Deglycosylated FSH is known to trigger poor Galphas coupling while efficiently binding its receptor. In the present study, we tested the possibility that a deglycosylated equine LH (eLHdg) might be able to selectively activate beta-arrestin-dependent signaling. We compared native eLH to an eLH derivative [i.e. truncated eLHbeta (Delta121-149) combined with asparagine56-deglycosylated eLHalpha (eLHdg)] previously reported as an antagonist of cAMP accumulation at the FSH receptor (FSH-R). We confirmed that, when used in conjunction with FSH, eLHdg acted as an antagonist for cAMP accumulation in HEK-293 cells stably expressing the FSH-R. Furthermore, when used alone at concentrations up to 1 nM, eLHdg had no detectable agonistic activity on cAMP accumulation, protein kinase A activity or cAMP-responsive element-dependent transcriptional activity. At higher concentrations, however, a weak agonistic action was observed with eLHdg, whereas eLH led to robust responses whatever the concentration. Both eLH and eLHdg triggered receptor internalization and led to beta-arrestin recruitment. Both eLH and eLHdg triggered ERK and ribosomal protein (rp) S6 phosphorylation at 1 nM. The depletion of endogenous beta-arrestins had only a partial effect on eLH-induced ERK and rpS6 phosphorylation. In contrast, ERK and rpS6 phosphorylation was completely abolished at all time points in beta-arrestin-depleted cells. Together, these results show that eLHdg has the ability to preferentially activate beta-arrestin-dependent signaling at the FSH-R. This finding provides a new conceptual and experimental framework to revisit the physiological meaning of gonadotropin structural heterogeneity. Importantly, it also opens a field of possibilities for the development of selective modulators of gonadotropin receptors.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 of 20A c c e p t e d M a n u s c r i p t ABSTRACT Follicle-stimulating hormone (FSH) controls the proliferation and differentiation of Sertoli cells of the testis. FSH binds a G protein-coupled receptor (GPCR) to stimulate downstream effectors of the phosphoinositide-3 kinase (PI3K)-dependent pathway, without enhancing PI3K activity. To clarify this paradox, we explored the activity of phosphatase and tensin homolog deleted in chromosome 10 (PTEN), the PI3K major regulator, in primary cultures of rat Sertoli cells. We show that, within minutes, FSH increases PTEN neosynthesis, requiring the proteasomal degradation of an unidentified intermediate, as well as PTEN enzymatic activity. Importantly, introducing an antisense cDNA of PTEN into differentiating Sertoli cells restores FSHdependent cell proliferation. In conclusion, these results provide a new mechanism of PTEN regulation, which could serve to block entry into S phase of Sertoli cells, while they are proceeding through differentiation in pre-pubertal animals.
The injection of equine chorionic gonadotropin (eCG) in dairy goats induces the production of anti-eCG antibodies (Abs) in some females. We have previously shown that Abs negatively modulate the LH and FSH-like bioactivities of eCG, in most cases, compromising fertility in treated females. Surprisingly, we found out that some anti-eCG Abs improved fertility and prolificity of the treated females, in vivo. These Abs, when complexed with eCG, enhanced LH and FSH ability to induce steroidogenesis on specific target cells, in vitro. In the present study, we analyzed the impact of three eCG/anti-eCG Ab-enhancing complexes on two transduction mechanisms triggered by the FSH receptor: guanine nucleotide-binding protein alphaS-subunit/cAMP/protein kinase A (PKA) and beta-arrestin-dependent pathways, respectively. In all cases, significant enhancing effects were observed on ERK phosphorylation compared with eCG alone. However, cAMP production and PKA activation induced by eCG could be differently modulated by Abs. By using a pharmacological inhibitor of PKA and small interfering RNA-mediated knock-down of endogenous beta-arrestin 1 and 2, we demonstrated that signaling bias was induced and was clearly dependent on the complexed Ab. Together, our data show that eCG/anti-eCG Ab-enhancing complexes can differentially modulate cAMP/PKA and beta-arrestin pathways as a function of the complexed Ab. We hypothesize that enhancing Abs may change the eCG conformation, the immune complex acquiring new "biased" pharmacological properties ultimately leading to the physiological effects observed in vivo. The modulation of ligand pharmacological properties by Abs opens promising research avenues towards the optimization of glycoprotein hormone biological activities and, more generally, the development of new therapeutics.
