after the initiation of FSH treatment to induce luteolysis from the previous cycle, and they were twice inseminated at 12 and 24h after the onset of estrus. Heifers in each treatment were assigned to be injected or not with GnRH at the time of first insemination with frozen/thawed semen to induce ovulation. About 7days after artificial insemination (AI), ova/embryos were collected and classified. The numbers of large follicles at the onset of estrus were not statistically significantly different; meanwhile, the maximum diameters of follicles at the time of first insemination in treatment C were smaller compared with the other treatment groups (p < 0.001). The administration of GnRH at the first insemination time resulted in a greater number of CLs and fewer unovulated follicles at the time of ova/embryo collection (p = 0.001), which subsequently resulted in a higher number of total ova/embryos recovered (p = 0.030). Among heifers treated with different superstimulation protocols, the ablation of all follicles ≥5mm in diameter before superstimulation (treatment C) resulted in significantly higher quality of fertilized ova and transferable embryos (p = 0.001). In summary, it could be inferred that GnRH treatment improved ovarian function rather than embryo quality. Dominant follicle ablation prior to superstimulation is preferable for collecting a greater number of transferable embryos.

ErbB4 cleavage by gonadotropin-releasing hormone receptor stimulation in cultured gonadotroph cells

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G-protein-coupled receptors, and its stimulation activates extracellular signal-regulated protein kinase (ERK). In the present study, we first examined the actions of GnRH on the ErbB family using two types of cultured gonadotroph cells. As reported previously, AG1478, an inhibitor of the ErbB family tyrosine kinase, inhibited GnRH-induced ERK activation in undifferentiated gonadotroph αT3-1 cells. However, AG1478 did not inhibit ERK activation in differentiated gonadotroph LβT2 cells, suggesting that transactivation of the ErbB family was not necessary for ERK activation in LβT2 cells. We found that ErbB4 was expressed in αT3-1 cells but not in LβT2 cells. GnRH induced the cleavage of ErbB4 and accumulation of an 80-kDa fragment in αT3-1 cells. Pharmacological experiments suggested that Gq/11 and tumor necrosis factor-α-converting enzyme (TACE) were essential for GnRH-induced ErbB4 cleavage. GnRH increased the phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), indicating that GnRH activated protein kinase C (PKC). Down-regulation of PKC and bisindolylmaleimide I, a PKC inhibitor, strongly inhibited the GnRH-induced cleavage of ErbB4. It was surprising that GnRH treatment of LβT2 cells after overexpression of ErbB4 induced ErbB4 cleavage in a TACE-dependent manner. ErbB4 cleavage was induced also by treatment of αT3-1 cells, ErbB4-overexpressing LβT2 cells, and immortalized GnRH neurons (GT1-7 cells) with leuprorelin acetate. These results may suggest that the pharmacological effects of leuprorelin acetate are conducted through TACE-mediated proteolysis of membrane proteins, including ErbB4, in gonadotroph cells and GnRH neurons.

Administration of gonadotropin-releasing hormone agonist on Day 5 increases luteal blood flow and improves pregnancy prediction accuracy on Day 14 in recipient Holstein cows.

This study assessed the effects of gonadotropin-releasing hormone (GnRH) treatment on Day 5 (Day 0 = estrus) on luteal blood flow and accuracy of pregnancy prediction in recipient cows. On Day 5, 120 lactating Holstein cows were randomly assigned to a control group (n = 63) or GnRH group treated with 100 μg of GnRH agonist (n = 57). On Days 3, 5, 7, and 14, each cow underwent ultrasound examination to measure the blood flow area (BFA) and time-averaged maximum velocity (TAMV) at the spiral arteries at the base of the corpus luteum using color Doppler ultrasonography. Cows with a corpus luteum diameter ≥ 20 mm (n = 120) received embryo transfers on Day 7. The BFA values in the GnRH group were significantly higher than those in the control group on Days 7 and 14. TAMV did not differ between these groups. According to receiver operating characteristic analyses to predict pregnancy, a BFA cutoff of 0.52 cm2 yielded the highest sensitivity (83.3%) and specificity (90.5%) on Day 7, and BFA and TAMV values of 0.94 cm2 and 44.93 cm/s, respectively, yielded the highest sensitivity (97.1%) and specificity (100%) on Day 14 in the GnRH group. The areas under the curve for the paired BFA and TAMV in the GnRH group were 0.058 higher than those in the control group (0.996 and 0.938, respectively; P < 0.05). In conclusion, GnRH treatment on Day 5 increased the luteal BFA in recipient cows on Days 7 and 14, and improved the accuracy of pregnancy prediction on Day 14.

MiR-361-3p regulates FSH by targeting FSHB in a porcine anterior pituitary cell model

FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.