Successful superovulation and embryo collection require donors to be managed for precise insemination intervals. At times, determinations of these intervals can become difficult because of oestrus detection subjectivity. The objective of this study was to determine the feasibility of performing a rapid cow-side LH assay (Predi′Bov®, ReproPharm, Nouzilly, France) on superovulated donors with emphasis on determining how to use the results in a commercial program to time inseminations. This study was conducted at Ovagenix (Bryan, TX, USA) on 24 superovulated donors. On Day 0 cows received an intravaginal progesterone-releasing device (CIDR®, Pfizer Animal Health, New York, NY, USA) and were administered 25 mg of progesterone and 2.5 mg of oestradiol 17β intramuscularly (IM, 2 mL). Superstimulation was induced using 150–300 mg of Folltropin-V® (FSH, Bioniche Animal Health Inc., ON, Canada) administered with 7 injections at 12-h intervals in decreasing dosages (Day 4–7). On Day 6, two doses of Estrumate® (equaling 1 mg, Intervet Inc., Merck Animal Health, Summit, NJ, USA) were given IM, concurrently with FSH injections 5 and 6 with CIDR removal on Day 7 (a.m.). HeatWatch® transmitters (Cow Chips LLC, Denver, CO, USA) were applied to donors on Day 7 to determine onset of standing oestrus. Blood samples were collected in heparinized tubes via tail vein puncture starting at CIDR removal continuing every 6 h until a positive Predi′Bov® test was acquired or 36 h after CIDR removal. Whole blood (0.5 mL–1) was submitted to a proprietary 3-step procedure using a tube-stick applicator (40 min) to determine whether increased concentrations of LH were present. Stick applicators that turned blue were positive for increased concentrations of LH. Donors were artificially inseminated 12 and 24 h after a positive test, with specified sires, and embryos were nonsurgically collected 7 days after insemination. The Predi′Bov® test determined 91.6% (22/24) of the LH peaks occurred within 12 to 24 h after CIDR removal; 45.8% (11/24) occurred at 18 h; and 4% (1/24) at 30 h. A total of 203 viable embryos (average 8.5/donor) was collected with 174 embryos (average 9.2 ± 4.7/donor) from donors that exhibited oestrus and produced a positive test (19/24; 79.2%) and 29 embryos (average 5.8 ± 6.3/donor) from donors exhibiting silent oestrus that produced a positive test (5/24; 20.8%). One donor exhibited oestrus and never produced a positive test, but contributed 8 viable embryos to the total. In conclusion, Predi′Bov® cow-side usage could offer commercial utility when identifying oestrus is difficult or nonexistent. Increased detection of ovulation could help to identify optimal AI intervals, thereby increasing embryo production and limiting the use of expensive semen for desired donors.
The LH peak in cattle is the most precise event for predicting ovulation beginning 24 h later, and thus, AI time. Previous studies demonstrated that embryo production was improved when AI was conducted 12 h before ovulation; that is, 12 h after LH peak. This study aimed to evaluate the benefit of LH peak monitoring with Predi′Bov® (ReproPharm®, Nouzilly, France) following superstimulation in order to optimize numbers of viable embryos (VE). Predi′Bov® is a rapid (40 min) and easy to use on-farm test allowing LH peak detection from a few drops of blood. The test was also used to estimate the variability in the time of the LH peak and onset of oestrus. This study was conducted by the embryo transfer teams of 3 French cooperatives, in collaboration with UNCEIA. Forty heifers in stations (Creavia, Midatest) and 23 cows on farms (GEN′Iatest) were superstimulated by 8 injections IM of Stimufol® or Pluset® (FSH1 to FSH8) over 4 days in 2011–2012. Donor station heifers were treated twice in a Latin square design with a reference protocol where AI was conducted 12 and 24 h after onset of oestrus, and in an experimental protocol where AI was conducted 12 and 24 h after a positive Predi′Bov® test. Semen of different sires was used for both protocols. The Predi′Bov® test was carried out on 3 blood samples (BS1,2,3) collected every 6 h beginning at FSH7 in stations and FSH8 on farms to detect the earliest LH peaks. To determine late LH peaks, Predi′Bov® test was carried out on BS4 collected 24 h after FSH8. Univariate statistical analysis was performed to look at the relationship between qualitative (chi-square) and quantitative (t-test) variables. The difference was considered significant when P < 0.05. The Predi′Bov® test showed that 37.5% (15/40) of LH peaks occurred during the last day of FSH treatment (BS1 or BS2) in stations and 26.1% (6/23) at BS1 on farms. At Creavia station (n = 24), the LH peak was detected anytime from 24 h before to 9 h after the onset of oestrus. In stations, the VE percentage did not differ whether AI was done following oestrus or LH peak detection (63.1% and 61.8% in reference and experimental protocols, respectively). In stations, the VE percentage from 9 females with an LH peak detected at FSH7 (BS1; 41%) in the reference protocol did not differ from the experimental protocol (50%). On farms, the VE percentage was numerically higher but not significant in the experimental protocol (65.4%, n = 16) compared to the reference protocol (47.2%, n = 7). Further investigations are needed, taking into account the effect of collection rank, sire, and female effects, to confirm the trends shown by these results. In conclusion, Predi′Bov® can be used as easily on farms as in stations. Its use allows the detection of animals that have early or late LH peaks, which in turn provides the opportunity of carrying out AI at the optimal time for such females.
Insemination of superovulated bovine donors in due time is of central importance for fertilization and embryo viability. A preliminary test focusing on LH surge detection during superovulation (unpublished datas) indicated that one quarter of the donors present LH surges 12 to 24 h before heat observation (which could correspond, in the case of AI after heat observation, to post ovulation AI). Therefore, it was hypothesised that the average number of embryos per flush could be improved by inseminating donors with early LH surge 12 h after the beginning of the LH peak whenever the heat occurs.In a donor herd station, a trial was performed with 54 Holstein heifers, equipped with Heatime® tags (system detecting the peak activity linked to the heat) collected twice or 3 times after the following superovulation protocol: D-6 to D-11 = reference heat; D0 = input of an implant of norgestomet (Crestar®); D2 8:00 = first FSH (Stimufol®) injection (FSH1); D4 8:00 = cloprostenol (Estrumate®) injection; D4 16:00 = implant removal; D5 8:00 = FSH7 and first LH surge detection test (Predi'Bov®); D5 16:00 = FSH8 and 2nd Predi'Bov® test. Two AI's at interval of 8 to 16h were done (AI's were performed either at 9:00, or between 17:00 and 19:00). For the standard protocol (= STA), thefirst AI occurred after heat observation or activity peak detection by Heatime (whatever the Predi'Bov® test results were). For the adjusted protocol (= ADJ), the first AI occurred from 11 to 16 h after the first positive Predi'Bov® test result or like STA protocol if both results were negative. Heifers followed alternately the 2 protocols, 27 beginning with the ADJ protocol, 27 others with the STA one. LH surge precocity was not repeatable among donors. In the case of an early LH surge detection (one positive Predi'Bov® test), the heat activity peak occurred from 2 to 8 h after the FSH8 for 44% of the flushes, from 8 to 24 h after FSH8 for 54% of the flushes and never for 2% of the flushes. When no early LH surge was observed, the heat activity peak occurred more than 8 h after FSH8 for 78% of the flushes. Interval of heat activity peak-FSH8, IA1-heat activity peak and IA1-early LH surge were highly variable but did not effect the mean number of viable embryos. However, we observed a significant effect (P = 0.04) of the precocity of the heat on the average number of total embryos: 13.8 ± 8.4 v. 11.1 ± 8.1 when the interval heat activity peak-FSH8 had been respectively <8 h or ≥8 h, respecively. Among the 148 collections, 74 were done after the STA protocol, 74 after the ADJ protocol and 70 followed an early LH surge. The adjustment of the AI depending on the detection of an early LH surge (ADJ protocol) had a significant positive (P = 0.04) effect on the mean percentage of viable embryos per flush (52% ± 28 in STA group and 62% ± 31 in ADJ group). Nevertheless, regarding the mean number of viable embryos, this effect failed to reach significance (P = 0.23) (respectively 5.7 ± 5.1 in STA group and 6.7 ± 6.7 in ADJ group). A larger study on more animals is necessary to obtain a significant difference in the number of viable embryos.
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